bassianolide and Fish-Diseases

Haematological parameters are frequently used as physiological indicators in aquaculture studies. These parameters also have extended applications in clinical evaluation, diagnosis and prognosis in fish health status. However, no normal reference range of values has been demonstrated in depth for any of these haematological parameters for the European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus aurata). The main objective of this article is to present for the first time through extended literature review, the haematological parameters normal range values for the two most important aquaculture fish species farmed in Mediterranean Sea, D. labrax and S. aurata, and to demonstrate their similarities and their differences. In this article, we also discuss the environmental and external factors affecting their normal blood parameters values and we propose fundamental guidelines on the reporting units.

Topics: Animals; Bass; Fish Diseases; Hematology; Reference Values; Sea Bream

Good knowledge on the disease situation and its impact on production is a base mechanism for designing health surveillance, risk analysis and biosecurity systems. Mediterranean marine fish farming, as any aquaculture production, is affected by various infectious diseases. However, seabass and seabream, the main produced species, are not listed as susceptible host species for the notifiable pathogens listed in the current EU legislation, which generates a lack of systematic reporting. The results presented in this study come from a survey directly to fish farms (50 hatchery and on-growing units from 10 Mediterranean countries), with data from 2015 to 2017, conducted by the H2020 project MedAID. Seabass showed a higher survival rate (85%) through a production cycle than seabream (80%) in spite of equal mortality due to pathogen infections (10%). The differences in survival may be explained by mortality 'of other causes'. Seabream and seabass have different disease profiles, and the profile is slightly different between geographical regions. Among the most important diseases, tenacibaculosis and vibriosis were identified in seabass and Sparicotyle chrysophrii (a gill fluke) and nodavirus in seabream. Correlating mortality data to management variables showed that increasing density, buying fingerlings from external sources and treatments due to disease are factors that negatively influence mortality rate. Most of the surveyed farms did not keep sufficient quality data to implement good health status reports and perform detailed impact studies, which shows the necessity of updating the current legislative framework to provide the basis for better reporting of relevant pathogens in the Mediterranean basin.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Fisheries; Flavobacteriaceae Infections; Mediterranean Region; Sea Bream; Surveys and Questionnaires; Vibrio Infections

This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.

Topics: Animals; Bass; Fish Diseases; Immunity, Innate; Immunity, Mucosal; Nodaviridae; RNA Virus Infections; Vaccination

Among the topics related to invasion science, the least studied are parasite co-introduction and spillback. This leads to an uncertainty in invasion ecology theories and applications to management. Therefore, the present study brings a systematic review of published information on the metazoan parasite fauna of Micropterus salmoides, a widely introduced fish, with the aim of comparing information about the composition and richness of the associated parasite communities in its native and introduced regions. This review demonstrates that there were twice as many studies of M. salmoides in its native region in comparison with introduced regions, although most of the studies focused on the analysis of a single species or taxon of parasite. This bias impacts the number of parasite species observed and, consequently, the apparent importance of enemy release in introduced regions. The composition of the parasite community in the two regions showed high similarity, which indicates the introduction and acquisition of parasites in introduced regions. Otherwise, there was no pattern related to the geographic distance, highlighting the influence of the propagule pressure and vector strength on the introduction of novel parasites. This illustrates the importance of vector strength on fish-parasite co-introduction and the necessity of new research examining host-parasite interactions with the parasite community of the invaded ecosystems. We still do not know the major influences of the composition of the parasite fauna of M. salmoides or how we can manage to develop a more restrictive vector pathway of introduction. The future of our ecosystems depends on how to account for current and future interactions among novel interactions, habitat, and climate change.

Topics: Animals; Bass; Ecology; Ecosystem; Fish Diseases; Helminths; Host-Parasite Interactions; Introduced Species; Parasite Load

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

Topics: Animals; Aquaculture; Bass; Biomarkers; Electrophoresis, Polyacrylamide Gel; Fish Diseases; Melanins; Monophenol Monooxygenase; Nephrocalcinosis; Peroxidase; Pigments, Biological; Tyrosine 3-Monooxygenase; X-Ray Absorption Spectroscopy

Mycobacterium species have long been recognised as a significant source of morbidity and mortality in finfish aquaculture, as well as in wild finfishes. Mycobacteria infecting fishes also include zoonotic pathogens that can cause protracted illness, especially in immunocompromised individuals. Several basic aspects of mycobacterial pathobiology in aquatic animals remain poorly understood, although a number of important recent developments have been made, especially with respect to identification of novel Mycobacterium spp. infecting fishes and a new group of mycobacteria closely related to the human pathogen Mycobacterium ulcerans. This review will encompass important aspects of mycobacterial disease in fishes, discuss recent research including studies of mycobacteriosis in striped bass (Morone saxatilis) of Chesapeake Bay, USA, and suggest directions for future work.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Fishes; Mycobacterium; Mycobacterium Infections; Phylogeny; Species Specificity; Zoonoses

European sea bass (Dicentrarchus labrax L.) is a marine species of great economic importance, particularly in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases and thereby leading to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in marker-assisted selection of fish resistant to a range of infections. To date, genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is relatively poor but tends to accumulate rapidly. In this review, we summarize and update current knowledge on the immune system and immune regulatory genes of the sea bass.

Topics: Animals; Bass; Cytokines; Fish Diseases; Histocompatibility Antigens; Immunity, Innate; Immunoglobulins; T-Lymphocytes

Fish mariculture has dramatically expanded in recent years in Mediterranean countries. In this scenario, several pathological problems have logically arisen and parasitological etiologies are increasingly being reported, either as primary or secondary pathogens. Myxozoa is the most diverse and economically important group of fish parasites, and several species are known to cause or contribute to losses in mariculture. Species of the genus Enteromyxum currently constitute the most serious parasitological threat. Some unusual biological characters, such as wide host spectrum and direct fish-to-fish transmission, together with high virulence for some host species, combine a dangerous cocktail which is emerging in recent years. Closed-system (recirculation) and heated-water locations are especially sensitive to chronic infections by these parasites, which can cause serious mortality and even discourage culture of some fish species at certain locations (i.e, Diplodus puntazzo). The presentation presents an overview of recent advances in research of marine myxozoans, focusing mainly in the most pathogenic, Enteromyxum spp. The incidence of these and other emerging infections, and the design of potential strategies for control will be introduced.

Topics: Animals; Bass; Fish Diseases; Fisheries; Fishes; Glomerulonephritis; Mediterranean Sea; Protozoan Infections, Animal; Sea Bream

This paper reviews the occurrence of cymothoid isopod parasitism in aquaculture, reports the first case of infection by a cymothoid isopod (Ceratothoa oestroides) in Turkish aquaculture, and analyses its effects on sea bass Dicentrarchus labrax. Analyses revealed that C. oestroides negatively affects the weights and lengths of sea bass hosts. These effects have been previously underestimated because host age has not been accounted for. The analysis of condition factors as a means of assessing parasite effects is therefore likely to be misleading. Infection of fish of all ages by all cymothoid stages indicates that sea bass are not intermediate hosts but that C. oestroides has effected a complete host shift.

Topics: Animals; Aquaculture; Bass; Crustacea; Ectoparasitic Infestations; Fish Diseases; Incidence; Sea Bream; Turkey

Diseases of striped bass, their hybrids, and redfish (red drum) are important constraints to the culture of these two species. Since striped bass have been cultured for years the organisms that cause most diseases of these fish are well known, but very little specific disease information exists for redfish. However, it appears that the organisms that cause diseases of striped bass and redfish do not differ greatly from those of other fishes. The most significant viral disease is lymphocystis, but infectious pancreatic necrosis has occurred in striped bass. Vibriosis (Vibrio sp.) and motile Aeromonas septicemia (Aeromonas hydrophila) are the most frequently encountered bacterial diseases. Both species of fish are affected by fungi (usually Saprolegnia) when the fish are injured or stressed. Amyloodinium ocellatum is the most serious protozoan that infects striped bass and redfish, but the other common protozoans (Trichodina, Ichthyophthirius, Cryptocaron, etc.) have also been reported. Treatment of any of these diseases is a problem because of the absence of approved drugs or chemicals for use on striped bass or redfish. The most common therapeutics used on striped bass and redfish are copper sulfate, formalin, salt (in freshwater) and Terramycin.

Topics: Animals; Anti-Infective Agents; Bacterial Infections; Bass; Fish Diseases; Fishes; Mycoses; Parasitic Diseases; Parasitic Diseases, Animal; Virus Diseases

An experiment was performed to study the effects of dietary levels of black soldier fly larva meal (BSFLM) on the growth performance, immunity and disease resistance of juvenile grouper (Epinephelus coioides). Four isoproteic and isoenergetic diets were formulated with dietary BSFLM levels of 0 g/kg (T0), 25 g/kg (T2.5), 50 g/kg (T5) and 100 g/kg (T10). Each diet was randomly fed to triplicate groups, each containing 40 fish. The results of the 30-day study indicated that fish growth performance was not affected in the T2.5 and T5 groups compared with the T0 group. In the group with a dietary BSFLM level of 100 g/kg, the feed coefficient was significantly higher than that in the other three groups. The superoxide dismutase, catalase, glutathione peroxidase, lysozyme activity, and malondialdehyde content in the liver, and the interleukin-1 beta (IL-1β), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α) and heat shock protein 70 (HSP70) expression in the gills, head kidney, liver and spleen remained consistent in all groups. In addition, no significant differences in the cumulative mortality or parasite abundance in groupers after Vibrio harveyi and Cryptocaryon irritans infection were observed. These results suggested that BSFLM supplemented diets did not inhibit disease resistance in groupers.

Topics: Animal Feed; Animals; Bass; Diet; Diptera; Disease Resistance; Fish Diseases; Larva

European seabass (Dicentrarchus labrax) production is often hampered by bacterial infections such as photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp). Since diet can impact fish immunity, this work investigated the effect of dietary supplementation of 5% Gracilaria sp. aqueous extract (GRA) on seabass antioxidant capacity and resistance against Phdp. After infection, mortality was delayed in fish fed GRA, which also revealed increased lysozyme activity levels, as well as decreased lipid peroxidation, suggesting higher antioxidant capacity than in fish fed a control diet. Dietary GRA induced a down-regulation of hepatic stress-responsive heat shock proteins (grp-78, grp-170, grp-94, grp-75), while bacterial infection caused a down-regulation in antioxidant genes (prdx4 and mn-sod). Diet and infection interaction down-regulated the transcription levels of genes associated with oxidative stress response (prdx5 and gpx4) in liver. In head-kidney, GRA led to an up-regulation of genes associated with inflammation (il34, ccr9, cd33) and a down-regulation of genes related to cytokine signalling (mif, il1b, defb, a2m, myd88). Additionally, bacterial infection up-regulated immunoglobulins production (IgMs) and down-regulated the transcription of the antimicrobial peptide leap2 in head kidney. Overall, we found that GRA supplementation modulated seabass resistance to Phdp infection.

Topics: Animal Feed; Animals; Antimicrobial Cationic Peptides; Aquaculture; Bass; Blood Glucose; Cytokines; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression Regulation; Gracilaria; Gram-Negative Bacterial Infections; Head Kidney; Heat-Shock Proteins; Immunoglobulin M; Lipid Peroxidation; Liver; Muramidase; Photobacterium; Triglycerides

Mycobacteriosis is a chronic progressive disease affecting teleost fishes all over the world. No vaccine is commercially available against its main etiological agent, Mycobacterium marinum. The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to modulate M. marinum pathology. The innate and adaptive immune responses in sea bass (Dicentrarchus labrax) vaccinated with M. marinum iipA::kan mutant with (and without) the use of adjuvant, with (and without) a booster vaccination were monitored. The adjuvanted vaccine induced enhanced immune responses. TNF-α transcription levels were extremely high in spleen of the fish vaccinated with the addition of adjuvant in both fish vaccinated once and twice, followed by an IgM response highly specific for M. marinum. Also, histologically, granulomas started appearing in spleen and head-kidney tissues (but with no visible bacteria) within a month after vaccination, mainly with the adjuvanted vaccine. This was followed by reduction in pathology, as demonstrated by the lower number of granulomas (with visible bacteria), indicating that even heat-killed bacteria were able to elicit granulomatous formations. Adhesion of the internal organs and moderate pigmentation were observed in the perivisceral adipose tissue of nearly all vaccinated fish. Although the adjuvanted heat-killed avirulent iipA::kan mutant clearly induced a strong humoral and adaptive immune response, the booster treatment did not seem to have produced a significantly higher degree of protection from the disease compared to fish that received a single vaccination.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Bacterial Vaccines; Bass; Fish Diseases; Immunity, Innate; Immunization, Secondary; Mycobacterium Infections; Mycobacterium marinum; Random Allocation; Vaccination

Amyloodinium ocellatum, the causative agent of amyloodiniosis (marine velvet, velvet disease), affects marine and brackish fish in various warm and temperate habitats. We recorded disease outbreaks with high morbidity and mortality rates in marine-cultured European seabass Dicentrarchus labrax fry at 2 locations in northwest Egypt. The sudden outbreak, high morbidity and mortality rates, and skin lesions with a velvety appearance in affected fish all indicated A. ocellatum infection. This was further confirmed by microscopic findings of the parasitic stage (trophonts) in skin and gill smears. While ecological factors including water temperature and salinity were all amenable to parasite establishment and propagation, mortality rates differed between the 2 farms, with rates of mortality well correlated with prevalence and intensity of A. ocellatum infections. Characterization by PCR targeting rDNA gene fragments and subsequent DNA sequencing and phylogenetic analysis further confirmed the molecular identity of the A. ocellatum isolate, which was genetically similar to isolates from other geographical locations. Finally, an improved treatment method using dual hyposalination and copper sulfate exposure to increase the efficiency and decrease the toxicity of copper sulfate was tested. The gradual reduction in water salinity coupled with copper sulfate treatment was more efficient at controlling the disease than only applying copper sulfate. To our knowledge, this is the first parasitological and molecular characterization of A. ocellatum in marine cultures in Egypt. The high molecular identity and close phylogenetic relationship further confirmed the monophyletic nature of A. ocellatum isolates.

Topics: Animals; Aquaculture; Bass; Copper Sulfate; Dinoflagellida; Disease Outbreaks; Ectoparasitic Infestations; Fish Diseases; Phylogeny; Protozoan Infections, Animal; Salinity; Water

In order to elucidate the immune-protective mechanisms of inactivated Cryptocaryon irritans vaccine, different doses of C. irritans theronts were used to immunize orange-spotted grouper (Epinephelus coioides). We measured serum immobilization titer, blood leukocyte respiratory burst activity, serum alternative complement activity, and serum lysozyme activity weekly. In addition, the expression levels of immune-related genes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), major histocompatibility complexes I and II (MHC I and II), and transforming growth factor-β1 (TGF-β1) were determined in spleen and gills. The results showed that the immobilization titer, respiratory burst activity, and alternative complement activity of immunized fish were significantly increased, and the levels of the last two immune parameters in the high-dose vaccine group were significantly higher than in the low-dose vaccine group. Serum lysozyme activity in the high-dose vaccine group was significantly higher than in the PBS control group. Vaccination also regulated host immune-related gene expression. For example, at 2- and 3- weeks post immunization, IL-1β expression in the high-dose vaccine group spleen was significantly increased. At 4-weeks post immunization, the fish were challenged with a lethal dose of parasite, and the survival rates of high-dose vaccine group, low-dose vaccine group, PBS control group, and adjuvant control group were 80%, 40%, 0%, and 10% respectively. These results demonstrate that inactivated C. irritans vaccination improves specific and nonspecific immune responses in fish, enhancing their anti-parasite ability. These effects are vaccine antigen dose-dependent.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Cytokines; Fish Diseases; Gene Expression Profiling; Hematologic Tests; Major Histocompatibility Complex; Protozoan Vaccines; Time Factors; Transcriptome; Vaccines, Inactivated

Flavobacterium columnare is an aquatic bacterium that is responsible for columnaris disease. This aquatic pathogen has a worldwide distribution and is highly infectious to both warm and cold water fish. A modified live F. columnare vaccine was developed by repeated passage of a virulent strain on increasing concentrations of rifampicin that resulted in attenuation. Here we report vaccination/challenge trials to evaluate efficacy and safety. In separate laboratory trials, immersion vaccination of channel catfish (Ictalurus punctatus) fry between 10 to 48 days post hatch (DPH) with experimental vaccine or licensed product resulted in relative percent survival (RPS) between 57-94% following challenge. Similarly, a vaccination/challenge trial using largemouth bass (Micropterus salmoides) fry at 10 DPH was performed using various doses of licensed product under laboratory conditions. Results demonstrated safety of the vaccine and significant protection following challenge with RPS values between 74-94%, depending on vaccine dose. Together, these trials demonstrate the vaccine administered to early life-stage channel catfish and largemouth bass is safe and reduces mortality following challenge with F. columnare.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Ictaluridae

We conducted a field trial to evaluate the effectiveness of Aquaflor (50% florfenicol) for controlling mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass (female white bass Morone chrysops X male striped bass M. saxatilis). Bacterial samples collected from moribund fish representing a reference population were presumptively identified microbiologically and were later confirmed to be S. iniae by biochemical characterization and polymerase chain reaction. The trial comprised a 1-d acclimation period, 10-d treatment period, and 14-d posttreatment period. During the treatment period, Aquaflor-medicated feed was administered to treated tanks (N = 3) at a target dose of 10 mg of florfenicol x kg of fish(-1) x d(-1), and nonmedicated feed was administered to control tanks (N = 3). At the end of the posttreatment period, mean (+/- SD) cumulative mortality in treated tanks (9 +/- 11%) was significantly (P = 0.040) less than that in control tanks (52 +/- 13%). Analysis of medicated feed samples revealed that treated tanks had received an actual dose of 8.3 mg florfenicol x kg fish(-1) x d(-1) (83% of target). No florfenicol was detected in control feed samples. Although the actual florfenicol dose administered to treated tanks was less than the target dose, the trial was accepted by the U.S. Food and Drug Administration Center for Veterinary Medicine as demonstrating the efficacy of Aquaflor to control mortality associated with S. iniae in cultured sunshine bass populations.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Fish Diseases; Fresh Water; Streptococcal Infections; Streptococcus; Thiamphenicol; Time Factors

Brown-marbled grouper Epinephelus fuscoguttatus which had been fed diets containing sodium alginate and kappa (kappa)-carrageenan at 5, 10 and 20gkg(-1), respectively after 0, 2, 4, 6, 8 and 14 weeks were examined for survival, growth, innate cellular and humoral responses as compared to the fish that fed control non-supplemented diet. Survival was 100% for the fish that fed all diets after 14 weeks and no significant difference in growth was observed among seven diets. The fish that fed a diet containing sodium alginate at 10gkg(-1), and fed a diet containing kappa-carrageenan at 5gkg(-1) over 2-8 weeks showed significantly increased leucocyte count, respiratory burst, phagocytic activity and phagocytic index, The fish that fed a diet containing sodium alginate at 5gkg(-1), and fed a diet containing kappa-carrageenan at 5gkg(-1) over 2-8 weeks showed significantly increased alternative complement activity (ACH50) and lysozyme activity. In another experiment, E. fuscoguttatus, which had been fed control diet, and all diets containing sodium alginate, and kappa-carrageenan after 14 weeks were challenged with Vibrio alginolyticus at 5.0x10(9) colony-forming units (cfu) fish(-1) and then placed in seawater of 34 per thousand. The survival of fish that fed a diet containing sodium alginate at 10gkg(-1) and kappa-carrageenan at 5gkg(-1) was significantly higher than that of fish which fed the control diet over 96-168h. It was concluded that E. fuscoguttatus which fed a diet containing sodium alginate at 10gkg(-1) or less, or fed a diet containing kappa-carrageenan at 5gkg(-1) enhance the innate immunity, and increase the resistance from V. alginolyticus infection.

Topics: Alginates; Animals; Bass; Carrageenan; Complement Pathway, Alternative; Dietary Supplements; Fish Diseases; Glucuronic Acid; Hexuronic Acids; Immunity, Innate; Leukocyte Count; Phagocytosis; Respiratory Burst; Vibrio alginolyticus; Vibrio Infections

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Topics: Administration, Oral; Animals; Bacterial Outer Membrane Proteins; Bass; Cell Line; Chitosan; Escherichia coli; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immune Sera; Listonella; Nanoparticles; Plasmids; Recombinant Proteins; Tetrazolium Salts; Thiazoles; Vaccination; Vaccines, DNA

Little information is available until now about the copepods infecting different fish species. Therefore, this study aimed to provide light on siphonostomatoids infecting Epinephelus chlorostigma. Twenty fish specimens were taken from the Red Sea coast (Jeddah, Saudi Arabia), and ectoparasitic copepods were investigated. Light microscopy and molecular tools were used to examine copepods isolated from fish. Parasitological indexes were calculated and showed that 60% of the examined fish were infected with a mean intensity of 12 parasite/fish. Morphological examination revealed that this copepod species is characterized by all unique features of the genus Hatschekia with special reference to Hatschekia sargi. The taxonomic position of the recovered species in the Hatschekiidae family within Siphonostomatoida was confirmed using phylogenetic analysis based on partial mitochondrial cytochrome c oxidase subunit I (mt COI) gene sequences. The mt COI gene query revealed that the recovered Hatschekia species is closely related to Hatschekia maculatus (gb| JQ664005.1). This study discovers a new host for Hatschekia species isolated from Saudi Arabia and conducts the first genomic investigation of the mt COI gene.

Topics: Animals; Bass; Copepoda; Fish Diseases; Genes, Mitochondrial; Parasites; Phylogeny

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

Topics: Animals; Bass; Capsid Proteins; DNA Virus Infections; Fish Diseases; Polymerase Chain Reaction; Ranavirus; Reproducibility of Results

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1β, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

Topics: Animals; Bass; Fish Diseases; Polysaccharides; Reishi; Vaccines, DNA; Vibrio; Vibrio Infections

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-β, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Topics: Animals; Baculoviridae; Bass; Capsid Proteins; DNA Virus Infections; Fish Diseases; Iridoviridae; Vaccines, Synthetic; Viral Vaccines

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (

Topics: Animals; Base Sequence; Bass; Cloning, Molecular; Disulfides; DNA; Fish Diseases; Fish Proteins; Gene Expression Regulation; Hydrogen Peroxide; Immunity; Oxidoreductases; Phylogeny; RNA, Messenger; Thioredoxins

Mandarinfish ranavirus (MRV), also known as a variant of largemouth bass virus (LMBV), is an emerging pathogen in mandarinfish aquaculture. In this study, monoclonal antibodies (mAbs) against MRV were produced and characterized, and 7 mAbs were obtained through Western blotting screening and all 7 mAbs specifically recognized MRV/LMBV but not several piscine iridoviruses as ISKNV, GIV and TFV. By LC MS/MS analysis, the recognized viral proteins by seven mAbs were identified as MRV-pORF47L, MRV-pORF55R, MRV-pORF57L, MRV-pORF77L and MRV-pORF78L, respectively, and all five viral proteins are late expression structural proteins by Western blotting. Based on mAb 1C4, immuno-histochemistry and immuno-histo-fluorescence were performed to re-assess the tissue tropism of MRV. The result showed that abundant reactive signals were observed in infected spleen, kidney as well as intestine and pyloric caecum. Real-time quantitative PCR also demonstrated that spleen as well as pyloric caecum and intestines are the major target tissue upon MRV infection. In infected intestines and pyloric caecum, numerous enlarged, multinucleated cells with intracytoplasmic inclusions were identified as the target cells of MRV, suggesting that MRV serves as a digestive tract pathogen to mandarinfish, which may explain why acute infection of MRV can cause the typical clinicopathology featured by severe ascites.

Topics: Animals; Antibodies, Monoclonal; Bass; Cecum; Fish Diseases; Iridoviridae; Ranavirus; Tandem Mass Spectrometry; Viral Proteins

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression Regulation; Mammals; Phylogeny; Receptors, Melanocortin

In this study, we evaluated to reveal the effects of aqueous methanolic extract of celery (Apium graveolens) on the growth performance, immune responses, and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). For this purpose, twenty fish (initial mean weight of 4.80 ± 0.06 g) were placed into twelve tanks (400 L) in triplicate and fish were fed with control (C) and three different levels (0.01, 0.05, and 0.1 g/kg) of A. graveolens (AG) extract-containing diets (AG0.01, AG0.05, and AG0.1) for 30 days. Blood and tissue (kidney, spleen, and intestine) samples were taken from the fish every 10 days during the study to determine the immune responses of the fish. Respiratory burst activity (RBA) was significantly decreased in the AG0.1 group compared to all other groups on the 10th day of the study (P < 0.05). Significance was noticed in the RBA of fish in all AG groups compared to the C group (P < 0.05) on the 30th day of the experiment Lysozyme activity (LYS) was raised on the 10th day of the study in all celery groups compared to the C group (P < 0.05). No differences in the myeloperoxidase activity (MPO) were observed among the experimental groups (P > 0.05). The final mean weight (FMW) was not affected in any experimental groups (P > 0.05). However, in the AG0.05 group, the specific growth rate (SGR) increased, and the feed conversion ratio (FCR) decreased compared to other groups (P < 0.05). IL-1β in the kidney was highly elevated in the AG0.01 group on the 20th day of the study (P < 0.05). Similar results were observed on IL-6, IL-8, and TNF-α expression in the kidney (P < 0.05). Anti-inflammatory responses (IL-10 and TGF-β) also increased in all experimental groups and tissues compared to the C group (P < 0.05). COX-2 was upregulated on the 20th day of the study in all tissues (P < 0.05). At the end of the feeding trial, the survival rate of the AG0.1 group in fish infected with Vibrio anguillarum infection was higher than the C group. Dietary celery extract did not affect growth performance directly but increased innate immune responses and a high survival rate. Overall, compared to the control group, the growth, immunity, and resistance of European seabass fed with a diet containing 0.05 g/kg celery aqueous methanolic extract has been improved, and this could be used as an immunostimulant feed additive.

Topics: Animal Feed; Animals; Apium; Bass; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Immunity, Innate; Vibrio Infections

Topics: Animals; Antiviral Agents; Aquaculture; Bass; DNA Virus Infections; Fish Diseases; Viral Proteins; Virus Replication

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Base Sequence; Bass; Beclin-1; Fish Diseases; Fish Proteins; Hydrogen Peroxide; Mammals; Nodaviridae; Oxidative Stress; Phylogeny

Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Hymenostomatida; Superoxide Dismutase

Topics: Animals; Bass; Fish Diseases; Nocardia; Nocardia Infections; RNA, Ribosomal, 16S; Virulence; Virulence Factors

Antimicrobial peptides (AMPs) are a potent arm of the innate immune system that can directly kill pathogens and induce immunomodulation. In the marine aquaculture, European sea bass (Dicentrarchus labrax L.) is one of the most prosperous species but is highly susceptible to nodavirus (NNV), which produces high rates of mortality in larvae and juvenile stages. Thus, we aimed to evaluate whether AMPs exert immunomodulatory and/or NNV-preventive actions in sea bass. To do this, plasmids encoding the sea bass AMPs dicentracin (pDIC), beta-defensin (pDB1), hepcidin (pHAMP2) or NK-lysin (pNKL) were generated and intramuscularly injected into sea bass juveniles to evaluate their immunomodulatory and anti-NNV roles. Sea bass muscle transcribes the AMPs and produces an increase in their circulating levels, along with an increase of the antibacterial activity. Immune-related gene analysis revealed a great activation of the inflammatory response and the recruitment of neutrophilic granulocytes at the site of injection. However, AMP-encoding plasmids, namely pHAMP2, negatively affected to NNV disease by increasing fish mortality. In conclusion, plasmids encoding AMPs show immunostimulatory effects on European sea bass but do not improve the resistance to NNV.

Topics: Animals; Antimicrobial Peptides; Bass; Fish Diseases

As a pathogen of cultured teleosts, Pseudomonas plecoglossicida has caused significant economic losses. flgC plays an important role in encoding flagellar basal-body rod proteins. Our previous studies revealed the high expression of P. plecoglossicida flgC in infected Epinephelus coioides. To explore the role of flgC in the virulence of P. plecoglossicida and the immune response of E. coioides to the infection of P. plecoglossicida, flgC gene of P. plecoglossicida was knocked down by RNA interference (RNAi). The results showed that the flgC gene in all four mutants of P. plecoglossicida was significantly knocked down, and the mutant with the best knockdown efficiency of 94.3% was selected for subsequent studies. Compared with the NZBD9 strain of P. plecoglossicida, the flgC-RNAi strain showed a significantly decrease in chemotaxis, motility, adhesion, and biofilm formation. Furthermore, compared with the E. coioides infected with the NZBD9 strain, the infection of flgC-RNAi strain resulted in the infected E. coioides a 1.5-day delay in the time of first death and an 80% increase in survival rate, far fewer white nodules upon the spleen surfaces, and lower pathogen load in the spleens. RNAi of flgC significantly influenced the metabolome and transcriptome of the spleen in infected E. coioides. KEGG enrichment analysis exhibited that the Toll-like receptor signaling pathway was the most enriched immune pathway; the most significantly enriched metabolic pathways were associated with Linoleic acid metabolism, Choline metabolism in cancer, and Glycerophospholipid metabolism. Further combined analysis of transcriptome and metabolome indicated significant correlations among pantothenate and CoA biosynthesis, beta-alanine metabolism, lysosome metabolites, and related genes. These results suggested that flgC was a pathogenic gene of P. plecoglossicida; flgC was associated with the regulation of chemotaxis, motility, biofilm formation, and adhesion; flgC influenced the immune response of E. coioides to the infection of P. plecoglossicida.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Immunity, Innate; Pseudomonas Infections; Virulence

Three of the most important diseases of Mediterranean intensive European sea bass farming are, viral nervous necrosis (VNN) caused by the red grouper nervous necrosis virus (RGNNV) genotype of b-nodavirus, photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp) and vibriosis caused mainly by the O1 serotype of Vibrio anguillarum (VaO1). Prevention against these diseases is performed through vaccination with a monovalent vaccine against the viral disease and, usually, with bivalent vaccines against the bacterial diseases. However, it is very difficult to program two vaccinations during the same season for the same fish stock and producers are forced to either vaccinate for the viral or the bacterial diseases or to perform double vaccination with both vaccines, without any prior knowledge on any interactions that may occur due to the plethora of antigens (Ag) injected. Ideally, therefore, a trivalent vaccine should be developed against all three diseases. The objective of this work was to analyse the immune response of sea bass against combinations of Ags from all three pathogens, namely viral particles, Phdp whole cells (WC), lipopolysaccharide (LPS), capsular polysaccharide (CPS) and extracellular products (ECPs) and VaO1 WC and ECPs in respect to the identification of any phenomena of immunodominance/immunosuppression between Ags with a view to select candidate Ags for inclusion in a trivalent vaccine formulation. Eight triplicate groups of fish were immunized with different combinations of the aforementioned Ags and another triplicate group served as negative control. Blood serum was isolated at various time-points post-immunization for the measurement of specific antibodies against each Ag and, in addition, leucocytes were isolated at day 29 post-immunization for analysis of various cellular activities. Results indicated that best levels of specific a-NNV virus antibodies (Abs) were produced when VaO1 ECPs were not included in the Ag combinations, in contrast to the leucocytes proliferation assay where best stimulation against NNV Ags was measured when VaO1 ECPs were present in Ag combinations. VaO1 ECPs apparently is a strong immunogen for both humoral and cellular responses but suppresses immunological reactions against the other Ags.VaO1 WC, Phdp LPS and ECPs raised good humoral immune responses in the groups with best responses against VNN Ags, but only VaO1 WC and Phdp ECPs provided good stimulation of leucocytes, with Phdp WC a

Topics: Animals; Bass; Fish Diseases; Immunity, Humoral; Lipopolysaccharides; Necrosis; Vaccines

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP,

Topics: Animals; Bass; Capsid Proteins; Epitopes, B-Lymphocyte; Fish Diseases; Immunodominant Epitopes; Necrosis; Nodaviridae; RNA Virus Infections

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

Topics: Animals; Bass; Fish Diseases; Inflammation; Liver; Nocardia; Nocardia Infections; Pyroptosis

Anisakidosis is a foodborne zoonotic infection induced by members of the family Anisakidae via the consumption of raw or undercooked fish such as sushi and sashimi. Identifying anisakid larval species is critical for the epidemiology and diagnosis of diseases caused by them. This study aimed at identifying Anisakis larvae collected from marine fish in Egyptian waters based on morphological characteristics and molecular analysis. Thirty marine fish coral trout, Plectropomus areolatus, were collected from Hurghada, Red Sea, Egypt, to investigate larval nematodes of the genus Anisakis. The larvae were detected encapsulated in the peritoneal cavity and muscle of the fish host. This examination revealed that anisakid larvae naturally infected 19 fish specimens with a prevalence of 63.33% and a mean intensity of 4.1 ± 0.40. Most of them (68 larvae: 71.57%) were found in the musculature. Morphological and morphometric analyses using light and scanning electron microscopy revealed a head region with a prominent boring tooth, inconspicuous lips, and a characteristic protruded cylindrical mucron. All larvae in this study possessed the same morphology as Anisakis Larval type I. Molecular analysis based on ITS region using maximum likelihood and Bayesian phylogenetic methods confirmed them as Anisakis typica. This is the first study to identify A. typica larvae from the commercial fish coral trout P. areolatus in Egyptian waters using morphological and molecular methods.

Topics: Animals; Anisakiasis; Anisakis; Ascaridoidea; Bass; Bayes Theorem; Fish Diseases; Fishes; Indian Ocean; Larva; Phylogeny; Trout

Peroxiredoxin 1 is a member of the typical 2-Cys peroxiredoxin family, which serves diverse functions in gene expression, immune and inflammatory responses, and tumor progression. In this study, we aimed to analyze the structural, functional, and immunomodulatory properties of peroxiredoxin 1 from Epinephelus akaara (EaPrx1). The open reading frame of EaPrx1 is 597 base pairs in length, encoding 198 amino acids, with a molecular weight of approximately 22 kDa. The in silico analysis revealed that EaPrx1 shares a conserved thioredoxin fold and signature motifs that are critical for its catalytic activity and oligomerization. Further, EaPrx1 is closely related to Epinephelus lanceolatus Prx1 and clustered in the Fishes group of the vertebrate clade, revealing that EaPrx1 was conserved throughout evolution. In terms of tissue distribution, a high level of EaPrx1 expression was observed in the spleen, brain, and blood tissues. Likewise, in immune challenge experiments, significant transcriptional modulations of EaPrx1 upon lipopolysaccharide, polyinosinic:polycytidylic acid, and nervous necrosis virus injections were noted at different time points, indicating the immunological role of EaPrx1 against pathogenic infections. In the functional analysis, rEaPrx1 exhibited substantial DNA protection, insulin disulfide reduction, and tissue repair activities, which were concentration-dependent. EaPrx1/pcDNA™ 3.1 (+)-transfected fathead minnow cells revealed high cell viability upon arsenic toxicity, indicating the heavy metal detoxification activity of EaPrx1. Taken together, the transcriptional and functional studies imply critical roles of EaPrx1 in innate immunity, redox regulation, apoptosis, and tissue-repair processes in E. akaara.

Topics: Animals; Antioxidants; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Oxidation-Reduction; Peroxiredoxins; Phylogeny

Topics: Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Tea; Virus Diseases

Cryptocaryon irritans is a parasitic ciliate of marine fish, causing serious mortality and economic loss of grouper. In this study, the orange-spotted grouper (Epinephelus coioides) were separately exposed to C. irritans infection for 72 h at a dose of 5000 or 10000 active theronts per fish, and we evaluated the changes in histopathology, oxidative stress, immune response, and intestinal microbiota composition. The results showed that C. irritans infection caused pathological alteration on the skin, gills, and liver of E. coioides. Oxidative stress responses occurred in the liver and gills, reflected in the corresponding antioxidant enzyme and gene indexes. The mRNA expression levels of inflammation-related genes (IL-1β, IL-6, and IL-8) and the mediators of apoptosis (casp3, casp9, and cytc) were increased in the liver and gills of the fish. C. irritans infection also affected the diversity and composition of intestinal microbiota. Specifically, the relative abundance of Firmicutes was increased, whereas that of Proteobacteria was decreased. Several potentially beneficial bacteria (Pandoraea, Clostridium sensu stricto 1, Christensenellaceae R-7 group, and Weissella) were decreased, whereas pathogenic bacteria (Streptococcus and Acinetobacter) were increased. In conclusion, this study reveals that C. irritans infection caused histopathology, immune disorders, and intestinal microbial community variation in E. coioides.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gastrointestinal Microbiome; Hymenostomatida; Immunity; Oxidative Stress; Phylogeny

Protein kinase C (PKC) constitutes the main signal transduction pathway, and participates in the signal pathway of cell proliferation and movement in mammals. In this study, PKC-ɑ was obtained from Epinephelus coioides, an important marine fish cultivated in the coastal areas of southern China and Southeast Asia. The full length cDNA of PKC-ɑ was 3362 bp in length containing a 23 bp 5'UTR, a 1719 bp 3'UTR, and a 1620 bp open reading frame encoding 539 amino acids. It contains three conservative domains including protein kinase C conserved region 2 (C2), Serine/Threonine protein kinases, catalytic domain (S_TKc) and ser/thr-type protein kinases (S_TK_X). Its mRNA can be detected in all 11 tissues examined of E. coioides, and the expression was significantly upregulated response to Singapore grouper iridovirus (SGIV) infection, one of the important pathogens of marine fish. Upregulated E. coioides PKC-ɑ significantly inhibited the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and SGIV-induced cell apoptosis. The results indicated that the PKC-ɑ may play an important role in pathogenic stimulation.

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Mammals; Phylogeny; Protein Kinase C; Ranavirus; Singapore

Gilthead sea bream (Sparus aurata) is considered an asymptomatic carrier for the nodavirus genotype affecting European sea bass (Dicentrarchus labrax), RGNNV. Only larvae and juveniles of sea bream have been found to be susceptible to the RGNNV/SJNNV reassortant. Nevertheless, the molecular bases of the high resistance of sea bream against RGNNV are not known, and the overall transcriptome response to the virus remains unexplored. In this work, we conducted the first RNA-Seq analysis of sea bream infected with RGNNV to elucidate the immune mechanisms involved in their resistance. Since we recently published the transcriptome response of sea bass infected with RGNNV, we wanted to take the same tissues (brain and head kidney) at the same time points (24 and 72 h postinfection) to conduct comparative analyses. Sea bream responded to RGNNV challenge with a powerful immune arsenal characterized by the high expression of a multitude of type I interferon-related genes, immune receptors and antigen presentation-related genes in both tissues. Moreover, complement-, coagulation- and angiogenesis-related genes were highly enriched in the head kidney at the earlier sampling point. Interestingly, despite the strong immune response found in the brain, inflammation seems to have been restrained, resulting in a neuroprotective scenario. While the response in sea bass was characterized by the activation of the stress axis, which could lead to immunosuppression and neuronal damage, genes involved in these processes were not modulated in sea bream. An efficient antiviral response accompanied by low inflammation and the absence of stimulation of the stress response seem to play a role in the success of sea bream in resisting RGNNV infection.

Topics: Animals; Bass; Fish Diseases; Genotype; Inflammation; Perciformes; Sea Bream; Sequence Analysis, RNA

Nervous necrosis virus (NNV) could infect more than 200 fish species worldwide, with almost 100% mortality in affected larvae and juvenile fish. Among different genotypes of NNV, the red-grouper nervous necrosis virus (RGNNV) genotype is the most widely reported with the highest number of susceptible species. Interferon (IFN) is a crucial antiviral cytokine and RGNNV needs to develop some efficient strategies to resist host IFN-stimulated antiviral immune. Although considerable researches on RGNNV, whether RGNNV B1 protein participates in regulating the host's IFN response remains unknown. Here, we reported that B1 protein acted as a transcript inhibition factor to suppress fish IFN production. We firstly found that ectopic expression of B1 protein significantly decreased IFN and IFN-stimulated genes (ISGs) mRNA levels and IFNφ1 promoter activity induced by polyinosinic:polycytidylic acid [poly (I:C)]. Further studies showed that B1 protein inhibited the IFNφ1 promoter activity stimulated by the key RIG-I-like receptors (RLRs) factors, including MDA5, MAVS, TBK1, IRF3, and IRF7 and decreased their protein levels. Moreover, B1 protein significantly inhibited the activity of constitutively active cytomegalovirus (CMV) promoter, which suggested that B1 protein was a transcription inhibitor. Western blot indicated that B1 protein decreased the Ser5 phosphorylation of RNA polymerase II (RNAP II) C-terminal domain (CTD). Together, our data demonstrated that RGNNV B1 protein was a host transcript antagonist, which intervened RNAP II Ser5-phosphorylation, inhibiting host IFN response and facilitating RGNNV replication.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Necrosis; Nodaviridae; Poly I-C; RNA Polymerase II; RNA Virus Infections; Sequence Alignment; Virus Replication

Largemouth bass (

Topics: Animals; Bass; Fish Diseases; Gene Expression Profiling; Iridoviridae; Iridovirus; Phylogeny

The largemouth bass virus (LMBV) isolate of Santee-Cooper ranavirus showed evidence of widespread infection in adult fish, but disease presentation caused by different viral strains exhibited considerable difference. In this study, a highly pathogenic LMBV-like resembling Santee-Cooper ranavirus was isolated and identified from juvenile largemouth bass. The pathogenicity and dynamic distribution of LMBV-like strain, histopathological analysis and host immune response of juvenile largemouth bass infected with LMBV-like were investigated. The results show that LMBV-like was highly pathogenic to juvenile fish, and the infected fish showed typical signs of acute haemorrhages and visceral enlargement. LMBV-like positive cells were found in the liver, spleen, kidney, gills, and intestinal tissue, and the virus content in spleen was the highest. Histopathological analysis showed different pathological changes in major tissues of diseased fish, mostly manifested as infiltration of inflammatory cell and histiocyte necrosis. In addition, humoral immune factors such as superoxide dismutase (SOD), catalase (CAT) and acid phosphatase (ACP) were used as serum indicators to evaluate the immune response of juvenile fish after infection. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression patterns of immune-related genes (CD40, IFN-γ, IgM, IL-1β, IL-8, IL-12a, Mxd3, TGF-β, and TNFα) in liver, spleen, and head kidney tissues. The results showed that immunological activity of the juvenile largemouth bass was significantly enhanced by LMBV-like infection. This research comprehensively systematically revealed the pathogenic characteristics of LMBV-like separated from juvenile largemouth bass and properties of the host's immune response caused by the virus infection, which providing a basis for further exploring the interaction between the virus and the host, and prevention and treatment of disease caused by Santee-Cooper ranavirus.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Ranavirus; Virulence

Singapore grouper iridovirus (SGIV), with various mechanisms for evading and modulating host, has inflicted heavy economic losses in the grouper aquaculture. MAP kinase phosphatase 1 (MKP-1) regulates mitogen-activated protein kinases (MAPKs) to mediate the innate immune response. Here, we cloned EcMKP-1, an MKP-1 homolog from the orange-spotted grouper Epinephelus coioides, and investigated its role in the infection of SGIV. In juvenile grouper, EcMKP-1 was highly upregulated and peaked at different times after injection with lipopolysaccharide, polyriboinosinic polyribocytidylic acid and SGIV. EcMKP-1 expression in heterologous fathead minnow cells was able to suppress SGIV infection and replication. Furthermore, EcMKP-1 was a negative regulator of c-Jun N-terminal kinase (JNK) phosphorylation early in SGIV infection. EcMKP-1 decreased the apoptotic percentage and caspase-3 activity during the late stage of SGIV replication. Our results demonstrate critical functions of EcMKP-1 in antiviral immunity, JNK dephosphorylation and anti-apoptosis during SGIV infection.

Topics: Animals; Antiviral Agents; Bass; DNA Virus Infections; Dual Specificity Phosphatase 1; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Ranavirus; Singapore

Groupers are important mariculture fish in South China and Southeast Asian countries. However, the increasing frequency of infectious disease outbreaks has caused great economic losses in the grouper industry. Among these pathogens, Singapore grouper iridovirus (SGIV) infection causes high mortality in larval and juvenile stages of grouper. However, the mechanism underlying the action of viral manipulation on cellular immune response still remained largely uncertain. Here, using RNA-seq technology, we investigated the regulatory roles of SGIV infection on synthetic RNA duplex poly I:C induced immune response in vitro. Using reporter gene assays, we found that SGIV infection decreased poly I:C induced interferon promoter activation. Transcriptomic analysis showed that the mRNA expression levels of 2238 genes were up-regulated, while 1247 genes were down-regulated in poly I:C transfected grouper spleen (GS) cells. Interestingly, SGIV infection decreased the expression of 1479 up-regulated genes and increased the expression of 297 down-regulated genes in poly I:C transfected cells. The differentially expressed genes (DEGs) down-regulated by SGIV were directly related to immune, inflammation and viral infection, and JUN, STAT1, NFKB1, MAPK14A, TGFB1 and MX were the 6 top hub genes in the down-regulated DEGs' protein-protein interaction (PPI) network. Furthermore, quantitative real-time PCR (qPCR) analysis confirmed that the interferon signaling and inflammatory-related genes, including cGAS, STING, TBK1, MAVS, TNF, IRAK4 and NOD2 were up-regulated by poly I:C stimulation, but all significantly down-regulated after SGIV infection. Thus, we speculated that SGIV infection counteracted poly I:C induced antiviral immune response and this ability helped itself to escape host immune surveillance. Together, our data will contribute greatly to understanding the potential immune evasion mechanism of iridovirus infection in vitro.

Topics: Animals; Antiviral Agents; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Interferons; Iridovirus; Poly I-C; Ranavirus; Singapore

Largemouth bass virus (LMBV) infections resulting in enormous loss are becoming an increasing problem in the largemouth bass industry. Oral vaccination is considered to be an effective and economical measure because of the advantages of non-invasion, no size limitation, lower cost and easily-operated. Based on Bacillus subtilis (B. subtilis) spores, this study successfully constructed the CotC-LMBV recombinant B. subtilis spores and its protective efficacy and immune responses were evaluated. After challenged, the survival rate of largemouth bass orally vaccinated with CotC-LMBV spores was 53.3% and the relative percent survival (RPS) was 45.0% compared to the PBS group. In addition, the specific IgM level in serum in the CotC-LMBV group was significantly higher than in the control groups. In the spleen, the immune-related genes expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the CotC-LMBV group, suggesting that innate and adaptive immune responses were activated. This study indicated that oral administration of CotC-LMBV recombinant spores could stimulate an effective immune response and enhance fish immunity against LMBV infection. Therefore, oral vaccination could be an effective approach for the prevention of largemouth bass virus disease.

Topics: Administration, Oral; Animals; Bacillus subtilis; Bass; Capsid Proteins; Fish Diseases; Spores, Bacterial

Cells are important in the study of virus isolation and identification, viral pathogenic mechanisms and antiviral immunity. The spotted knifejaw (Oplegnathus punctatus) is a significant farmed fish in China that has been greatly affected by diseases in recent years. In this study, a new cell line derived from the spotted knifejaw brain (SKB) was established and characterized. SKB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. Chromosome analysis revealed that modal chromosome number was 48 for SKB. SKB cells exhibit susceptibility to several fish viruses, such as a largemouth bass virus, red grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV), Singapore grouper iridovirus (SGIV) and spotted knifejaw iridovirus isolate (SKIV-TJ), as shown by cytopathic effect and increased viral titers. Electron microscopy results showed that the cytoplasm contained a large number of vacuoles, and many virus particles existed at the edge of the vacuoles in RGNNV-infected cells and numerous viral particles were scattered throughout the cytoplasm in both ISKNV- and SKIV-TJ-infected cells. These results suggest that SKB is an ideal tool for studying host-virus interactions and potential vaccine development.

Topics: Animals; Bass; Brain; Cell Line; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridoviridae

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper β-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper β-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.

Topics: Adjuvants, Immunologic; Animals; Bass; beta-Defensins; Escherichia coli; Fish Diseases; Fish Proteins; Necrosis; Nodaviridae; Recombinant Proteins; RNA Virus Infections; Viral Vaccines

Topics: Animals; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Photobacterium; Vaccines; Virulence

Largemouth bass (Micropterus salmoides) is one of the important economical freshwater aquaculture species in China. However, the outbreak of viral diseases always caused great economic losses in the largemouth bass aquaculture industry. Largemouth bass virus (LMBV), a double-stranded DNA (dsDNA) virus belonging to genus Ranavirus, family Iridoviridae causes high mortality in cultivated largemouth bass. However, host responses, especially the molecular events involved in LMBV infection still remained largely uncertain. Here, we established an in vivo model of LMBV infection, and systematically investigated the mRNA expression profiles of host genes in liver and spleen from infected largemouth bass using RNA sequencing (RNA-seq). Histopathological analysis indicated that necrotic cells and the formed necrotic focus were present in spleen, while numerous basophilic cells, hepatocytes volume shrinkage, nucleus pyknosis, and the disappeared boundary of hepatocytes were observed in the liver of infected largemouth bass. Transcriptomic analysis showed that transcription levels of 5128 genes (2804 up-regulated genes and 2324 down-regulated) in liver and 7008 genes (2603 up-regulated and 4405 down-regulated) in spleen were altered significantly. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that numerous co-regulated differentially expressed genes (DEGs) in liver and spleen were enriched in the pathways related to cell death and immune signaling, such as apoptosis, necroptosis, cytokine-cytokine receptor interaction and JAK-STAT signaling. Moreover, the DEGs specially regulated by LMBV infection in liver were significantly enriched in the KEGG pathways related to metabolism and cell death, while those in spleen were enriched in the immune related pathways. In addition, the expression changes of several randomly selected genes, such as SOCS1, IL-6, CXCL2, CASP8, CYC and TNF from qPCR were consistent with the transcriptomic data. Taken together, our findings will provide new insights into the fundamental patterns of molecular responses induced by LMBV in vivo, but also contribute greatly to understanding the host defense mechanisms against iridoviral pathogens.

Topics: Animals; Bass; Fish Diseases; Gene Expression Profiling; Immunity; Ranavirus; Transcriptome; Virus Diseases

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acids protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection.

Topics: Amino Acid Sequence; Animals; Annexin A2; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Nodaviridae; RNA Virus Infections; Sequence Alignment

We identified two tripartite motif (TRIM) genes, LcTRIM21 and LcTRIM39, from the Asian Seabass Lates calcarifer, and examined their responses to experimental betanodavirus infection and stimulation with microbial pathogen-associated molecular patterns.. Genes encoding LcTRIM21 and LcTRIM39 were identified, cloned, and sequenced from the Asian Seabass. We analyzed the sequence using a variety of bioinformatics tools to determine protein structure, localization, and establish a phylogenetic tree. By using quantitative real-time PCR, we analyzed expression profiles of the LcTRIM21 and LcTRIM39 genes in response to betanodavirus challenge as well as molecular pathogen-associated molecular patterns like poly(I:C) and Zymosan A. The tissue distribution pattern of these genes was also examined in healthy animals.. Asian Seabass homologues of the TRIM gene, LcTRIM21 and LcTRIM39, were cloned, both encoding proteins with 547 amino acids. LcTRIM21 is predicted to have an isoelectric point of 6.32 and a molecular mass of 62.11 kilodaltons, while LcTRIM39 has an isoelectric point of 5.57 and a molecular mass of 62.11 kilodaltons. LcTRIM21 and LcTRIM39 homologues were predicted to be localized in cytoplasm by in silico protein localization. Structurally, both proteins contain an N-terminal really interesting new gene (RING) zinc-finger domain, B-box domain, coiled-coil domain and C-terminal PRY/SPRY domain. Most tissues and organs examined showed constitutive expression of LcTRIM21 and LcTRIM39. Upon poly(I:C) challenge or red-spotted grouper nervous necrosis virus infection, LcTRIM21 and LcTRIM39 mRNA expression was significantly upregulated, suggesting that they may play a critical antiviral role against fish viruses. LcTRIM21 and LcTRIM39 expression were also upregulated by administration of the glucan Zymosan A.. The TRIM-containing gene is an E3 ubiquitin ligase that exhibits antiviral activity by targeting viral proteins via proteasome-mediated ubiquitination. TRIM proteins can be explored for the discovery of antivirals and strategies to combat diseases like viral nervous necrosis, that threaten seabass aquaculture.

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Necrosis; Pathogen-Associated Molecular Pattern Molecules; Perciformes; Phylogeny; Poly I-C; Virus Diseases; Zymosan

Viral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass.. We generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A.. We have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

Topics: Animals; Bass; Bayes Theorem; Fish Diseases; Humans; Interferons; Membrane Proteins; Mitochondrial Proteins; Necrosis; Quantitative Trait Loci

Topics: Animals; Bass; Fish Diseases; Immunity; Vibrio; Vibrio Infections

Piscidins participate in the innate immune response of fish, which aims to eliminate recognized foreign microbes and restore the homeostasis of immune system. We characterized two piscidin-like antimicrobial peptides (LjPL-3 and LjPL-2) isolated from Japanese sea bass (Lateolabrax japonicus). LjPL-3 and LjPL-2 showed different expression patterns in tissues. After Vibrio harveyi infection, the mRNA expression of LjPL-3 and LjPL-2 was upregulated in the liver, spleen, head kidney, and trunk kidney. The synthetic mature peptides LjPL-3 and LjPL-2 exhibited different antimicrobial spectra. Furthermore, LjPL-3 and LjPL-2 treatments decreased inflammatory cytokine production while promoting chemotaxis and phagocytosis in monocytes/macrophages (MO/MФ). LjPL-2, but not LjPL-3, displayed bacterial killing capability in MO/MФ. LjPL-3 and LjPL-2 administration increased Japanese sea bass survival after V. harveyi challenge, which was accompanied by a decline in bacterial burden. These data suggested that LjPL-3 and LjPL-2 participate in immune response through direct bacterial killing and MO/MФ activation.

Topics: Animals; Anti-Infective Agents; Antimicrobial Peptides; Bass; Fish Diseases; Fish Proteins; Macrophages; Monocytes; Vibrio Infections

Specimens of two undescribed and one known gonad-infecting species of Philometra Costa, 1845 (Nematoda: Philometridae) were collected in some marine fishes from off the southern coast of Iraq. Based on light and scanning electron microscopy, the following new species are described: Philometra tayeni n. sp. (males and nongravid females) from ovaries of the purple-spotted bigeye Priacanthus tayenus Richardson (Priacanthidae, Acanthuriformes), and Philometra nibeae n. sp. (males and gravid female) from the ovary of the blotched croaker Nibea maculata (Bloch et Schneider) (Sciaenidae, Acanthuriformes). Philometra tayeni is mainly characterised by a pair of postanal papillae and a V-shaped caudal mound in males and by their body lengths (2.42-2.99 mm), whereas P. nibeae differs from its gonad-infecting congeners parasitising scienids mainly based on the body length of males (2.29-2.49 mm) and their spicules (96-117 µm), absence of a pair of postanal papillae and shape of caudal mound consisting of two parts. Philometra piscaria Moravec & Justine, 2014 (males and nongravid females), a parasite of the orange-spotted grouper Epinephelus coioides (Hamilton) (Epinephelidae, Perciformes), is recorded from the Arabian (= Persian) Gulf for the first time; previously unknown females of this species are described.

Topics: Animals; Bass; Dracunculoidea; Female; Fish Diseases; Fishes; Gonads; Iraq; Male; Microscopy, Electron, Scanning; Nematoda; Perciformes; Species Specificity

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

Topics: Animals; Bacillus subtilis; Bass; DNA Virus Infections; Fish Diseases; Iridovirus; Ranavirus; Singapore; Spores, Bacterial; Vaccination

Largemouth bass (Micropterus salmoides) is an important economic freshwater aquaculture fish originating from North America. However, the frequent outbreaks of Micropterus salmoides rhabdovirus (MSRV) have seriously limited the healthy development of Micropterus salmoides farming industry. In the present study, a strain of MSRV was isolated and identified from infected largemouth bass by PCR, transmission electron micrograph observation and genome sequences analysis, and tentatively named MSRV-HZ01 strain. Phylogenetic analyses showed that the MSRV-HZ01 presented the highest similarity to MSRV-2021, followed by MSRV-FJ985 and MSRV-YH01. The various tissues of juvenile largemouth bass exhibited significant pathological damage following MSRV-HZ01 immersion infection, and the mortality reached 90%. We also found that intestine was the key organ for MSRV to enter the fish body initially by dynamic analysis of viral infection, and the head kidney was the susceptible tissue of virus. Moreover, the MSRV was also transferred to the external mucosal tissue in later stage of viral infection to achieve horizontal transmission. In addition, the genes of IFN γ and IFN I-C were significantly up-regulated after MSRV infection to exert antiviral functions. The genes of cGAS and Sting might play an important role in the regulation of interferon expression. In conclusion, we investigated the virus infection dynamics and fish response following MSRV immersion infection, which would promote our understanding of the interaction between MSRV and largemouth bass under natural infection.

Topics: Animals; Bass; Fish Diseases; Immersion; Phylogeny; Rhabdoviridae; Virus Diseases

Type I interferons (IFNs) play a significant role in antiviral innate immunity. But, the antiviral function of IFNd is controversial in teleosts. Here, we identified three IFNd receptors belonging to cytokine receptor family B (LmCRFB1, LmCRFB2, and LmCRFB5) in spotted seabass (Lateolabrax maculatus). LmIFNd and its receptors were highly expressed in gill, spleen and head kidney tissues. Additionally, LmIFNd, its receptors, and their downstream signal genes (LmTYK2, LmJAK1, LmSTAT1, and LmSTAT2) were induced by infectious spleen and kidney necrosis virus (ISKNV) infection. Injection of recombinant protein (LmIFNd-His) in vivo and incubation with the LmIFNd-His in vitro both induced expressions of IFN-stimulated genes (LmISGs). IFNd-His had a dose-dependent protective effect on the activity of brain cells infected by ISKNV and reduced the number of ISNKV copies. LmIFNd-His also bound to extracellular domains of the three receptors in vitro in the pull-down assay. LmIFNd-His preferentially induced ISG expression through receptor complex LmCRFB1 and LmCRFB5, followed by LmCRFB2 and LmCRFB5, to induce the expressions of LmISGs. Our results show that LmIFNd can enhance the antiviral immune response of spotted seabass, and it uses receptor complex LmCRFB1 and LmCRFB5 as well as LmCRFB2 and LmCRFB5 to induce LmISG expression. It is the first study about the antiviral function of LmIFNd and its receptor complex in spotted seabass, and it provides a reference for further studies of the controversial anti-viral function of IFNd in teleosts.

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Iridoviridae

Grouper culture has been expanding in Malaysia due to the huge demand locally and globally. However, due to infectious diseases such as vibriosis, the fish mortality rate increased, which has affected the production of grouper. Therefore, this study focuses on the metabolic profiling of surviving infected grouper fed with different formulations of fatty acid diets that acted as immunostimulants for the fish to achieve desirable growth and health performance. After a six-week feeding trial and one-week post-bacterial challenge, the surviving infected grouper was sampled for GC-MS analysis. For metabolite extraction, a methanol/chloroform/water (2:2:1.8) extraction method was applied to the immune organs (spleen and liver) of surviving infected grouper. The distribution patterns of metabolites between experimental groups were then analyzed using a metabolomics platform. A total of 50 and 81 metabolites were putatively identified from the spleen and liver samples, respectively. Our further analysis identified glycine, serine, and threonine metabolism, and alanine, aspartate and glutamate metabolism had the most impacted pathways, respectively, in spleen and liver samples from surviving infected grouper. The metabolites that were highly abundant in the spleen found in these pathways were glycine (20.9%), l-threonine (1.0%) and l-serine (0.8%). Meanwhile, in the liver l-glutamine (1.8%) and aspartic acid (0.6%) were found to be highly abundant. Interestingly, among the fish diet groups, grouper fed with oleic acid diet produced more metabolites with a higher percent area compared to the control diets. The results obtained from this study elucidate the use of oleic acid as an immunostimulant in fish feed formulation affects more various immune-related metabolites than other formulated feed diets for vibriosis infected grouper.

Topics: Animal Feed; Animals; Bass; Diet; Fish Diseases; Oleic Acid; Vibrio Infections; Vibrio vulnificus

Singapore grouper iridovirus (SGIV) is a highly pathogenic Iridoviridae that causes hemorrhage and spleen enlargement in grouper. Despite previous genome annotation efforts, many open reading frames (ORFs) in SGIV remain uncharacterized, with largely unknown functions. In this study, we identified the protein encoded by SGIV ORF122, now referred to as VP122. Notably, overexpression of VP122 promoted SGIV replication. Moreover, VP122 exhibited antagonistic effects on the natural antiviral immune response through the cGAS-STING signaling pathway. It specifically inhibited the cGAS-STING-triggered transcription of various immune-related genes, including IFN1, IFN2, ISG15, ISG56, PKR, and TNF-α in GS cells. Additionally, VP122 significantly inhibited the activation of the ISRE promoter mediated by EccGAS and EcSTING but had no effect on EccGAS or EcSTING alone. Immunoprecipitation and Western blotting experiments revealed that VP122 specifically interacts with EcSTING but not EccGAS. Notably, this interaction between VP122 and EcSTING was independent of any specific domain of EcSTING. Furthermore, VP122 inhibited the self-interaction of EcSTING. Interestingly, VP122 did not affect the recruitment of EcTBK1 and EcIRF3 to the EcSTING complex. Collectively, our results demonstrate that SGIV VP122 targets EcSTING to evade the type I interferon immune response, revealing a crucial role for VP122 in modulating the host-virus interaction.

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity; Interferon Type I; Iridovirus; Ranavirus; Singapore

Methylation at the N

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Interferon Type I; Methyltransferases; Necrosis; Nodaviridae; Novirhabdovirus; Perches; RNA Virus Infections

As one of the important members of the autophagy-related protein family, Atg14 plays a key role in the formation and maturation of autophagosomes. However, little is known about the potential roles of fish Atg14 and its roles in virus infection. In the present study, the homolog of Atg14 (EcAtg14) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) of EcAtg14 consists of 1530 nucleotides, encoding 509 amino acids, with a predicted molecular weight of 56.9 kDa. EcAtg14 was distributed in all tested tissues, with higher expression in liver, blood and spleen. The expression of EcAtg14 was increased in grouper spleen (GS) cells after Singapore grouper iridovirus (SGIV) infection. EcAtg14 was distributed in the cytoplasm of GS cells. Overexpression of EcAtg14 promoted SGIV replication in GS cells and inhibited IFN3, ISRE and NF-κB promoter activities. Co-immunoprecipitation results showed that there was an interaction between EcAtg14 and EcBeclin. EcAtg14 also promoted the synthesis of LC3-II in GS cells. These findings provide a basis for understanding the innate immune mechanism of grouper against viral infection.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Phylogeny; Ranavirus; Singapore

Nuclear factor-κB (NF-κB)/Rel is a group of transcription factors that can be activated and regulates various aspects of innate and adaptive immune functions, which play a crucial role in mediating inflammatory responses. Interleukin-10 (IL-10) is a highly pleiotropic cytokine that has a central role in limiting the immune response to pathogens during infection and thereby alleviating damage to the host. This study aims to investigate the function of the Rel gene in virus infection and its regulatory effect on IL-10 in the largemouth bass (Micropterus salmoides). The ORF sequence of MsRel was 1941 bp, containing 646 amino acids with two conserved functional domains, including RHD and IPT domain. In healthy largemouth bass, the mRNA of MsRel was detected in all the tested tissues, including gill, liver, kidney, heart, spleen, intestine, stomach, skin, brain, fin and muscle. The expression of MsRel was induced by challenge with largemouth bass virus (LMBV) or red grouper nervous necrosis virus (RGNNV), as well as treatment with lipopolysaccharide (LPS) or poly (I:C) in vivo. As evidenced by the detection of viral gene mRNA levels, the infectivity of LMBV and morphological cytopathic effect (CPE), we found that overexpression of MsRel inhibited the infection and replication of LMBV, suggesting its antiviral roles in fish. Besides, the promoter analysis was carried out to determine whether MsRel was a regulator of MsIL-10. The results of the luciferase reporter assay indicated that MsRel has a positive regulatory role in MsIL-10 expression. Further analysis revealed that the potential binding sites of MsIL-10 may be located in the MsIL10-5-M (-42 to +8 bp) region of the MsIL-10 promoter. Furthermore, we observed that MsRel enhanced IFN-I and IFN-III promoter activities. Taken together, our findings demonstrated that MsRel affect LMBV infection by regulating the immune responses, and providing a new idea of the mechanisms how Rel regulate the expression of IL-10 in bony fish.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Interleukin-10; Poly I-C; Ranavirus; RNA, Messenger

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Perciformes; Ranavirus; RNA, Circular; RNA, Messenger; Singapore

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens, respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo, implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Guanosine Triphosphate; Immunity, Innate; Nodaviridae; RNA Virus Infections; Virus Internalization

As a key regulator of the innate immune system, FoxO1 has a variety of activities in biological organisms. In the present study, grouper FoxO1 (EcFoxO1) was cloned and the antiviral activity in red grouper neuron necrosis virus (RGNNV) and Singapore grouper iridescent virus (SGIV) was examined. The open reading frame (ORF) of EcFoxO1 contains 2,034 base pairs that encode a protein of 677 amino acids with a predicted molecular weight of 73.21 kDa. EcFoxO1 was shown to be broadly distributed in healthy grouper tissues, and was up-regulated in vitro in response to stimulation by RGNNV and SGIV. EcFoxO1 has a whole-cell distribution in grouper spleen (GS) cells. EcFoxO1 decreased the replication of RGNNV and SGIV, and activated interferon (IFN) 3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB) promoter activities. EcFoxO1 could interact with EcIRF3. Together, the results demonstrated that EcFoxO1 might be an important regulator of grouper innate immune response against RGNNV and SGIV infection.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Neurons; Ranavirus

The largemouth bass virus (LMBV) is a commonly encountered pathogen in aquaculture and presents significant challenges to development of the largemouth bass industry due to the lack of effective treatment methods. Here, the inhibitory potential and underlying mechanisms of adamantoyl chloride (AdCl) against LMBV were assessed both in vitro and in vivo. The results showed that AdCl (IC

Topics: Animals; Apoptosis; Bass; Chlorides; DNA Virus Infections; Fish Diseases

Tripartite motif 21 (TRIM21), a member of the TRIM family, plays an important role in apoptosis, autophagy and ubiquitination in human, and has been proven to play antiviral roles in different organisms. In this study, the TRIM21 gene of Micropterus salmoides (MsTRIM21) was cloned, and it encoded 376 amino acids, which showed 89.3% similarity with Micropterus dolomieu and 38.3% with homo sapiens. Bioinformatics analysis revealed MsTRIM21 contained four domains: C4HC3-type RING-variant (RINGv), coiled coil, PRY and SPRY. The high expression level of MsTRIM21 could be detected in liver, stomach and muscle of healthy Micropterus salmoides, and it was significantly upregulated in head kidney, muscle, gill and brain and significantly down-regulated in the stomach of Micropterus salmoides infected with largemouth bass ulcer syndrome virus (LBUSV). The overexpression of MsTRIM21 could significantly inhibit the viral replication in vitro, evidenced by the reduction of CPE severity and the downregulation of the viral gene transcription. In addition, the overexpression of MsTRIM21 could significantly increase the expression level of interferon regulatory factor (IRF) 3, IRF7, myxovirus resistance 1 (Mx1), interferon stimulated gene 15 (ISG15), double-stranded RNA-activated protein kinase (PKR) and tumor necrosis factor α (TNF-α) in vitro, indicating the enhancement of innate immune response and inflammatory response, which may directly affect the replication of LBUSV. Thus, these results provide new lights on the roles of fish TRIM21 in innate immune response against iridovirus.

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Humans; Immunity, Innate; Interferons; Ulcer

Largemouth bass ranavirus (LMBV) is highly contagious and lethal to largemouth bass, causing significant economic losses to the aquaculture industry. Oral vaccination is generally considered the most ideal strategy for protecting fish from viral infection. In this study, the fusion protein MCP-FlaC, consisting of the main capsid protein (MCP) as the antigen and flagellin C (FlaC) as the adjuvant, was intracellularly expressed in Pichia pastoris. Subsequently, the recombinant P. pastoris was freeze-dried to prepare the oral vaccine P-MCP-FlaC. Transmission electron microscopy and scanning electron microscopy analysis showed that the morphology and structure of the freeze-dried recombinant P. pastoris vaccine remained intact. The experiment fish (n = 100) was divided into five groups (P-MCP-FlaC, P-MCP, P-FlaC, P-pPIC3.5K, control) to evaluate the protective efficacy of the recombinant vaccine. Oral P-MCP-FlaC vaccine effectively up-regulated the serum enzymes activity (total superoxide dismutase, lysozyme, total antioxidant capacity, and complement component 3). The survival rate of P-MCP-FlaC group was significantly higher than that of the other groups. The mRNA expression of crucial immune genes (IL-1β, TNF-α, MHC-II, IFN-γ, Mx, IgM, IgT) was also signally elevated in P-MCP-FlaC group. Vaccine P-MCP-FlaC markedly inhibited the replication of LMBV in the spleen, head kidney, and intestine, while reducing the degree of lesion in the spleen. These results suggest that the oral P-MCP-FlaC vaccine could effectively control LMBV infection, proving an effective strategy for viral diseases prevention in aquaculture.

Topics: Adjuvants, Immunologic; Animals; Bass; Capsid Proteins; Fish Diseases; Flagellin; Ranavirus; Vaccines, Synthetic

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.

Topics: Animals; Bass; Fish Diseases; Genomics; Genotype; Lipopolysaccharides; Nodaviridae; Poly I-C; RNA Virus Infections

The TRAF family member-associated nuclear factor kappa B (NF-κB) activator (TANK) regulates the NF-κB activation through the TRAF-mediated signaling pathway and is involved in the antiviral pathway by inducing the interferon (IFN) production. In the present study, we identified a TANK ortholog from the red-spotted grouper (Epinephelus akaara) and analyzed its immunological functions. The coding sequence of EaTANK consists of 1047 base pairs and encodes a 348 amino acids protein. The predicted molecular weight and theoretical isoelectric point (pI) were 38.92 kDa and 5.39, respectively. According to the phylogenetic analysis, EaTANK was closely clustered with fish TANK orthologs, exhibiting the highest identity (97.1 %) and similarity (97.1 %) to that of Epinephelus lanceolatus. A highly conserved TBK1/IKKi binding domain (TBD) was identified between 110 and 164 residues. Our tissue distribution analysis showed that EaTANK mRNA was ubiquitously expressed in 12 tested tissues, with the highest expression in the spleen and peripheral blood cells (PBCs). According to the immune challenge experiments, EaTANK mRNA expression in PBCs was significantly elevated following stimulation with polyinosinic:polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS), or nervous necrosis virus (NNV). We also observed a significant elevation in the mRNA expression of downstream antiviral pathway-related genes (ISG15, IRF3, and IRF7) in EaTANK-overexpressing fathead minnow (FHM) cells against poly (I:C) stimulation. Moreover, the replication of 6 genes in the VHSV genome was inhibited by the overexpression of EaTANK. Finally, we confirmed that the expression of NFKB1 mRNA and promoter binding activity of NF-κB was significantly increased in poly (I:C)-stimulated EaTANK-overexpressing FHM cells. In conclusion, the results of this study suggest that TANK significantly contributes to the antiviral response and regulation of NF-κB activity in red-spotted grouper.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; NF-kappa B; Phylogeny; RNA, Messenger

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1β, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

Topics: Animals; Bass; Cholesterol Oxidase; Fish Diseases; Nocardia Infections; Nucleic Acid-Based Vaccines; Vaccines, DNA

Citrobacter freundii, a common pathogen of freshwater fish, causes significant commercial losses to the global fish farming industry. In the present study, a highly pathogenic C. freundii strain was isolated and identified from largemouth bass (Micropterus salmoides). The pathogenicity and antibiotic sensitivity of the C. freundii strain were evaluated, and the histopathology and host immune response of largemouth bass infected with C. freundii were investigated. The results showed that C. freundii was the pathogen causing disease outbreaks in largemouth bass, and the infected fish showed typical signs of acute hemorrhages and visceral enlargement. Antimicrobial susceptibility testing showed that the C. freundii strain was resistant to Kanamycin, Medimycin, Clindamycin, Penicillin, Oxacillin, Ampicillin, Cephalexin, Cefazolin, Cefradine and Vancomycin. Histopathological analysis showed different pathological changes in major tissues of diseased fish. In addition, humoral immune factors such as superoxide dismutase (SOD), catalase (CAT) and lysozyme (LZM) were used as serum indicators to evaluate the immune response of largemouth bass after infection. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression pattern of immune-related genes (CXCR1, IL-8, IRF7, IgM, CD40, IFN-γ, IL-1β, Hep1, and Hep2) in liver, spleen, and head kidney tissues, which demonstrated a strong immune response induced by C. freundii infection in largemouth bass. The present study provides insights into the pathogenic mechanism of C. freundii and immune response in largemouth bass, promoting the prevention and treatment of diseases caused by C. freundii infection.

Topics: Animals; Bass; Citrobacter freundii; Fish Diseases; Immunity

Rab32 is a member of the Rab GTPase family that is involved in membrane trafficking and immune response, which are crucial for controlling pathogen infection. However, the role of Rab32 in virus infection is not well understood. In this study, we focused on the regulation of Rab32 on virus infection and the host immunity in orange-spotted grouper, Epinephelus coioides. EcRab32 encoded a 213-amino acid polypeptide, which shared a high sequence identity with other Rab32 proteins from fishes to mammals. In healthy orange-spotted grouper, the mRNA of EcRab32 was expressed in all the detected tissues, with the more expression levels in the head kidney, liver and gill. Upon SGIV infection, the expression of EcRab32 was significantly up-regulated in vitro, indicating its potential role in viral infection. EcRab32 was observed to be distributed in the cytoplasm as punctate and vesicle-like structures. EcRab32 overexpression was found to notably inhibit SGIV infection, while the interruption of EcRab32 significantly promoted SGIV infection. In addition, using single particle imaging analysis, we found that EcRab32 overexpression prominently reduced the attachment and internalization of SGIV particles. Furthermore, the results demonstrated that EcRab32 played a positive role in regulating the interferon immune and inflammatory responses. Taken together, these findings indicated that EcRab32 influenced SGIV infection by regulating the host immune response, providing an overall understanding of the interplay between the Rab32 and innate immunity.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Mammals; Phylogeny; rab GTP-Binding Proteins; Ranavirus; Virus Diseases

Singapore grouper iridovirus (SGIV) is a virus with high fatality rate in the grouper culture industry. The outbreak of SGIV is often accompanied by a large number of grouper deaths, which has a great impact on the economy. Therefore, it is of great significance to find effective drugs against SGIV. It has been reported that edaravone is a broad-spectrum antiviral drug, most widely used clinically in recent years, but no report has been found exploring the effect of edaravone on SGIV infections. In this study, we evaluated the antiviral effect of edaravone against SGIV, and the anti-SGIV mechanism of edaravone was also explored. It was found that the safe concentration of edaravone on grouper spleen (GS) cells was 50 µg/mL, and it possessed antiviral activity against SGIV infection in a dose-dependent manner. Furthermore, edaravone could significantly disrupt SGIV particles and interference with SGIV binding to host cells, as well as SGIV replication in host cells. However, edaravone was not effective during the SGIV invasion into host cells. This study was the first time that it was determined that edaravone could exert antiviral effects in response to SGIV infection by directly interfering with the processes of SGIV infecting cells, aiming to provide a theoretical basis for the control of grouper virus disease.

Topics: Animals; Antiviral Agents; Bass; DNA Virus Infections; Edaravone; Fish Diseases; Fish Proteins; Iridovirus; Ranavirus

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.

Topics: Animals; Antimicrobial Peptides; Bass; Biofilms; Escherichia coli; Fish Diseases

Mammalian studies have shown that the nuclear transcription factor Y (NFYC) regulates the expression of major histocompatibility complex (MHC) by binding to CCAAT-box on promoters. However, few studies have focused on the regulatory mechanisms of NFYC in MHC pathway in fish. To explore the transcriptional regulatory mechanism of MHCIa in fish, we characterized NFYC and MHCIa of red-spotted grouper (Epinephelus akaara) (named EaNFYC and EaMHCIa, respectively). The EaNFYC genome sequence is 13,796 bp and contains 1,065 bp open reading frame. It is composed of ten exons and nine introns and encode a 354 amino acid sequence. The putative EaNFYC protein sequence shared 67.2-99.4% identity to vertebrate NFYC and possesses a typically conserved domain (histone- or haem-associated protein 5 domain (HAP5)) at the N-terminus. Transcripts of both EaNFYC and EaMHCIa were ubiquitously expressed in all detect tissues, and higher mRNA levels were detected in immune-relevant tissues (middle-kidney). EaNFYC expression increased after treatment with polyinosinic: polycytidylic acid, lipopolysaccharide, nervous necrosis virus, zymosan A, and Singapore grouper iridovirus. Analysis of subcellular localization indicated that EaNFYC was localized at the cell nucleus only. Furthermore, overexpression of EaNFYC significantly stimulated the expression of EaMHCIa, interferon signalling molecules and inflammatory cytokine. The region -878 bp to +82 bp of EaMHCIa promoter was identified to be the core promoter which EaNFYC take effect on. Additionally, point mutations and electrophoretic mobility shift assays verified that NFYC activate MHCIa expression by binding at the M1 and M2 binding sites that do not contain CCAAT-box. These results contribute to elucidating the function of fish NFYC on MHC transcriptional mechanisms, and provide the first evidence of positive regulation of MHCIa expression by NFYC in fish.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Mammals; Phylogeny; Sequence Alignment; Transcription Factors

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.

Topics: Animals; Bass; Fish Diseases; Iridoviridae; Nucleic Acid Amplification Techniques; Perciformes; Sensitivity and Specificity

Histopathological assessments of young-of-the-year (age-0) Smallmouth Bass Micropterus dolomieu in the Susquehanna River drainage identified a high prevalence of the myxozoan Myxobolus inornatus. This myxozoan infects the connective tissue of the muscle below the skin but is sometimes observed in the esophagus and buccal cavity. In some instances, shallow infections cause breaks in the skin, which could increase the chance of opportunistic bacterial infections. Several microbial pathogens, including Flavobacterium columnare, Aeromonas spp., and Largemouth Bass virus, have also been cultured from clinically diseased young of year. A multiplex fluorescence in situ hybridization (FISH) assay was developed to determine potential colocalization of M. inornatus, Flavobacterium spp., and Aeromonas spp. infections. With FISH, 75% of age-0 Smallmouth Bass exhibited M. inornatus infections, 10% had Aeromonas spp. infections, and 5% had Flavobacterium spp. infections, while 3% had coinfections with both bacterial species and M. inornatus. The results of the multiplex FISH assay revealed a low occurrence of coinfections of Flavobacterium spp. and/or Aeromonas spp. with M. inornatus in randomly sampled individuals.

Topics: Animals; Bass; Coinfection; Fish Diseases; In Situ Hybridization, Fluorescence; Myxobolus; Rivers

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%-20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus-yellowfin sea bream interaction.

Topics: Animals; Bass; Cell Line; Fish Diseases; Fish Proteins; Liver; Nodaviridae; RNA Virus Infections; Sea Bream

Largemouth Bass virus (LMBV) first became a concern in Kansas when it was identified as a potential cause of decreased catch rates at Crawford State Fishing Lake in 2007. The discovery of LMBV in eight additional impoundments from 2008 to 2017 increased concern about the prevalence and effects of LMBV in Kansas. In response, the Kansas Department of Wildlife, Parks, and Tourism tested 25 Largemouth Bass Micropterus salmoides impoundments for the presence of LMBV. The objectives of this study were to quantify the incidence of LMBV and examine differences in population metrics (i.e., body condition, relative abundance, and growth). A total of 1,260 Largemouth Bass were collected by using standard spring electrofishing surveys, and sagittal otoliths were collected from all of the sampled fish to estimate growth rates. Of the 25 study impoundments, 14 tested positive for LMBV. There was no evidence of LMBV effects on body condition, relative abundance of quality-length fish, or growth rates. The initial dates of LMBV infection of Largemouth Bass in these impoundments are unknown. The LMBV-positive populations in Kansas may have been exposed to the virus many years ago, and the fish may be in the process of rebounding from any potential negative effects.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Incidence; Kansas; Lakes; Prevalence; Ranavirus

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the. 神经坏死病毒（nervous necrosis virus, NNV）可感染多种重要海淡水经济鱼类，给水产养殖业造成巨大经济损失。自噬是一种进化上保守的依赖于溶酶体的细胞物质降解途径，参与多种病毒感染过程。研究表明，NNV能够诱导鱼类细胞发生自噬促进自身复制，但NNV在入侵阶段诱导细胞自噬的分子机制尚不清楚。在该研究中，我们发现在2种鱼类细胞中赤点石斑鱼神经坏死病毒（red-spotted grouper NNV，RGNNV）都能够在感染1.5和3 h时诱导自噬小体形成，表明RGNNV入侵阶段即可诱发细胞自噬。进一步研究发现RGNNV感染能够抑制自噬溶酶体和自噬小体融合，表明RGNNV入侵阶段诱导细胞发生不完全自噬。随后研究发现RGNNV表面的唯一结构蛋白衣壳蛋白（capsid protein，CP）通过与RGNNV受体海鲈热休克蛋白90ab1(heat shock protein HSP90ab1，LjHSP90ab1)结合抑制AKT-MTOR信号通路，从而诱导细胞自噬发生。机制研究表明CP蛋白竞争性结合于LjHSP90ab1的NM结构域以阻碍AKT与LjHSP90ab1的结合，从而抑制AKT-MTOR信号通路，诱导细胞自噬发生。综上所述，RGNNV在入侵阶段利用CP结合其受体HSP90ab1，进而抑制AKT-MTOR信号通路，诱导细胞不完全自噬。该研究揭示了NNV受体在病毒感染诱导细胞自噬中的功能，为深入解析NNV感染的致病机制提供了新视角。.

Topics: Animals; Autophagy; Bass; Capsid Proteins; Fish Diseases; Fish Proteins; Necrosis; Nodaviridae; Proto-Oncogene Proteins c-akt; RNA Virus Infections; TOR Serine-Threonine Kinases; Virulence

Microcotylid monogeneans can cause considerable health problems in cultured fish, and several Microcotyle species are reported from scorpaenid fish, an economically important aquaculture target species in Korea. We developed a PCR-RFLP assay targeting the mitochondrial cox1 gene, for discriminating Microcotyle sebastis and M. caudata from cultured Korean rockfish Sebastes schlegelii and dark-banded rockfish S. inermis. AseI enzyme treatment of the PCR products showed that M. sebastis sequence was cleaved while M. caudata was not. A total of 95.2% (118/124) of monogeneans from S. schlegelii were identified as M. sebastis, and 96.2% (126/131) of monogeneans from S. inermis were identified as M. caudata by PCR-RFLP. However, the remaining parasites from each host showed the opposite digestion pattern. Additional analyses of these specimens by targeting the ITS region by PCR-RFLP showed the same results, suggesting that cross-species infection by the parasites may have occurred. In Korea, S. inermis net cages are commonly located nearby S. schlegelii net cages, and this encaged proximity might have provided the opportunity for cross-infection to occur. Further examination of wild host populations and experimental cross-infection will be necessary to explain this phenomenon. The PCR-RFLP method in this study will help investigate the epidemiology and infection dynamics of Microcotyle species in S. inermis.

Topics: Animals; Bass; Fish Diseases; Perciformes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Republic of Korea; Urodela

Interleukin-8 (IL-8 or C-X-C motif chemokine ligand 8, CXCL8) is a cytokine secreted by numerous cell types and is best known for its functional roles in inflammatory response by binding to specific receptors (the interleukin-8 receptors, IL-8Rs). From the transcriptomic data of largemouth bass (Micropterus salmoides), we identified an IL-8R that is highly homologous to the functionally validated teleost IL-8Rs. The M. salmoides IL-8 receptor (MsCXCR2) was further compared with the C-X-C motif chemokine receptor 2 subfamily by phylogenetic analysis. Briefly, the full-length CDS sequence of MsCXCR2 was cloned into the pEGFP-N1 plasmid, and the membrane localization of fusion expressing MsCXCR2-EGFP was revealed in HEK293 cells. To determine the functional interaction between IL-8 and MsCXCR2, secretory expressed Larimichthys crocea IL-8 (LcIL-8) was used to stimulate MsCXCR2 expressing cells. MsCXCR2 was demonstrated to be activated by LcIL-8, leading to receptor internalization, which was further revealed by the detection of extracellular regulated protein kinase (ERK) phosphorylation. Quantitative real-time PCR was used to evaluate the expressional distribution and variation of MsCXCR2 in healthy and Nocardia seriolae infected fish. Based on our findings, MsCXCR2 was constitutively expressed in all examined tissues, despite at different levels. Furthermore, gene expression was found to be significantly upregulated in the liver and head kidney of diseased fish. Collectively, our findings reveal the molecular activity of MsCXCR2 and indicate the functional involvement of this IL-8R in the immune response induced by N. seriolae in M. salmoides.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; HEK293 Cells; Humans; Interleukin-8; Nocardia; Nocardia Infections; Phylogeny; Receptors, Interleukin-8B

Heat shock proteins (Hsps) are important for maintaining protein homeostasis and cell survival. In this study, Hsp27 of Epinephelus coioides, an economically important marine fish in China and Southeast Asian countries, was characterized. E. coioides Hsp27 contains the consered ACD_HspB1_like domain and three p38 MAPK phosphorylation sites, located at Thr-13, Thr-60 and Ser-167. E. coioides Hsp27 was distributed in both the cytoplasm and nucleus, its mRNA was detected in all 14 tissues examined, and its expression was up-regulated after challenge with Singapore grouper iridovirus (SGIV), an important E. coioides pathogen. Over-expression of E. coioides Hsp27 significantly upregulated the expressions of the key SGIV genes (VP19, LITAF, MCP, and ICP18), downgraded the expressions of the E. coioides immune factors (IRF3, IRF7, ISG15, and TRAF6) and proinflammatory factors (TNF-α, IL-8), downgraded the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and substantially inhibited the cell apoptosis induced by SGIV infection. These data illustrated that E. coioides Hsp27 might be involved in SGIV infection by negatively regulating the innate immune response.

Topics: Animals; Apoptosis; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Heat-Shock Proteins; Immunity, Innate; Iridovirus

Pseudomonas plecoglossicida is a well-known pathogen of viscera granulomas disease in fish, which has led to severe economic losses. In our previous study, L321_RS13075 was predicted to be a key virulence gene of P. plecoglossicida during the host-pathogen interaction with Epinephelus coioides. To investigate the role of L321_RS13075 in the regulation of virulence in P. plecoglossicida, a L321_RS13075 knock-down strain was constructed. And a significant reduction in the ability of colonization, intracellular survival, motility, biofilm formation, and adhesion was detected in the L321_RS13075 knock-down strain. Compared with the wild-type strain, the silence of L321_RS13075 in P. plecoglossicida resulted in a significant change in the transcriptome of infected Epinephelus coioides (E. coioides). Results of COG and GO analysis on E. coioides showed that genes related to immune responses and inorganic ion transport were significantly affected by L321_RS13075 of P. plecoglossicida. Meanwhile, the interactions of the genes related to immune responses and inorganic ion transport were predicted, and the important hub genes were identified. Taken together, the results indicated that L321_RS13075 was a virulent gene of P. plecoglossicida, which significantly affected the immune responses and inorganic ion transport in E. coioides.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Immunity; Ion Transport; Pseudomonas; Pseudomonas Infections

To study the roles of the exbB gene in Pseudomonas plecoglossicida during interactions with Epinephelus coioides, five short hairpin RNAs (shRNAs) were designed and synthesized to silence the exbB gene in P. plecoglossicida which resulted in significant reductions in exbB mRNA expression. The mutant with the best silencing efficiency (89.3%) was selected for further study. Silencing exbB in the exbB-RNA interference (RNAi) strain resulted in a 70% increase in the survival rate and a 3-day delay in the onset of infection in E. coioides. Silencing of the exbB gene also resulted in a significant decrease in the number of white spots on the spleen surface and in the spleen pathogen load. The results of dual RNA-seq showed that exbB silencing in P. plecoglossicida also resulted in a significant change in both the pathogen and host transcriptomes in the spleens of infected E. coioides. Comparative transcriptome analysis showed that silencing exbB caused significant changes in multiple signaling molecules and interaction- and immune system-related genes in E. coioides. Gene silencing also resulted in the differential expression of flagellar assembly and the bacterial secretion system in P. plecoglossicida during the infection period, and most of the DEGs were down-regulation. These host-pathogen interactions may make it easier for E. coioides to eliminate the exbB-RNAi strain of P. plecoglossicida, suggesting a significant decrease in the pathogenicity of this strain. These results indicated that exbB was a virulence gene of P. plecoglossicida which contributed a lot in the pathogen-host interactions with E. coioides.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Gene Silencing; Immunity, Innate; Pseudomonas; RNA, Small Interfering; Spleen; Transcriptome; Virulence

Largemouth bass (Micropterus salmoides) is an important freshwater-cultured species in China. Recently, a lethal and epidemic disease caused by Micropterus salmoides rhabdovirus (MSRV) results in huge economic losses to the largemouth bass industry. Current diagnostics for detecting MSRV are limited in sensitivity and speed and are inconvenient to be used for non-laboratory detection. In this study, three rapid and convenient detection assays of MSRV by recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD), targeting the conserved sequences of the MSRV-SS N gene, are described. With these RPA methods, the detection could achieve within 50 min at 38°C. Both methods of RPA-AGE and RPA-LFD could detect the viral DNA as low as 170 copies/μl of the MSRV standard plasmid and were 100-fold more sensitive than that in the method of routine PCR. Meanwhile, these RPA methods were highly specific for the detection of MSRV and can be feasibly applied to the diagnostic of MSRV infection. In brief, RPA-AGE, RPA-LFD and RT-RPA-LFD provide convenient, rapid, sensitive and reliable methods that could improve field diagnosis of MSRV with limited machine resources, and would enhance the production of largemouth bass.

Topics: Animals; Bass; Fish Diseases; Nucleic Acid Amplification Techniques; Recombinases; Rhabdoviridae; Rhabdoviridae Infections; Sensitivity and Specificity

Galectin-8 is a typical β-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Galectins; Gene Expression Regulation; Gram-Negative Bacteria; Gram-Positive Bacteria; Immunity, Innate; Phylogeny; Sequence Alignment; Wound Healing

In the present study, we studied the effect of β-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of β-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1β production were reduced in β-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the β-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with β-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that β-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Topics: Animals; Antiviral Agents; Bass; beta-Glucans; Fish Diseases; Nodaviridae; RNA Virus Infections

A new myxozoan species, Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae), was found in the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam. Myxospores were observed floating free in the gall bladder of 3 out of 20 fish examined (15%). Mature myxospores were elongate and slightly crescent-shaped and measured 12.2 ± 1.3 (10.8-16.0) μm in thickness and 5.8 ± 0.6 (4.8-6.9) μm in length, with two smooth equal shell valves. The two polar capsules were spherical and equal in size, measuring 2.6 ± 0.3 (2.3-2.9) μm in diameter. The posterior angle was slightly concave, 153.7° ± 5.6° (148.9°-166.0°). Molecular analysis of SSU rDNA sequence showed that Ceratomyxa binhthuanensis n. sp. differs from other Ceratomyxa spp. available in GenBank. Phylogenetic analysis indicated that C. binhthuanensis n. sp. was closely related to three species, Ceratomyxa nolani, Ceratomyxa yokoyamai, and Ceratomyxa cutmorei, which also infect fish hosts of the genus Epinephelus.

Topics: Animals; Bass; DNA, Ribosomal; Fish Diseases; Gallbladder; Myxozoa; Parasitic Diseases, Animal; Perciformes; Phylogeny; Vietnam

Matrix metalloproteinases (MMPs) are highly expressed in leukocytes and macrophages, which play a role in the innate immune response. Here, the cDNA sequence of MMP25 from Japanese sea bass (Lateolabrax japonicus) (LjMMP25) was identified. Phylogenetic analysis revealed that LjMMP25 was most closely related to large yellow croaker MMP25. Multiple sequence alignment of LjMMP25 with MMP25 sequences from other teleosts revealed that regions of known functional importance were highly conserved. Expression analysis revealed that LjMMP25 was highly expressed in the head kidney and widely expressed in other tissues including gill, spleen, and liver. LjMMP25 was found to regulate inflammatory cytokine production and promote phagocytosis and bacterial killing in monocytes/macrophages (MO/MФ). Furthermore, LjMMP25 regulated the inflammatory response by modulating NF-κB signaling. These findings reveal new information about the role of LjMMP25 in regulating pro-inflammatory responses in this species.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; GPI-Linked Proteins; Immunity, Innate; Leukocytes; Liver; Macrophages; Matrix Metalloproteinases, Membrane-Associated; Monocytes; Phagocytosis; Phylogeny; Sequence Alignment; Vibrio Infections

The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40-369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Nodaviridae; Phylogeny; Sequence Alignment; Ubiquitin-Specific Proteases

Topics: Animals; Bass; Fish Diseases; Necrosis; Nodaviridae; Sequence Alignment

The classical major histocompatibility complex class I (MHC-Ⅰ) molecule plays a key role in vertebrate immune response for its important functions in antigen presentation and immune regulation. MHC pathway is closely related to many diseases involving autoimmunity, antigen intrusion and inflammation. However, rare literatures about the effect of MHC-I on fish cells apoptosis were reported. In this study, a novel type of MHC-Ⅰα genotype from orange-spotted grouper (named EcMHC-ⅠA*01) were cloned and characterized. It shared a 77% identity to its Epinephelus coioides MHC-Iα homology that has been uploaded to NCBI (ACZ97571.1). Molecular characterization analysis showed that EcMHC-ⅠA*01 encodes a 357-amino-acid protein, containing a signal peptide，α1，α2，α3, Cytoplasmic (Cyt) and Transmembrane (TM) domains. Tissue expression pattern showed that EcMHC-ⅠA*01 was extensively distributed in twelve selected tissues, with higher expression in the gill, intestine and skin. The expression of EcMHC-ⅠA*01 in grouper liver and spleen tissues were significantly induced by different stimuli (Zymosan A, LPS, Ploy I:C, RGNNV and SGIV). Comparing with the EcMHC-ⅠA*01 expression levels induced by Zymosan A, Ploy I:C and RGNNV, the effects induced by SGIV and LPS were more significant. Subcellular localization analysis showed that EcMHC-ⅠA*01 localizes throughout the cytoplasm appeared both diffuse and focal intracellular expression pattern. Overexpression of EcMHC-ⅠA*01 inhibited the CPE progression, the mRNA expression of the SGIV related genes (MCP, LITAF, ICP-18 and VP19) and the protein expression of MCP. Meanwhile, qRT-PCR result showed that EcMHC-ⅠA*01 overexpression upregulated the expression of interferon signaling molecules (IFN-γ, ISG56, MDA5 and MXI) and inflammatory cytokines (IL-1β, IL-6, TNF-α and TRAF6). In addition, our results showed that overexpression of EcMHC-ⅠA*01 promoted the apoptosis of normal fathead minnow (FHM) cells as well as the apoptosis of FHM cells induced by SGIV. However, there was no significant change in the activity of caspase 3 between control group and EcMHC-ⅠA*01 overexpression group, suggesting that EcMHC-ⅠA*01-induced apoptosis may not depend on the caspase 3 pathway. Taken together, these data in our study provide new insights into the role of MHC-I in antiviral immune response and apoptosis in fish.

Topics: Animals; Apoptosis; Bass; Caspase 3; DNA Virus Infections; Fish Diseases; Fish Proteins; Genes, MHC Class I; Genotype; Iridovirus; Lipopolysaccharides; Phylogeny; Zymosan

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Necrosis; Nodaviridae; RNA Virus Infections; T-Cell Intracellular Antigen-1

Nanoplastics (NPs) might cause different negative effects on aquatic organisms at different biological levels, ranging from single cells to whole organisms, including cytotoxicity, reproduction, behavior or oxidative stress. However, the impact of NPs on disease resistance is almost unknown. The objective of this study was to assess whether exposure to 50 nm functionalized polystyrene NPs impacts fish susceptibility to viral diseases both in vitro and

Topics: Animals; Bass; Cell Line; Fish Diseases; Microplastics; Nodaviridae; RNA Virus Infections

Cathepsin L (CTSL) is a cysteine endopeptidase involved in protein degradation mainly in lysosomes. Following activation in an acidic environment, it plays a key role in a variety of physiological, immunological, and pathological processes. The biological function of CTSL in teleost remains unclear. Immunohistochemical analysis revealed that CTSL was expressed mainly in lymphoid organs, head kidney, trunk kidney, and liver, which particularly was expressed in leukocyte-like cells. We performed two forms of recombinant CTSL (rCTSL and rTCTSL) derived from orange-spotted grouper (Epinephelus coioides) to elucidate the role of CTSL in teleost innate immunity, based on differences in immune-related gene expression. We determined that rCTSL has a proteolytic function whereas rTCTSL does not. Under CTSL activation, we observed increases in IL-1β, IL-6, IL-12, IFNγ, CCL-1, CCL-3, epinecidin-1, lysozyme, and IgM. The bacteriolytic activity of rCTSL was more pronounced against Gram-positive bacteria than Gram-negative bacteria. Our findings indicate CTSL plays multiple roles in the reactions of innate immunity.

Topics: Amino Acid Sequence; Animals; Bacteria; Bass; Cathepsin L; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Proteolysis

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1β and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Nodaviridae; RNA Virus Infections; Sequence Alignment; Virus Diseases

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response.

Topics: Animals; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Phylogeny; Receptors, Scavenger

Ceratomyxa marginati n. sp. and C. aenei n. sp. are two new coelozoic myxosporean species infecting the gallbladder of the dusky grouper (Epinephelus marginatus) and the white grouper (Epinephelus aeneus), respectively. These two ceratomyxids were described using morphological characteristics and molecular analysis of the SSU rDNA. Ceratomyxa marginati n. sp. exhibits disporic plasmodia measuring 12-14 µm long and 11-12 µm wide and mature myxospores which are slightly crescent-shaped, measuring 6.0 ± 0.3 (5.6-6.8) µm in length and 12.9 ± 0.9 (11.5-14.0) μm in thickness. Plasmodia of C. aenei n. sp. were disporic and ellipsoidal and measured 28-32 μm in length and 19-22 μm in width. Mature myxospores of C. aenei n. sp. were elongated with unequal shell valves and measured 7.4 ± 0.6 (6.9-9.0) μm in length and 26.9 ± 2.4 (23.2-30.0) μm in thickness. Based on the SSU rDNA sequences, Ceratomyxa marginati n. sp. and C. aenei n. sp. are distinct from all other Ceratomyxa sequences available in GenBank. Phylogenetic analysis reveals that the two new species branched together within a clade with other Ceratomyxa species from different host families and different geographical localities with maximum support (100%).

Topics: Animals; Bass; Cnidaria; DNA, Ribosomal; Fish Diseases; Gallbladder; Humans; Myxozoa; Parasitic Diseases, Animal; Phylogeny

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

Topics: Animals; Anti-Infective Agents; Bass; Chemokines; Chemokines, CC; Fish Diseases; Fish Proteins; Gene Expression Regulation; Mammals; Phylogeny

Viral nervous necrosis (VNN) is now endemic in the Mediterranean basin and the RGNNV genotype betanodavirus has caused frequent epidemics in European sea bass for a long time. Unexpected and increasing VNN epidemics have been reported in gilthead sea bream (GSB) farms in the last few years, from which the RGNNV/SJNNV genotype has been mostly isolated. The aim of this study was to perform a molecular characterization of the betanodavirus isolated from GSB (weighing 90-100 g) in a marine fish farm in the Aegean Sea and also, as an early warning exercise, to investigate the presence/absence of the virus in associated nearby farms (n:20) and in hatcheries (n:3). No virus was detected in any of the nearby farms or two hatcheries. However, in one hatchery, betanodavirus was detected in a 160-day-old GSB. The identified betanodavirus was genotyped as reassortant RGNNV/SJNNV and was phylogenetically related to the virus detected in the farm located in the Aegean sea. There have been multiple detections of the RGNNV genotype in Turkish coastal waters; however, the RGNNV/SJNNV genotype has been detected for the first time and it should be an early warning to focus attention on betanodaviruses in Turkish aquaculture.

Topics: Animals; Bass; Fish Diseases; Genotype; Nodaviridae; RNA Virus Infections; Sea Bream

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies that transduce signals from various immune receptors. To investigate the interaction of TRAF3 and other proteins in signaling pathways and to identify its antiviral function in teleosts, we cloned and characterized a TRAF3 homolog from orange-spotted grouper (Epinephelus coioides) (EcTRAF3). The open reading frame of EcTRAF3 consists of 1767 base pairs encoding a 588 amino acid protein, and the predicted molecular mass is 66.71 kDa EcTRAF3 shares 99.83% identity with TRAF3 of Epinephelus lanceolatus. Expression analysis revealed that EcTRAF3 was broadly distributed in examined tissues and was up-regulated under polyinosinic-polycytidylic acid and red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vivo. EcTRAF3 was identified as a cytosolic protein based on fluorescence microscopy analysis. Overexpression of EcTRAF3 inhibited RGNNV replication in grouper spleen cells, and it interacted with the coat protein of RGNNV. Overexpression of EcTRAF3 also induced the activation of interferon β (IFN-β), IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). EcTRAF3 co-transfected with Stimulator of Interferon Genes (STING) of grouper (EcSTING) induced a significantly higher level of IFN-β promoter activity. Moreover, EcTRAF3 interacted with EcSTING, implying that EcTRAF3 may function as an enhancer in EcSTING-mediated signaling. Taken together, our results suggest that EcTRAF3 negatively regulates the RGNNV-induced cellular antiviral response and plays an important role in the immune response system of fish.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Interferon-beta; Nodaviridae; RNA Virus Infections; Signal Transduction; TNF Receptor-Associated Factor 3

Cystatin B is a cysteine protease inhibitor that plays a crucial role in immune response. Nevertheless, the molecular mechanism of fish Cystatin B in virus replication remains obscure. In this study, we identified and characterized Cystatin B (Ec-CysB) in the orange-spotted grouper (Epinephelus coioides). The Ec-CysB encoded a 100-amino acid protein with the conserved QXVXG motif, PC motif and cysteine protease inhibitory motif, which shared high identities with reported Cystatin B. The abundant transcriptional level of Ec-CysB was found in gill, intestine and head kidney. And the Ec-CysB expression was significantly up-regulated in spleen after infection with Singapore grouper iridovirus (SGIV) in vitro. Subcellular localization analysis revealed that Ec-CysB was distributed mainly in the cytoplasm and nucleus. Further studies showed that overexpression of Ec-CysB in vitro significantly increased SGIV replication and virus-induced cell apoptosis, but replication of SGIV was inhibited by knockdown or mutant of Ec-CysB. Moreover, overexpression of Ec-CysB significantly inhibited the interferon (IFN), interferon-stimulated response element (ISRE) promoter activities, and enhanced apoptosis-related transcription factors p53 promoter activities. Collectively, our results suggest that Ec-CysB affect viral replication and virus-induced cell apoptosis, which will help us to explore its potential functions during SGIV infection.

Topics: Animals; Base Sequence; Bass; Cystatin B; DNA Virus Infections; Fish Diseases; Fish Proteins; Interferons; Iridovirus; Phylogeny; Ranavirus; Transcription Factors

Aeromonas veronii bv. sobria is an emerging pathogen for the European seabass cultured in the Aegean Sea (Mediterranean) causing significant problems in the Greek and Turkish aquaculture industry since no licensed vaccine is currently available for the disease. A bivalent vaccine was developed based on two phenotypically distinct strains of the pathogen, PDB (motile, pigment-producing strain) and NS (non-motile, non-pigment-producing). The two strains comprising the bivalent vaccine were evaluated as monovalent products in zebrafish before the seabass trials. Challenges using the homologous or the heterologous strain showed that both vaccines were protective with RPS values ranging between 66 and 100% in zebrafish. The bivalent vaccine was then tested in European seabass following dip or intraperitoneal administration. Efficacy was evaluated separately against both strains comprising the bivalent vaccine. Dip vaccination applied to juvenile seabass of 2.5 g average weight provided protection following challenge tests 30 days post vaccination only in one of the two strains tested (strain PDB, RPS: 88%). This was also the case in the injection vaccination of adult seabass of 60 g average weight where the vaccine was effective only against the PDB strain (RPS: 63%). High antibody titers against both strains were found at 30 and 60 days after intraperitoneal vaccination in the adult seabass. The use of zebrafish as a model for vaccine development for aquaculture species is discussed.

Topics: Aeromonas; Aeromonas veronii; Animals; Autovaccines; Bacterial Vaccines; Bass; Fish Diseases; Vaccines, Combined; Zebrafish

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1

Topics: Animals; Antibodies, Monoclonal; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Phylogeny

Autophagy and apoptosis play important roles in the occurrence and development of diseases. Largemouth bass virus (LMBV) is a primary agent that causes infectious skin ulcerative syndrome in largemouth bass and threatens the aquaculture of the species. We investigated the relationship between LMBV and autophagy, as well as the effect of autophagy on apoptosis induced by LMBV. Results showed that LMBV could induce autophagy in epithelioma papulosum cyprinid (EPC) cells. There was also an increase in LC3-II protein and decrease in p62 protein, along with autophagosome-like membranous vesicles and punctate autophagosomes fluorescent spots being observed in EPC cells. Enhancing autophagy inhibited the replication of LMBV and apoptosis in EPC cells while inhibiting autophagy produced the opposite effect. These results offer new insights into the pathogenesis of LMBV and anti-LMBV strategies.

Topics: Animals; Apoptosis; Autophagy; Bass; Carcinoma; Cyprinidae; DNA Virus Infections; DNA Viruses; Fish Diseases; Virus Replication

A previous study confirmed that spotted sea bass (Lateolabrax maculatus), an economically important cultured species in East Asia, is a new host of Aeromonas veronii, which can cause acute death in these fish, but there is little in-depth understanding of this disease. In the present study, the virulence of 10 isolates of A. veronii derived from spotted sea bass was determined. It was found that the 18BJ181 isolate was a virulent strain and led to the fastest death of spotted sea bass. Death was determined to be within in 2-12 h, and resulted in abdominal effusion and varying degrees of hemorrhage in internal organs. Bacterial colonization analysis showed that the bacterial load in the spleen was highest, and was up to 3.1 × 10

Topics: Aeromonas veronii; Animals; Bass; Fish Diseases; Gene Expression Profiling; Immunity; Sepsis

Humpback grouper (Cromileptes altivelis), one kind of commercial fish with considerable economic value, has been recognized as a promising candidate for mariculture. In the wake of the development of aquaculture industry, the breeding density of C. altivelis has increased gradually, which gave rise to the occurrence of various pathogenic diseases. In our research, we established a new kidney cell line (designated as CAK) from humpback grouper and evaluated its susceptibility to bacteria and heavy metals. The results of our study showed that the optimal growth temperature was 26 °C, and optimal medium was L-15 supplemented with 20% fetal bovine serum (FBS). The sequencing of 18S rRNA gene indicated that CAK cell line was derived from C. altivelis. Chromosome analysis showed that the number of chromosome in CAK was 48. After being transfected of pEGFP-N3 plasmid, high transfection efficiency of CAK was observed, suggesting the potential to be used for the study of foreign functional genes. Moreover, the bacterial susceptibility results revealed that CAK cells were sensitive to Vibrio harveyi and Edwardsiella tarda, especially V. harveyi. Meanwhile, three heavy metals (Hg, Cu, and Cd) had toxic effects on the CAK cells with a dose-dependent manner. To sum up, the CAK cell line might be an ideal tool in vitro for analyzing the function of exogenous genes, bacterial susceptibility, and toxicity assay of heavy metals.

Topics: Animals; Bass; Cell Line; Fish Diseases; Kidney; Metals, Heavy; Salmon

Transforming growth factor-β activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase family. It is an upstream factor of the IκB kinase, which activates IKKα and IKKβ. TAK1 is a key factor in the induction of nuclear factor κB (NF-κB) and plays a crucial role in the activation of inflammatory responses. However, the roles of TAK1 during viral infection in teleost fish are largely unknown. In this study, we cloned a TAK1 homolog (HgTAK1) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀). The open reading frame of HgTAK1 consists of 1728 nucleotides encoding 575 amino acids, and the predicted molecular weight is 64.32 kDa HgTAK1 has an S_TKc domain, which consists of a serine/threonine protein kinase and a catalytic domain. Expression pattern analysis showed that HgTAK1 was distributed in all tested tissues, with abundant contents in the heart, head kidney, and blood. Additionally, HgTAK1 was distributed in the cytoplasm of grouper spleen (GS) cells. After Singapore grouper iridovirus (SGIV) infection, the expression of HgTAK1 increased in GS cells. Overexpression of HgTAK1 could promote the replication of SGIV in GS cells and inhibit the activation of NF-κB and IFN stimulated response elements (ISRE) in reporter assay. When co-expressed with IRF3 or HgIRF7 in GS cells, HgTAK1 obviously down-regulated IRF3- or IRF7-mediated the NF-κB and ISRE promoter induction. The interaction between HgTAK1 and IRF3 or IRF7 has been identified by co-immunoprecipitation assay. These findings provide a basis for understanding the innate immune mechanism of the grouper response to viral infection.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; NF-kappa B; Ranavirus; Sequence Alignment; Singapore

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Topics: Animals; Bacillus; Bass; Dietary Supplements; Disease Resistance; Fish Diseases; Lipopolysaccharides; Vibrio; Vibrio Infections

Grouper iridovirus is a devastating pathogen that belongs to the genus Ranavirus. Based on the previous results that natural ingredient quercetin isolated from Illicium verum Hook. f. could effectively inhibit Singapore grouper iridovirus (SGIV) replication, suggesting that quercetin could serve as potential antiviral agent against grouper iridovirus. To know about whether quercetin has indirect antiviral activity against SGIV, this study made the investigation in vitro and in vivo, and the potential mechanism was also explored. Pretreating the cells with quercetin (12.5 μg/mL) significantly inhibited the replication of SGIV, similar results were also confirmed in vivo. Importantly, quercetin pretreatment could induce the expression of genes involved in type I interferon (IFN) system (IFN, STAT1, PKR, MxI and ISG15) and TLR9. It suggested that quercetin exerted the indirect antiviral activity against SGIV infection through promoting the recognition of SGIV and activating the IFN pathway to establish the antiviral status of host cell. Taken together, our results shedded light on the indirect antiviral function of natural ingredient quercetin, and clearly demonstrated that natural ingredient quercetin will be an excellent potential agent against SGIV infection in grouper aquaculture.

Topics: Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Iridovirus; Plants, Medicinal; Quercetin; Ranavirus

Largemouth bass (LMB, 265-475 mm) were collected to document whether changes in fish condition and reproductive status influenced the concentration of total mercury (Hg) and selenium (Se) in axial muscle by season and sex. The fatty acid (FA) composition of fish was also examined to describe seasonal and sexual differences and identify whether arachidonic acid (ARA) could be used as a biomarker of Hg toxicity. There was a trend for females to have lower (p < 0.062) Se concentrations than males. The concentration of Se for females during spring (mean ± SD, 686 ± 51 ng/g dw) was 15% lower than males (806 ± 67 ng/g dw). Lower Se concentrations in females than males continued through summer and fall. Concentration of Hg for females during spring (152 ± 39 ng/g ww) was also 59% lower than males (373 ± 303 ng/g ww), but the difference was not significant (p > 0.2). The percent of lipids was greatest in fall and winter (3%) and comprised primarily of omega-3 fatty acids (35 g/100 g lipid). Fish condition as measured by percent lipids and relative weight was negatively (p < 0.02) related to Hg concentration for females and males. Lipid content for both sexes was also positively (p < 0.05) related to the Se:Hg ratio. Relative weight was positively related to the Se:Hg ratio for females during all seasons (p = 0.014), but only during spring and summer for males (p < 0.007). A low Se:Hg value was associated with an elevation in ARA for both sexes and a reduced hepatosomatic index in males. Data suggested that females transferred muscle stores of Se and Hg to developing oocytes during spring. This study generates hypotheses regarding the physiological drivers of seasonal and sexual variability in Hg, Se, and FA in LMB that may be applicable to other species and have implications for fisheries health and management.

Topics: Animals; Bass; Environmental Monitoring; Fatty Acids; Female; Fish Diseases; Fishes; Male; Mercury; Muscles; Seasons; Selenium; Water Pollutants, Chemical

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC

Topics: Animals; Bass; Carps; Catalytic Domain; Female; Fish Diseases; Oxyquinoline; Quinolines; Rhabdoviridae; Rhabdoviridae Infections

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Phosphoric Diester Hydrolases; Vaccines, Attenuated; Vibrio Infections

Interleukin 10 (IL-10), a pleiotropic cytokine, plays an essential role in multiple immunity responses. In the current study, the sequences of IL-10 family were identified from spotted knifejaw (Oplegnathus punctatus) whole genome, and O. punctatus IL-10 (OpIL-10) was cloned and characterized. OpIL-10 encodes 187 amino acids with a typical IL-10 family signature motif and predicted α-helices. It shared high identities with Notolabrus celidotus IL-10 and Epinephelus Lanceolatus IL-10. OpIL-10 was widely detected in healthy tissues, with the abundant expression in liver and skin. It was significantly up-regulated in the six immune-related tissues (liver, spleen, kidney, intestine, gill and skin) after infection against Vibrio harveyi and spotted knifejaw iridovirus (SKIV). Dual-luciferase analysis showed that OpIL-10 overexpression could suppress the activity of NF-κB. Meanwhile, OpIL-10 knockdown caused the down-regulation of five immune-related genes in JAK2/STAT3 signaling pathway and NF-κB signaling pathway, including IL-10R2, TYK2, STAT3, NOD2, and IκB. In addition, LPS and poly I:C stimulated expression of pro-inflammatory cytokines, including IL-6, IL-1β, IL-8, and IL-12, were lower with recombinant OpIL-10 (rOp IL-10) than the control group, indicating the anti-inflammatory roles of rOpIL-10. Taken together, these results indicated OpIL-10 as a negative regulator in the inflammatory responses of spotted knifejaw against bacterial and viral infection, which would help us better understand the role of IL-10 in teleost immunity.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity; Interleukin-10; NF-kappa B; Phylogeny; Virus Diseases

Largemouth bass ranavirus disease (LMBVD) caused by largemouth bass ranavirus (LMBV) has resulted in severe economic losses in the largemouth bass (Micropterus salmoides) farming industry in China. Early and accurate diagnosis is the key measure for the prevention and control of LMBVD. In this study, a quantitative polymerase chain reaction (qPCR) and a real-time recombinase-aided amplification (real-time RAA) assay were established for the detection of LMBV. The sensitivity and specificity of these two methods, and the efficacy for detection of LMBV from clinical samples were also evaluated. Results showed that the real-time RAA reaction was completed in <30 min at 39℃ with a detection limit of 58.3 copies, while qPCR reaction required 60 min with a detection limit of 5.8 copies. Both methods were specific for LMBV, where no cross-reactions observed with the other tested fish pathogens. Comparing the amplification results of both assays to the results obtained by virus isolation using 53 clinical tissue samples, results showed that the clinical sensitivity of real-time RAA and qPCR were 93.75% and 100% respectively, and the clinical specificity of both were 100%. Our results showed that qPCR is more suitable for quantitative analysis and accurate detection of LMBV in the laboratory, while real-time RAA is more suitable as a point-of-care diagnostic tool for on-site detection and screening of LMBV under farm conditions and in poorly equipped laboratories.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Ranavirus; Recombinases; Sensitivity and Specificity

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Mammals; Phylogeny; Ranavirus

Vibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of high-throughput sequencing and in silico analyses. This study also presents the first whole genome sequences of V. harveyi isolated from European sea bass. A large number of nonsynonymous variations were detected among sequences of the three strains. The prediction analysis of resistance genes did not correspond with the in vitro antimicrobial susceptibility tests. Six virulence genes previously unrelated to virulence of vibrios were detected in all three studied strains. The results show that differences were detected at every level of comparison among the three studied strains isolated from the same fish species originating from a small geographic area.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Vibrio; Vibrio Infections

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

Topics: Animals; Bass; Fish Diseases; Nocardia; Ulcer

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (

Topics: Adenosine Triphosphate; Animals; Bass; Connexin 43; Connexins; Cysteine; Fish Diseases; Fish Proteins; Immunity, Innate; Inflammation

An 8 week feeding trial was carried out to investigate the effects of dietary nucleotides on growth performance, intestinal morphology, immune response and disease resistance of juvenile largemouth bass, Micropterus salmoides. Five grades of dietary nucleotide levels were designed as 0, 0.2, 0.4, 0.8 and 1.2 g kg

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Intestines; Nucleotides

A dominant bacterium, ZYL-12, isolated from the liver of a diseased orange-spotted grouper Epinephelus coioides, was identified as Vibrio sinaloensis, based on phenotypic and molecular analysis. The median lethal dosage of ZYL-12 was calculated as 1.6 × 105 CFU g-1 fish weight. The infection experiment indicated that ZYL-12 caused noticeable histological lesions to the liver, kidney and spleen of the fish. Growth characteristics showed that ZYL-12 possessed strong environmental adaptability. This note is the first report about the pathogenicity of V. sinaloensis isolated from diseased fish.

Topics: Animals; Bass; Cloning, Molecular; Fish Diseases; Vibrio; Vibrio Infections

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1β, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Mammals; Proprotein Convertase 9; Ranavirus

Topics: Animals; Bass; DNA Viruses; Fish Diseases; Viruses, Unclassified

Chemical contaminants are one of the causes of the ongoing degradation of coastal and estuarine nurseries, key functional habitats in which the juveniles of many marine species grow. As chemical contaminants can cause a decrease in the energy available and induce defence mechanisms reducing the amount of energy allocated to life history traits, quantifying their effect on the fitness of juvenile fish is key to understand their population-level consequences. However, these effects are primarily estimated experimentally or in the wild but on a limited number of contaminants or congeners that do not reflect the wide variety of chemical contaminants to which juvenile fish are exposed. To address this issue, we measured concentrations of 14 trace metal elements (TMEs) and bioaccumulative organic contaminants (OCs) in European sea bass juveniles (1-year-old) from three major French nurseries (Seine, Loire and Gironde estuaries). We tested the hypotheses that (i) levels and profiles of contaminants differed among studied nurseries, and ii) fish growth and body condition (based on morphometric measurements and muscle C:N ratio) were lower in individuals with higher contaminant concentrations. Multivariate analyses showed that each nursery had distinct contaminant profiles for both TMEs and OCs, confirming the specific contamination of each estuary, and the large array of contaminants accumulated by sea bass juveniles. Increasing concentrations in some TMEs were associated to decreased growth, and TMEs were consistently related to lower fish body condition. The effect of OCs was more difficult to pinpoint possibly due to operational constraints (i.e., analyses on pooled fish) with contrasting results (i.e., higher growth and decreased body condition). Overall, this study shows that chemical contaminants are related to lower fish growth and body condition at an early life stage in the wild, an effect that can have major consequences if sustained in subsequent ages and associated with a decline in survival and/or reproductive success.

Topics: Animals; Bass; Ecosystem; Estuaries; Fish Diseases; Trace Elements; Water Pollutants, Chemical

Topics: Animals; Aquaculture; Bass; Cichlids; Fish Diseases; Fresh Water; Humans; Trypanosoma; Trypanosomiasis

Members of the order Trypanorhyncha are cestode parasites that are frequently found infecting the muscles of several marine fish species, affecting fish health and resulting in consumers' rejection. Seventy-five specimens of marine fish were freshly caught from boat landing sites at the Alexandria coast along the Mediterranean Sea in Egypt, including two Carangids, the greater amberjack Seriola dumerili and the gulley jack Pseudocarans dentex; two Serranids, the Haifa grouper Epinephelus haifensis and the mottled grouper Mycteroperca rubra. Forty-five fish were infected; the infection was recorded as blastocysts embedded in fish flesh. Blastocysts were isolated and ruptured; the generated plerocerci were described morphologically, where, four different species were recovered; Callitetrarhynchus gracilis, Callitetrarhynchus speciosus, Protogrillotia zerbiae, and Grillotia brayi. The taxonomic position of these parasites was justified by multiple-sequence alignment and a phylogenetic tree was constructed following maximum likelihood analysis of the 18s rRNA sequences of the recovered worms. The accession numbers MN625168, MN625169, MN611431and MN611432 were respectively assigned to the recovered parasites. The results obtained from the molecular analyses confirmed the morphological records of the recovered parasites. Since metacestodes are found in the musculature of infected fish specimens, it is necessary to remove these areas in the commercialization of fish.

Topics: Animals; Bass; Cestoda; Cysticercosis; Fish Diseases; Mediterranean Sea; Perciformes; Phylogeny

This study describes the etiological agent of Vibriosis along with its distribution and antimicrobial resistance profiles among farmed Asian sea bass (Lates calcarifer) in Thailand. The study isolated 283 Vibrionaceae from 15 Asian sea bass farms located around the provinces of the Andaman Sea and Gulf of Thailand coasts to uncover the distribution and antimicrobial resistance profiles. Bacterial identification based on a combination of the biochemical characteristics, Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, and the species-specific PCR demonstrated the predominant Vibrionaceae were Vibrio harveyi (n = 56), Photobacterium damselae (n = 35), and V. vulnificus (n = 31), respectively. According to a laboratory challenge experiment, among the six isolates, only V. harveyi was found to cause clinical signs of muscle necrosis and scale loss in Asian sea bass. Antibiotics resistance test results exhibited high resistance to antibiotics such as metronidazole (100%), streptomycin (97%), clindamycin (96%), colistin sulphate (70%) and amoxicillin (59%). Remarkably, 100% of Vibrionaceae isolates are susceptible to florfenicol. The 28 of 29 resistance profiles were multidrug resistances (MDR), with V. vulnificus having the highest MAR value (0.66). The findings of this study advise that a surveillance program, as well as preventive and control measures, be developed for Vibrionaceae to reduce production loss, pathogen proliferation, and antibiotic abuse, whereas AMR data indicate substantial health problems for aquatic animals and humans.

Topics: Animals; Anti-Bacterial Agents; Bass; Drug Resistance, Bacterial; Farms; Fish Diseases; Humans; Perciformes; Prevalence; Thailand; Vibrionaceae

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 μg/mL, green tea polyphenols (TP) ≤ 10 μg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 μg/mL, (-)-epigallocatechin (EGC) ≤ 10 μg/mL, (-)-epicatechin gallate (EGC) ≤ 5 μg/mL, and (-)-epicatechin (EC) ≤ 50 μg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.

Topics: Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Iridovirus; Ranavirus; Tea

Singapore grouper iridovirus (SGIV) causes high economic losses in mariculture. Effective drugs for managing SGIV infection are urgently required. Medicinal plant resources are rich in China. Medicinal plants have a long history and significant curative effects in the treatment of many diseases. Reverse-transcription quantitative real-time PCR is the most commonly used method for detecting virus infection and assessing antiviral efficacy with high accuracy. However, their applications are limited due to high reagent costs and complex time-consuming operations. Aptamers have been applied in some biosensors to achieve the accurate detection of pathogens or diseases through signal amplification. This study aimed to establish an aptamer-based high-throughput screening (AHTS) model for the efficient selection and evaluation of medicinal plants components against SGIV infection. Q2-AHTS is an expeditious, rapid method for selecting medicinal plant drugs against SGIV, which was characterized as being dram, high-speed, sensitive, and accurate. AHTS strategy reduced work intensity and experimental costs and shortened the whole screening cycle for effective ingredients. AHTS should be suitable for the rapid selection of effective components against other viruses, thus further promoting the development of high-throughput screening technology.

Topics: Animals; Antiviral Agents; Aptamers, Nucleotide; Bass; DNA Virus Infections; Fish Diseases; High-Throughput Screening Assays; Ranavirus

NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.

Topics: Animals; Antiviral Agents; Bass; Brain; Fish Diseases; Fish Proteins; Immunity, Innate; Inflammasomes; Necrosis; Nodaviridae; RNA Virus Infections; Virus Diseases

Lipid metabolism plays an important role in viral infections, and it can directly or indirectly affect various stages of viral infection in cells. As an important component of lipid metabolism, high-density lipoprotein (HDL) plays crucial roles in inflammation, immunity, and viral infections. Scavenger receptor B type 1 (SR-B1), a receptor of HDL, cannot be ignored in the regulation of lipid metabolism. Here, we investigate, for the first time, the role of Epinephelus coioides SR-B1 (Ec-SR-B1) in red-spotted grouper nervous necrosis virus (RGNNV) infection. Our results indicate that Ec-SR-B1 could promote RGNNV infection. We also demonstrate that Ec-SR-B1 could facilitate viral entry and interact with capsid protein (CP) of RGNNV. As the natural ligand of SR-B1, HDL significantly increased RGNNV entry in a dose-dependent manner. However, we observed no effect of HDL on Ec-SR-B1 expression. The results of the micro-scale thermophoresis assay did not reveal an association between HDL and CP, suggesting that RGNNV does not enter target cells by using HDL as a ligand to bind to its receptor. In addition, block lipid transport-1, a compound that inhibits HDL-mediated cholesterol transfer, reduced the HDL-induced enhancement of RGNNV infection, indicating a role for lipid transfer in facilitating RGNNV entry. Furthermore, HDL inhibited the expression of pro-inflammatory factors and antiviral genes in a dose-dependent manner. These findings suggest that the HDL-induced enhancement of RGNNV entry involves the complex interplay between Ec-SR-B1, HDL, and RGNNV, as well as the regulation of innate antiviral responses by HDL. In summary, we highlight the crucial role of HDL in RGNNV entry, identify a possible molecular connection between RGNNV and lipoprotein metabolism, and indicate the role of Ec-SR-B1 in RGNNV infection.

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Ligands; Lipoproteins, HDL; Necrosis; Nodaviridae; Receptors, Scavenger; Virus Internalization

Growing evidences have demonstrated that multiple TRIM (tripartite motif) proteins exert critical roles in host defense against different microbial pathogens. Although mammalian TRIM21 has been reported to function as an important regulatory factor in antiviral immune and inflammatory response, the role of fish TRIM21 against virus infection still remains largely unknown. In the present study, we investigated the characteristics of TRIM21 gene (EcTRIM21) from orange spotted grouper (Epinephelus coioides). The full-length EcTRIM21 cDNA encoded a 557 amino acid peptide with 92.1% and 31.14% identity with giant grouper (Epinephelus lanceolatus) and human (Homo sapiens), respectively. EcTRIM21 contained four conserved domains, including RING, B-Box, PRY and SPRY domain. EcTRIM21 expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM21 might be involved in host defense against fish virus infections. Subcellular localization showed that EcTRIM21 were distributed in the cytoplasm in a punctate manner. Overexpression of EcTRIM21 in vitro significantly inhibited RGNNV and SGIV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral core genes. Consistently, knockdown of EcTRIM21 by small interfering RNA (siRNA) promoted the replication of RGNNV and SGIV in vitro. Furthermore, EcTRIM21 overexpression increased both interferon (IFN) and interferon stimulated response element (ISRE) promoter activities. In addition, the transcription levels of IFN signaling related molecules were positively regulated by EcTRIM21 overexpression. Together, our data demonstrated that fish TRIM21 exerted antiviral activity against fish viruses through positive regulation of host interferon response.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Humans; Interferons; Iridovirus; Mammals; Nodaviridae; Phylogeny; Ranavirus; Sequence Alignment; Tripartite Motif Proteins

Pseudomonas plecoglossicida is a Gram-negative pathogenic bacterium that causes visceral white spot disease in several marine fish species, resulting in high mortality and financial loss. Based on previous RNA sequencing (RNA-seq) results, rpoD gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that rpoD may contribute to bacterial pathogenicity. To investigate the role of this gene, five specific short hairpin RNAs (shRNAs) were designed and synthesized based on the rpoD gene sequence, with all five mutants exhibiting a significant decrease in rpoD gene expression in P. plecoglossicida. The mutant with the highest silencing efficiency (89.2%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain NZBD9, silencing rpoD in the rpoD-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of rpoD also resulted in a 25% increase in the survival rate, a one-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface of infected orange-spotted groupers (Epinephelus coioides). In addition, rpoD expression and pathogen load were significantly lower in the spleens of E. coioides infected with the rpoD-RNAi strain than with the WT strain of P. plecoglossicida. We performed RNA-seq of E. coioides spleens infected with different P. plecoglossicida strains. Results showed that rpoD silencing in P. plecoglossicida led to a significant change in the infected spleen transcriptomes. In addition, comparative transcriptome analysis showed that silencing rpoD caused significant changes in complement and coagulation cascades and the IL-17 signaling pathway. Thus, this study revealed the effects of the rpoD gene on P. plecoglossicida pathogenicity and identified the main pathway involved in the immune response of E. coioides.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Immunity, Innate; Pseudomonas; Pseudomonas Infections; Virulence

Columnaris is a bacterial disease, found in freshwater fish, caused by Flavobacterium oreochromis. The disease has a devastating impact on a range of cultured and wild freshwater fish species e.g. Lates calcarifer (Asian sea bass), which is a serious economic losses to the freshwater aquaculture in Thailand. The disease can be prevented by an efficacious vaccine, however, no licensed effective vaccine is available to date. Current study was based on the development of a novel mucoadhesive nano-encapsulated vaccine (EncapFlavoNP

Topics: Animals; Bacterial Vaccines; Bass; Cichlids; Fish Diseases; Flavobacterium; Gram-Negative Bacterial Infections; Immersion; Lipids; Vaccination

The most convincing species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae) are known overwhelmingly from groupers (Serranidae: Epinephelinae). Six species of Allopodocotyle have been reported, collectively, from species of Cromileptes Swainson, 1839, Epinephelus Bloch, 1793 and Plectropomus Oken, 1817. These are A. epinepheli (Yamaguti, 1942), A. heronensis Downie & Cribb, 2011, A. manteri (Saoud & Ramadan, 1984), A. mecopera (Manter, 1940), A. plectropomi (Manter, 1963) and A. serrani (Yamaguti, 1952). In addition, a not yet fully described and unnamed seventh species, morphologically and phylogenetically close to A. epinepheli, was isolated from the orange-spotted grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia in 2016. An eighth species, again from E. coioides off Bali is described herein.. Morphological and phylogenetic analyses justify the recognition of A. palmi sp. nov., which is also genetically different from the as yet unnamed congener from the same host and locality. For the first time, 3D confocal laser scanning microscopy was applied to study and distinguish Digenea taxonomically. We introduce the 'Palm pattern', a new simplified way to visualise morphometric differences of related digenean taxa.. Allopodocotyle palmi sp. nov. is distinguished from its congeners that infect groupers by its elongate body with a size > 2.7 mm and diagonal testes. The ovary is located mainly, and the anterior testis completely, in the posterior half of the body; the uterine coils are in the fourth eighth of the body. The cirrus-sac is 0.75-1.4 (1.1) mm long, its posterior extremity is well separated from the anterior extent of the vitelline fields, just reaching the anterior border of uterine coils. In addition, Prosorhynchus maternus Bray & Justine, 2006 (Bucephalidae) was isolated from E. coioides, representing the first record in Indonesia and the third record for this fish species.. The biodiversity research in Indonesia is enhanced with a new species description based on modern and newly applied techniques.

Topics: Animals; Bass; Female; Fish Diseases; Indonesia; Male; Phylogeny; Seafood; Trematoda

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 μg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 μg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Topics: Allyl Compounds; Animals; Anti-Bacterial Agents; Bass; Biphenyl Compounds; Fish Diseases; Magnolia; Nocardia; Nocardia Infections; Phenols; Plant Extracts; Plants, Medicinal

Two new species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) are described from the gill and scales of smallmouth bass (Micropterus dolomieu Lacepède, 1802 [Centrarchiformes: Centrarchidae]) from the Watauga River, French Broad River Basin, North Carolina, United States. Myxobolus intralamina n. sp. infects the lumen of the lamellar arterioles and Myxobolus infrabractea n. sp. infects the inner surface of the scale. They differ from all congeners by a combination of myxospore dimensions, polar tubule coil count, and the presence or absence of an iodinophilic vacuole in the sporoplasm and an intercapsular process. A phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) recovered M. intralamina n. sp. sister to Myxobolus lepomis and Myxobolus branchiarum and M. infrabractea n. sp. sister to Myxobolus micropterii in a clade composed of five Myxobolus spp. infecting centrarchids and Henneguya spp. (Myxobolidae) infecting percids. Histological sections of infected gill revealed intra-lamellar plasmodia of M. intralamina n. sp. within the lumen of the lamellar arterioles and plasmodia of M. infrabractea n. sp. developing beneath the scales. These new species comprise the first species of Myxobolus reported from a black bass (Micropterus Lacepède, 1802) in the Southeast United States.

Topics: Animals; Bass; Fish Diseases; Gills; Myxobolus; Myxozoa; North Carolina; Parasitic Diseases, Animal; Perciformes; Phylogeny; Rivers

Major capsid protein (MCP) can be used as a subunit vaccine against largemouth bass virus (LMBV). However, subunit vaccines usually have low immunogenicity. Here, to identify the major immunogenicity determinant region of the MCP gene, we truncated the MCP of the LMBV gene into four parts (MCP-1, MCP-2, MCP-3 and MCP-4). Enzyme-linked immunosorbent assay (ELISA) was used to identify the antigenicity of these four truncated MCP proteins. Then, the highly antigenic truncated protein was modified with mannose and connected with functionalized single-walled carbon nanotubes (SWCNTs) as carriers. Largemouth basses were immunized by bath immersion, challenged with LMBV on the 28th day after immunization and evaluated for related immune indicators. The results indicated that the MCP-2 protein could induce a higher antibody titre than the other truncated MCP proteins. We found that the levels of immune-related genes (TNF-α, CD40, IgM, IFNγ and IL-10) in the spleen and kidney were significantly increased in the MCP-2 and MCP-2-Man groups. ELISA results showed that the antibody content in the serum increased significantly in the MCP-2 group 7 days post-vaccination and increased with days in all the vaccinated groups, with the highest observed on the 21st day. Notably, the MCP-2-Man vaccine (10 mg L

Topics: Animals; Bass; Capsid Proteins; DNA Virus Infections; Epitopes; Fish Diseases; Immunoglobulin M; Interleukin-10; Mannose; Nanotubes, Carbon; Tumor Necrosis Factor-alpha; Vaccines, Subunit

Autophagy-related gene 5 (Atg5), an essential component of autophagy machinery, is associated with innate immune responses. Here, the Atg5 of sea perch (Lateolabrax japonicus) (LjAtg5) was cloned and its role in regulating autophagy and interferon (IFN) response during red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. The LjAtg5 cDNA encoded a polypeptide of 275 amino acids with an APG5 domain, and had the closet genetic relationship with Micropterus salmoides Atg5. Autophagic detection showed LjAtg5 was conserved in inducing cell autophagy. Spatial expression analysis revealed LjAtg5 had a higher expression level in liver, brain, and kidney tissues of RGNNV-infected sea perch compared with the control group. In RGNNV-infected LJB cells, overexpression of LjAtg5 significantly increased the mRNA and protein levels of capsid protein, whereas knockdown of LjAtg5 led to the opposite effect, indicating LjAtg5 played a pro-viral role during RGNNV infection. Furthermore, dual luciferase reporter assay revealed LjAtg5 significantly suppressed the activation of sea perch type I IFN promoter in vitro, and overexpression of LjAtg5 strongly weaken the expression of genes related to the RIG-I-like receptors (RLRs) signaling pathway and IFN stimulated genes. These results suggested LjAtg5 promoted RGNNV infection by negatively regulating RLRs-IFN signaling pathway.

Topics: Animals; Autophagy; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Interferons; Nodaviridae; Perches; RNA Virus Infections; Signal Transduction

Vaccination is an effective method to prevent

Topics: Animals; Bass; Ciliophora Infections; Fish Diseases; Hymenostomatida; Immunization; Immunoglobulin M; Tetrahymena thermophila; Tubulin; Vaccination

Topics: Animals; Bass; Biphenyl Compounds; Female; Fish Diseases; Lignans; Rhabdoviridae

Largemouth bass virus (LMBV), belonging to the genus

Topics: Animals; Apoptosis; Bass; Cell Death; DNA Virus Infections; Fish Diseases; Ranavirus; Virus Diseases

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG "lysosome pathway" were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.

Topics: Animals; Bass; DNA Virus Infections; Extracellular Signal-Regulated MAP Kinases; Fish Diseases; Fish Proteins; Immunity, Innate; Interferons; Iridovirus; Necrosis; Nodaviridae; p38 Mitogen-Activated Protein Kinases; Purines; Pyrimidines; Ranavirus; Singapore; Transcriptome

As a highly infectious epidemic in aquaculture, Pseudomonas plecoglossicida infection results in high mortality of teleosts and serious economic losses. Host-pathogen interactions shape the outcome of an infection, yet we still understand little about the molecular mechanism of these pathogen-mediated processes. Here, a P. plecoglossicida strain (NZBD9) and Epinephelus coioides were investigated as a model system to characterize pathogen-induced host metabolic remodeling over the course of infection. We present a non-targeted metabolomics profiling of E. coioides spleens from uninfected E. coioides and those infected with wild-type and clpV-RNA interference (RNAi) strains. The most significant changes of E. coioides upon infection were associated with amino acids, lysophospatidylcholines, and unsaturated fatty acids, involving disturbances in host nutritional utilization and immune responses. Dihydrosphingosine and fatty acid 16:2 were screened as potential biomarkers for assessing P. plecoglossicida infection. The silencing of the P. plecoglossicida clpV gene significantly recovered the lipid metabolism of infected E. coioides. This comprehensive metabolomics study provides novel insights into how P. plecoglossicida shape host metabolism to support their survival and replication and highlights the potential of the virulence gene clpV in the treatment of P. plecoglossicida infection in aquaculture.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Pseudomonas; Pseudomonas Infections

Micropterus salmoides rhabdovirus (MSRV) is one of the common pathogens in the largemouth bass industry, which can cause lethal diseases in juvenile fish and enormous economic losses. To establish effective means to prevent MSRV infection, the pcDNA3.1-G plasmid containing the MSRV glycoprotein gene was successfully constructed and intramuscularly injected into the largemouth bass to evaluate the immune responses and protective effects in our study. As the results showed, the serum antibody levels of the fish vaccinated with different doses of pcDNA3.1-G were significantly higher compared with the control groups (PBS and pcDNA3.1). Meanwhile, the immune parameters (acid phosphatase and alkaline phosphatase) were also significantly up-regulated. Several immune-related genes (IgM, IL-8, IL-12p40 and CD40) were expressed in the pcDNA3.1-G groups at higher levels than in the control groups, which indicated that strong immune responses were induced. Besides, the survival percentages of fish in the control groups (PBS and pcDNA3.1) and pcDNA3.1-G groups (2.5, 5, 10 and 20 μg/fish) at 14 days after challenge experiment with MSRV were 0%, 0%, 6.1%, 15.2%, 29.0% and 48.5% respectively. This study indicated that pcDNA3.1-G was a prospective DNA vaccine candidate against MSRV-induced mortality.

Topics: Animals; Bass; Fish Diseases; Prospective Studies; Rhabdoviridae; Vaccines, DNA

Interferon (IFN)-induced protein 35 (IFI35, also known as IFP35), a member of IFN induced genes (ISGs), participates in virus infection, cancer progression and the chronic inflammatory diseases. However, its roles during fish nodavirus infection still remained largely unknown. In the present study, a homolog of IFI35 from orange spotted grouper (Epinephelus coioides) (EcIFI35) was cloned and characterized. The open reading frame of EcIFI35 was composed of 1,128 bp, and encoded a 375 amino acid polypeptide, which contained two conserved N-myc-interactor (Nmi)/IFP35 domains (NIDs). Homology analysis indicated that EcIFI35 shared 95.73% and 31.96% identity with homologs of giant grouper (E. lanceolatus) and human (Homo sapiens), respectively. The transcription of EcIFI35 was significantly up-regulated in grouper spleen (GS) cells after challenged with red-spotted grouper nervous necrosis virus (RGNNV), polyinosinic:polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS). The subcellular localization analysis showed that EcIFI35 encoded a cytoplasmic protein. The ectopic expression of EcIFI35 inhibited RGNNV replication by reducing viral genes transcription and protein synthesis. Co-immunoprecipitation (Co-IP) assay demonstrated that EcIFI35 interacted with RGNNV coat protein (CP), and partly co-localized with CP. EcIFI35 overexpression promoted the expression of IFN-related molecules and pro-inflammatory factors, including IFN regulatory factor 7 (IRF7), mitochondrial antiviral signaling protein (MAVS) and myxovirus resistance gene I (MxI), nuclear factor κB (NF-κB), interleukin 6 (IL-6) and IL-8. Together, our results revealed that EcIFI35 interacted with CP and inhibited fish nodavirus replication through positively regulated host innate immune response.

Topics: Amino Acid Sequence; Amino Acids; Animals; Antiviral Agents; Bass; DNA Virus Infections; Factor VII; Fish Diseases; Fish Proteins; Gene Expression Regulation; Humans; Immunity, Innate; Interferons; Interleukin-6; Interleukin-8; Lipopolysaccharides; NF-kappa B; Nodaviridae; Poly I-C; Sequence Alignment

Viral infection causes changes in the internal environment of host cells, and a series of stress responses are generated to respond to these changes and help the cell survive. Stress granule (SG) formation is a type of cellular stress response that inhibits viral replication. However, the relationship between red-spotted grouper nervous necrosis virus (RGNNV) infection and SGs, and the roles of the SG marker protein RAS GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) in viral infection remain unclear. In this study, RGNNV infection induced grouper spleen (GS) cells to produce SGs. The SGs particles co-located with the classic SG marker protein eIF3η, and some SGs depolymerized under treatment with the translation inhibitor, cycloheximide (CHX). In addition, when the four kinases of the eukaryotic translation initiation factor 2α (eIF2α)-dependent pathway were inhibited, knockdown of HRI and GCN2 with small interfering RNAs and inhibition of PKR with 2-aminopurine had little effect on the formation of SGs, but the PERK inhibitor significantly inhibited the formation of SGs and decreased the phosphorylation of eIF2α. G3BP1 of

Topics: Animals; Bass; DNA Helicases; Fish Diseases; Fish Proteins; Humans; Immunity, Innate; Necrosis; Nodaviridae; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; Stress Granules; Virus Replication

Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.

Topics: Animals; Base Sequence; Bass; Cloning, Molecular; Cystatin A; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Phylogeny

As a member of the γc family, interleukin 15 plays an important function in the immune response. In this study, we cloned an IL15 from Epinephelus coioides (named Ec-IL15). The open reading frame of Ec-IL15 is 528 bp, encoding 175 amino acids. Sequence alignment analysis showed that EcIL-15 has a conserved Pfam: IL15 domain and four cysteine residues. Subcellular localization studies have shown that Ec-IL15 is distributed in whole cells. In healthy groupers, Ec-IL15 was expressed in all 11 tissues tested and the highest in liver. After ConA, PHA, LPS and poly I:C stimulation, Ec-IL15 expression of HKLs was significantly upregulated. After V. harveyi infection, the expression of Ec-IL15 in 9 tissues was significantly upregulated and peaked within 48 h. In addition, recombinant Ec-IL15 protein can not only stimulate HKLs proliferation and cytokine expression, but also has the potential as an immune enhancer.

Topics: Animals; Bass; Cloning, Molecular; Cysteine; Fish Diseases; Fish Proteins; Interleukin-15; Lipopolysaccharides; Phylogeny; Poly I; Vibrio

The disease caused by Micropterus salmoides rhabdovirus (MSRV) has brought substantial economic losses to the largemouth bass aquaculture industry in China. Vaccination was considered as a potential way to prevent and control this disease. As a kind of sustained and controlled release system, alginate and chitosan microspheres (SA-CS) are widely used in the development of oral vaccination for fish. Here, we prepared a king of alginate-chitosan composite microsphere to encapsulate the second segment of MSRV glycoprotein (G2 protein) and then evaluated the immune effect of the microsphere vaccine on largemouth bass. Largemouth bass were vaccinated via intragastric immunization by different treatments (PBS, SA-CS, G2 and SA-CS-G2). The results showed that a stronger immune response including serum antibody levels, immune-related physiological indexes (acid phosphatase, alkaline phosphatase, superoxide dismutase and total antioxidant capacity) and the expression of immune-related gene (IgM、IL-8、IL-1β、CD4、TGF-β、TNF-α) can be induced obviously with SA-CS-G2 groups compared with G2 groups when fish were vaccinated. Furthermore, fish were injected with a lethal dose of MSRV after immunization for 28 days, and the highest relative percentage survival (54.8%) was observed in SA-CS-G2 group (40 μg per fish), which is significantly higher than that of G2 group (25.8%). This study showed that alginate-chitosan microspheres as the vaccine carrier can effectively improve the immune effect of oral vaccination and induce better immune protection effect against MSRV infection.

Topics: Acid Phosphatase; Alginates; Alkaline Phosphatase; Animals; Antioxidants; Bass; Chitosan; Delayed-Action Preparations; Fish Diseases; Immunoglobulin M; Interleukin-8; Microspheres; Rhabdoviridae; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vaccines, Subunit; Vaccines, Synthetic

The teleost mucosal immune system consists mainly of the skin, gills and gut, which play crucial roles in local immune responses against invading organisms. Immunoglobulins are essential molecules in adaptive immunity that perform crucial biological functions. In our study, a mucosal immunity model was constructed in Epinephelus coioides groupers after Cryptocaryon irritans infection, according to previous experience. Total IgM and IgT in the groupers increased in the serum and mucus in the immune group, whereas only pathogen-specific IgM were detected existence. More critically, pathogen-specific IgM was detected in the head kidney, gill and skin supernatants, thus suggesting that the systematic immune and mucosal immune system secreted immunoglobulins. Furthermore, an early response in the skin was observed, on the basis of the detection of pathogen-specific IgM in the skin supernatant. In conclusion, this research characterized the grouper IgM and IgT in mucosal immune responses to pathogens in the gills and skin, thus providing a theoretical basis for future studies on vaccines against C. irritans.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Hymenostomatida; Immunoglobulin M; Phylogeny

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.

Topics: Animals; Antiviral Agents; Bass; Cytokines; Fish Diseases; Nodaviridae; RNA Virus Infections

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100−140 nm long and 50−100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.

Topics: Animals; Bass; Fish Diseases; Iridovirus; Novirhabdovirus; Rhabdoviridae

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cathepsin D; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Phylogeny

The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.

Topics: Animals; Bass; Fish Diseases; Necrosis; Nodaviridae; Reverse Genetics; RNA Virus Infections

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method's detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.

Topics: Animals; Bass; Fish Diseases; Necrosis; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections

Prevalence, morphology, and molecular characteristics of isopodiosis in the European seabass, Dicentrarchus labrax, in Egypt were assessed using light and electron microscopy and polymerase chain reaction targeting the mitochondrial COI (cytochrome oxidase c) gene.. Adult parasites were found mainly in the branchial cavity between gill arches and to a lesser extent in the buccal cavity. They were morphologically identified as the Cymothoidae Livoneca redmanii Leach, 1818 (Crustacea: Isopoda). Obviously, a 23% prevalence rate of isopods was reported in D. labrax from Egyptian Mediterranean waters. Destructive and degenerative necrotic alterations with complete sloughing of gill lamellae of the infested fish were observed. DNA sequencing of the mitochondrial COI gene confirmed the identification of the parasite which was deposited in the GenBank under accession numbers MW600099, MZ208984, and MZ208985. Furthermore, the phylogenetic analysis demonstrated that parasites emerged from a monophyletic clade closely affiliated with L. redmanii and were clearly distinguished from other isopod genospecies.. The present investigation addresses L. redmanii infestation in D. labrax in Egypt and affirmed morphological properties via the scanning electron microscopy (SEM) and molecular characteristics of this isopod species. The drastic effects of this parasite on the infected fish were proven both clinically and histopathologically.

Topics: Animals; Bass; Fish Diseases; Gills; Isopoda; Parasites; Phylogeny

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Topics: Animals; Bacterial Vaccines; Bass; Catfishes; Edwardsiella; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Ictaluridae

Topics: Animals; Bass; Fish Diseases; Immunity, Innate; MicroRNAs; RNA, Messenger; Vibrio Infections; Vibrio parahaemolyticus

Cystatin F (CyF), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyF and its latent molecular mechanism during virus infection in fish remain vacant. In our research, we cloned the open reading frame (ORF) of CyF homology from orange-spotted grouper (Ec-CyF) consisting of 342 nucleotides and encoding a 114-amino acid protein. Ec-CyF included two cystatins family sequences containing one KXVXG sequence without the signal peptide, and a hairpin ring containing proline and tryptophan (PW). Tissue distribution analysis indicated that Ec-CyF was highly expressed in spleen and head kidney. Besides, further analysis showed that the expression of Ec-CyF increased during SGIV infection in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyF was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyF demoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was impeded, as well as the restraint of caspase 3/7 and caspase 8. In addition, Ec-CyF overexpression up-regulated the expression of IFN related molecules including ISG15, IFN, IFP35, IRF3, IRF7, MYD88 and down-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyF-overexpressing increased the activity of IFN3 and ISRE promoter, but impeded NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyF was involved in innate immunity response and played a key role in DNA virus infection.

Topics: Amino Acid Sequence; Animals; Bass; Caspase 3; Caspase 8; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Interleukin-8; Myeloid Differentiation Factor 88; NF-kappa B; Nucleotides; Phylogeny; Proline; Protein Sorting Signals; RNA, Messenger; Tryptophan; Tumor Necrosis Factor-alpha

Cucullanus tunisiensis sp. nov., (Nematoda: Cucullanidae), collected from the intestine of the white grouper Epinephelus aeneus from waters off the coast of Tunisia is described based on light and scanning electron microscopic observations. The new species is characterized by the presence of lateral alae, ventral sucker, long unequal spicules (left spicule 2474-2789 μm long, right spicule 2357-2518 μm long). This is the sixth nominal species of the genus Cucullanus Müller, 1777 and the first representative of this genus infecting fishes of Serranidae family reported from Tunisian waters.

Topics: Animals; Ascaridida; Ascaridoidea; Bass; Fish Diseases; Nematoda; Parasites; Perciformes; Species Specificity; Tunisia

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Bass; Capsid Proteins; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Interferons; Iridovirus; Mammals; NF-kappa B; Nodaviridae; Ranavirus; Sequence Alignment

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Ranavirus; RNA, Long Noncoding; RNA, Messenger; Singapore

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (

Topics: Amino Acids; Animals; Antiviral Agents; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; GTP-Binding Proteins; Humans; Immunity, Innate; Interferons; Iridovirus; NF-kappa B; Nodaviridae; Ranavirus; Sequence Alignment; TNF Receptor-Associated Factor 4; TNF Receptor-Associated Factor 5; TNF Receptor-Associated Factor 6; Zebrafish

Singapore grouper iridovirus (SGIV) with high pathogenicity can cause great economic losses to aquaculture industry. Thus, it is of urgency to find effective antiviral strategies to combat SGIV. Curcumin has been demonstrated effective antiviral activity on SGIV infection. However, the molecular mechanism behind this action needs to be further explanations. In view of the fact that apoptosis (type I programmed cell death) and autophagy (type II programmed cell death) were key regulators during SGIV infection, we aimed to investigate the relevance between antiviral activity of curcumin and SGIV-associated programmed and clarify the role of potential signaling pathways. Our results showed that curcumin suppressed SGIV-induced apoptosis. At the same time, the activities of caspase-3/8/9 and activating protein-1 (AP-1), P53, nuclear factor-κB (NF-ΚB) promoters were inhibited. Besides, the activation of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen activate protein kinase (p38 MAPK) signal pathways were suppressed in curcumin-treated cells. On the other hand, curcumin down-regulated protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway to promote autophagy representing by increased LC3 II and Beclin1 expression. Curcumin also hindered the transition of cells from G1 to S phase, as well as down-regulating the expression of CyclinD1. Our findings revealed the resistance curcumin induced to the effects of DNA virus on cell apoptosis and autophagy and the insights gained from this study may be of assistance to understand the molecular mechanism of curcumin against DNA virus infection.

Topics: Animals; Antiviral Agents; Apoptosis; Autophagy; Bass; Curcumin; DNA Virus Infections; Fish Diseases; Iridovirus; Mammals; Ranavirus; Singapore

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Topics: Amino Acid Sequence; Amino Acids; Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Interferons; Nodaviridae; Phylogeny; Poly I-C; Ranavirus; Sequence Alignment; STAT2 Transcription Factor

The nervous necrosis virus (NNV) causes the viral nervous necrosis (VNN) disease in aquatic animals and has been a major threat in aquaculture. Thus, it is essential for the development of a prevention method to minimize economic losses caused by NNV such as the identification of NNV resistance genes and application of these genes in molecular breeding to increase disease resistance. gab3 is an important NNV resistance gene in Asian seabass. However, the mechanism of gab3 in NNV resistance has not been elucidated. In this study, knockdown of gab3 in NNV-infected Asian seabass cells resulted in a significant decrease in viral RNA and virus titers. Knockout of gab3 in zebrafish led to an increased survival rate and resistant time after NNV infection. Cellular localization of the GAB3 and NNV by immunofluorescence staining showed that the GAB3 was translocated from the nucleus to the cytoplasm, and finally reached the cell membrane of SB cells after 48 h post NNV infection. Our study suggests that gab3 plays an important role in NNV replication and silencing gab3 can inhibit virus replication.

Topics: Animals; Bass; Fish Diseases; Necrosis; Nodaviridae; Perciformes; RNA Virus Infections; Virus Replication; Zebrafish

Topics: Animals; Aquaculture; Bass; Coinfection; Fish Diseases; Prevalence; Vibrio; Vibrio Infections

Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is a crucial regulator of several signaling pathways and plays a vital role in cell proliferation, growth, apoptosis, and immune responses. However, the role of GSK3β during viral infection in teleosts remains largely unknown. In the present study, a GSK3β homologue from Epinephelus coioides (EcGSK3β) was cloned and characterized. The open reading frame of EcGSK3β consists of 1323 bp, encoding a 440 amino acid protein, with a predicted molecular mass of 48.23 kDa. Similar to its mammalian counterpart, EcGSK3β contains an S_TKc domain. EcGSK3β shares 99.77% homology with the giant grouper (Epinephelus lanceolatus). Quantitative real-time PCR analysis indicated that EcGSK3β mRNA was broadly expressed in all tested tissues, with abundant expression in the skin, blood, and intestines. Additionally, the expression of EcGSK3β increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that EcGSK3β is mainly distributed in the cytoplasm. EcGSK3β overexpression promoted SGIV replication during viral infection in vitro. In contrast, silencing of EcGSK3β inhibited SGIV replication. EcGSK3β significantly downregulated the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these findings are important for a better understanding of the function of GSK3β in fish and reveal its involvement in the host response to viral immune challenge.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Glycogen Synthase Kinase 3 beta; Immunity, Innate; Iridovirus; Mammals; Phylogeny; Ranavirus; Singapore

Pseudorhabdosynochus suratthaniensis n. sp. is described from the gills of Cephalopholis argus; P. cephalopholi n. sp., from the gills of C. sonnerati; and P. samaesarnensis n. sp., from the gills of Epinephelus lanceolatus. These fish were all caught in the Gulf of Thailand. Pseudorhabdosynochus suratthaniensis n. sp. is distinguished from congeneric species by the structure of its sclerotized vagina, which has a wide sclerotized trumpet and a single large primary chamber, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus cephalopholi n. sp. is also distinguished by the structure of its sclerotized vagina that, like the P. suratthaniensis n. sp., has a sclerotized trumpet, but it also has a long coiled or curved primary canal near its midlength, and a distal part with a primary chamber and a secondary chamber communicating with the primary chamber through a short secondary canal. In addition, P. cephalopholi n. sp. is distinguished by some sclerotized organs (ventral and dorsal hamuli, ventral bar, and quadriloculate organ) with different lengths, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus samaesarnensis n. sp. is distinguished by its sclerotized vagina that has an anterior cup-shaped trumpet and a short straight or curved primary canal. For Thailand, these are the first species of Pseudorhabdosynochus described from species of Cephalopholis and the second species of Pseudorhabdosynochus described from Epinephelus.. Trois nouvelles espèces de Pseudorhabdosynochus (Monogenea, Diplectanidae), parasites de plusieurs espèces de Cephalopholis et Epinephelus (Perciformes, Serranidae) de Thaïlande.. Pseudorhabdosynochus suratthaniensis n. sp. est décrit à partir des branchies de Cephalopholis argus, P. cephalopholi n. sp. des branchies de C. sonnerati, et P. samaesarnensis n. sp. des branchies d’Epinephelus lanceolatus. Ces poissons ont tous été pêchés dans le golfe de Thaïlande. Pseudorhabdosynochus suratthaniensis n. sp. se distingue des espèces congénères par la structure de son vagin sclérifié, qui possède une large trompette sclérifiée et une seule grande chambre primaire, et par le nombre des rangées de bâtonnets dans chacun de ses squamodisques. Pseudorhabdosynochus cephalopholi n. sp. se distingue également par la structure de son vagin sclérifié qui, comme P. suratthaniensis n. sp., a une trompette sclérifiée, mais a également un long canal primaire enroulé ou incurvé près de sa mi-longueur et une partie distale avec une chambre primaire et une chambre secondaire communiquant avec la chambre primaire par un court canal secondaire. De plus, P. cephalopholi n. sp. se distingue par certains organes sclérifiés (hamuli ventraux et dorsaux, barre ventrale et organe quadriloculé) de longueurs différentes, et par le nombre des rangées de bâtonnets dans chacun de ses squamodisques. Pseudorhabdosynochus samaesarnensis n. sp. se distingue par son vagin sclérifié qui a une trompette antérieure en forme de coupe et un court canal primaire droit ou courbe. Pour la Thaïlande, il s’agit de la première espèce de Pseudorhabdosynochus décrite à partir d’espèces de Cephalopholis et de la deuxième espèce de Pseudorhabdosynochus décrite à partir d’Epinephelus.

Topics: Animals; Bass; Female; Fish Diseases; Gills; Perciformes; Thailand; Trematoda; Trematode Infections

Natural killer lysin (Nklysin) is a small molecule antimicrobial peptide produced by natural killer cells and T lymphocytes and widely expressed in vertebrates. Homologues of Nklysin have been found in several fish, but only several of biological activity was identified. In this study, we characterized a Nklysin from grouper (Epinephelus coioides), and explored its expression pattern and biological function in bacterial infection. We also investigated the role of Nklysin in viral replication and maturation. The nklysin gene of grouper encodes a 169 amino acid, sharing 92.90% identity to H. septemfasciatus NKlysin protein, containing a saposin B domain and six well-conserved cysteine residues that necessary for antimicrobial activity by forming three intrachain disulfide bonds. Analysis of qRT-PCR revealed that nklysin gene widely expressed in all tested tissues with the higher expressions in spleen. After bacterial challenge, the nklysin gene expression significantly varied in different tissues. In addition, a large-scale of the recombinant Nklysin protein was secreted in Pichia pastoris strain GS115. The MIC assay showed that the Nklysin protein directly inhibited growth of several pathogens, including Proteus mirabilis, Bacillus subtilis, Salmonella typhi, Escherichia coli, Shigella sonnei and Streptococcus agalactiae. Further analysis showed the Nklysin protein over-expression might prevent viral genes transcriptions and replication in FHM cells. Our findings suggested that the Nklysin of grouper might be a potential agent for antibacterial and antiviral infection in the future.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antiviral Agents; Bass; DNA Virus Infections; Escherichia coli; Fish Diseases; Fish Proteins; Gene Expression Regulation; Phylogeny; Recombinant Proteins

We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 μg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.

Topics: Animals; Bass; Biosensing Techniques; Fish Diseases; Immunosorbents; Larva; Necrosis; Nodaviridae; Phosphates; Ponds; Water

Vibrio harveyi is the dominant pathogen in mariculture, and biocontrol of this pathogen using antagonistic probiotics is a long-standing biological challenge. Here, Pseudoalteromonas piscicida WCPW15003 as a probiotic effectively antagonized dominant pathogenic V. harveyi in a mariculture, with a growth-of-inhibition ratio of 6.3 h

Topics: Animals; Bass; Fish Diseases; Metabolomics; Proteomics; Vibrio; Vibrio Infections

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Mice; Spores, Bacterial; Technology; Vaccination; Vibrio Infections; Zebrafish

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFβ. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

Topics: Animals; Antiviral Agents; Bass; Cytokines; Epigenesis, Genetic; Fish Diseases; Fish Proteins; Necrosis; Nodaviridae; RNA Virus Infections; Temperature; Virus Diseases

Red-spotted grouper (Epinephelus akaara) is a popular aquaculture species with high commercial value in the food industry. However, some infectious diseases may cause mass mortality in cultural practice. Therefore, it is important to understand the immune responses of red-spotted groupers upon pathogenic invasion to develop successful disease prevention mechanisms. Here, we analyzed the transcriptomic profiles of red-spotted grouper head kidney stimulated with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) and identified differentially expressed genes (DEGs) using RNA-sequencing technology. Cluster analysis of the identified DEGs showed DEG distribution in nine separate clusters based on their expression patterns. However, significant upregulation of most DEGs was observed 6 h after poly I:C stimulation. The DEGs were functionally annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, which revealed significant expression of many immune-related signaling pathways, including antiviral, protein translation, cellular protein catabolic process, inflammatory responses, DNA repair, and cell division. Furthermore, selected DEGs were validated by quantitative real-time PCR, confirming the reliability of our findings. Collectively, this study provides insight into the immune responses of red-spotted groupers, thereby expanding the understanding of fish immunity.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Lipopolysaccharides; Necrosis; Nodaviridae; Poly I-C; Reproducibility of Results; RNA Virus Infections; RNA-Seq; Transcriptome

DNA vaccines, as an effective prophylactic technology to induce both humoral and cellular immune responses, have already been widely studied to prevent and control viral and bacterial infections in aquaculture. To find a more effective and safer way to control Micropterus salmoides rhabdovirus (MSRV) infection in largemouth bass, two different DNA vaccines expressing partial (pcDNA3.1-G2) and full-length (pcDNA3.1-G) of the MSRV G protein were developed and injected intramuscularly with different doses. The immune effect was comprehensively compared and evaluated by detecting immune-related parameters including serum antibody levels, immune-related physiological indexes, immune-related gene expression and relative survival rates in this study. The results showed that compared with the pcDNA3.1-G vaccine, the pcDNA3.1-G2 vaccine induced higher serum antibody levels, a lower nonspecific immune response in serum (ACP, SOD and T-AOC activities), higher immune-related gene expression and a higher relative survival rate. Moreover, the immune effect of pcDNA3.1-G2-vaccinated fish showed gradually higher with the increasing pcDNA3.1-G2 concentration, especially in pcDNA3.1-G2 (10μg/per fish) group, the relative survival rate reached to 82.5%, which was significant higher (p < 0.05) than pcDNA3.1-G (10μg/per fish) group. This study indicated that screening the potential core part of an antigen is an achievable strategy to improve the immunogenicity and immunoprotective effect of DNA vaccine.

Topics: Animals; Bass; Fish Diseases; GTP-Binding Proteins; Immunity, Innate; Rhabdoviridae; Vaccines, DNA

The microbiota of fish skin, the primary barrier against disease, is highly dynamic and modulated by several factors. In fish aquaculture, disease outbreaks occur mainly during early-life stages, with associated high economic losses. Antibiotic treatments sometimes remain the best option to control bacterial diseases, despite many reported negative impacts of its use on fish and associated microbiota. Notwithstanding, studies monitoring the effects of disease and antibiotic treatment on the microbiota of fingerlings are scarce. We sequenced the bacterial 16S rRNA V4 gene region using a metabarcoding approach to assess the impact of a mixed infection with Photobacterium damselae ssp. piscicida and Vibrio harveyi and subsequent antibiotic treatment with flumequine, on the skin microbiota of farmed seabass (Dicentrarchus labrax) fingerlings. Both infection and antibiotic treatment led to a significant increase in bacterial diversity and core microbial communities and impacted microbiome structure. Dysbiosis was confirmed by changes in the abundance of potential pathogenic and opportunistic bacterial taxa. Skin bacterial metabolic function was also significantly affected by flumequine administration, suggesting a detriment to fish skin health. Our results add to an increasing body of literature, showing how fish microbiome response to infection and antibiotics cannot be easily predicted.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Fish Diseases; Microbiota; Photobacterium; RNA, Ribosomal, 16S

Immunoglobulins (Igs) play a vital role in the adaptive immunity of gnathostomes. IgT, a particular Ig class in teleost fishes, receives much attention concerning the mucosal immunity. While, the characteristic and function of Epinephelus coioides IgT is still unknown. In our study, a polyclonal antibody was first prepared with grouper IgT heavy chain recombinant protein. IgT was revealed to be polymeric in serum and mucus. In normal groupers, IgT had high expression level in head kidney and spleen, while little amount in gills, thymus, gut and liver. The number of IgT-positive cells in different tissues was in line with their IgT expression. Furthermore, IgT could coat fractional bacteria in the mucus. In conclusion, this research revealed the protein characteristic, basal expression and bacterial coverage of grouper IgT. This is the first study to identify the characteristic of grouper IgT and demonstrate the capacity of coating microbes.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gills; Head Kidney; Immunoglobulins

Polystyrene nanoplastics (PS-NPs) are known to impair the function of the digestive system, intestinal flora, immune system, and nervous system of marine organisms. We tested whether PS-NPs influence viral infection of orange-spotted grouper (Epinephelus coioides). We found that grouper spleen (GS) cells took up PS-NPs at exposure concentrations of 5, 50, and 500 μg/mL and experienced cytotoxicity at 50 and 500 μg/mL concentrations. At 12 h after exposure to 50 μg/mL of PS-NPs, the replication of Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) increased in GS cells after their invasion. Juvenile fish exposed to 300 and 3000 μg/L of PS-NPs for 7 d showed PS-NPs uptake to the spleen and vacuole formation in brain tissue. Moreover, PS-NPs exposure accelerated SGIV replication in the spleen and RGNNV replication in the brain. PS-NP exposure also decreased the expression of toll-like receptor genes and interferon-related genes before and after virus invasion in vitro and in vivo, thus reducing the resistance of cells and tissues to viral replication. This is the first report that PS-NPs have toxic effects on GS cells and spleen and brain tissues, and it provides new insights into assessing the impact of PS-NPs on marine fish.

Topics: Animals; Bass; Brain; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Microplastics; Phylogeny; Polystyrenes; Spleen; Virus Replication

The purpose of this study was to determine the phylogenetic relationships among the primary betanodavirus strains circulating in Tunisian coastal waters. A survey was conducted to investigate nodavirus infections at 15 European sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata farming sites located along the northern and eastern coasts of Tunisia. The primary objective of the study was to create epidemiological awareness of these infections by determining phylogenetic relationships between the main betanodavirus strains circulating during the period 2012-2019, using RNA1 and/or RNA2 genome segments. Approximately 40% (118 of 294) tissue pools tested were positive for betanodavirus. Positive pools were distributed across all of the sampling sites. While fish mortalities were always correlated with the presence of virus in sea bass, a severe outbreak was also identified in sea bream larvae in 2019. Phylogenetic analysis revealed that almost all Tunisian strains from both sea bass and sea bream irrespective of outbreaks clustered within the RGNNV genotype. It is noteworthy that samples collected during the 2019 outbreak from sea bream contained both RNA1 and RNA2 fragments belonging to the RGNNV and SJNNV genotype, respectively, an indication of viral genome reassortment. To our knowledge, this is the first report of reassortant betanodavirus in Tunisia.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Genotype; Nodaviridae; Phylogeny; Phylogeography; RNA Virus Infections; Sea Bream

Heat shock protein 40 (Hsp40), a member of Heat shock proteins (Hsps) family, plays a crucial role in regulation of cell proliferation, survival and apoptosis in mammals. In this study, Hsp40, EcHsp40, was identified from Epinephelus coioides, an economically important marine-cultured fish in China and Southeast Asian counties. The full length of EcHsp40 was 2236 bp in length containing a 1026 bp open reading frame (ORF) encoding 341 amino acids, with a molecular mass of 37.88 kDa and a theoretical pI of 9.09. EcHsp40 has two conserved domains DnaJ and DnaJ_C. EcHsp40 mRNA was detected in all tissues examined, and the expression was significantly up-regulated response to challenged with Vibrio alginolyticus or Singapore grouper iridovirus (SGIV), one of the important pathogens of marine fish. EcHsp40 was distributed in both the cytoplasm and nucleus, over-expression of EcHsp40 can inhibit the activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), significantly promote SGIV-induced apoptosis, intracellular caspase-3 activity and viral replication, suggesting that the EcHsp40 may play an important role in pathogenic stimulation.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; HSP40 Heat-Shock Proteins; Iridovirus; Phylogeny; Ranavirus; Vibrio alginolyticus

Viral nervous necrosis (VNN) is one of the most important problems in sea bass culture. Although there have been many studies on detection and molecular characterization of betanodavirus, the causative agent of VNN, there has been little focus on understanding its prevalence to create epidemiological maps. The purpose of this study was to investigate the prevalence of betanodavirus in active sea bass hatcheries and on selected farms in Turkey by RT-qPCR. A total of 2460 samples, including fertilized eggs, prelarvae, postlarvae, fry, and fingerlings, were collected from 16 hatcheries to cover all production stages. A total of 600 sea bass were also collected from 20 farms. Betanodavirus was detected in one hatchery (1/16) in fingerling-sized sea bass, and the prevalence of betanodavirus at the hatchery level was calculated to be 6.25%. Betanodavirus was also detected on one farm (1/20) in fingerling-sized sea bass, and the prevalence of betanodavirus at the farm level was calculated to be 5%. Virus isolation initially could not be achieved in E-11 cells, but later, SSN-1 cells were used successfully. Partial genome sequence analysis of the RNA1 and RNA2 segments of the viruses revealed that they were of the red-spotted grouper nervous necrosis virus genotype, which is endemic in the Mediterranean basin. The absence of mortality related to VNN in the hatcheries and on the farms, the healthy appearance of the sea bass, the low viral load detected, and the results of retrospective epidemiological studies indicated that the infection was subclinical. Not detecting betanodavirus in other age groups where biosecurity was implemented indicates that there was no active infection. In light of these findings, it can be concluded that there was no betanodavirus circulating in hatcheries, and the virus might have been of seawater origin.

Topics: Animals; Aquaculture; Bass; Biosecurity; Fish Diseases; Nodaviridae; Retrospective Studies; RNA Virus Infections; Turkey

Hybridization is an artificial breeding strategy for generating potentially desirable offspring. Recently, a novel Hulong grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus) yielded significant growth superiority over its parent. Improved innate immunity is considered as another desirable feature during hybridization. However, whether this Hulong grouper achieved disease resistance has not yet been revealed. In this study, we first examine the infection intensity of C. irritans in the Hulong grouper, and found that the Hulong grouper is less susceptible to C. irritans primary infection. A higher immobilization titer was found in the infected Hulong grouper at Day 2 when compared with the control grouper. Furthermore, severe hyperplasia was observed in the orange-spotted grouper, but not in the Hulong grouper's skin epidermis. To further understand the innate immune mechanism against C. irritans, we conducted a comparative transcriptome analysis of the Hulong grouper during the infection. There are 6464 differentially expressed genes (DEGs) identified in the skin between the control and infected Hulong grouper. This indicates that the innate immune components, such as the complement system, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Interleukin 17 (IL-17) signaling pathway, and Toll-like receptor (TLR) signaling pathway were up-regulated during the infection. These results show that the C. irritans infection can induce a remarkable inflammatory response in the Hulong grouper. Moreover, a total of 75 pairs of orthologs with the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions ＞1, considered rapidly evolving genes (REGs), was identified between the Hulong and orange-spotted grouper. More critically, most REGs were enriched in the immune system, suggesting that rapid evolution of the immune system might occur in the Hulong grouper. These results provide a more comprehensive understanding of the innate immunity mechanism of the hybrid Hulong grouper.

Topics: Animals; Bass; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Immunity, Innate; Parasites; Transcriptome

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Host-Pathogen Interactions; Pseudomonas; Pseudomonas Infections; Virulence

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (

Topics: Amino Acid Sequence; Animals; Antimicrobial Peptides; Apoferritins; Bacterial Load; Bass; Cation Transport Proteins; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Hepcidins; Iron; Iron Overload; Liver; Photobacterium

CC motif chemokine ligand 25 (CCL25) is a key chemokine that attracts various types of leukocytes, such as activated peritoneal macrophages. However, information on CCL25 in fish is limited. Here, a CCL25 gene (LjCCL25) was identified from Japanese sea bass (Lateolabrax japonicus), showing upregulation in multiple tissues against Vibrio harveyi infection. The recombinant LjCCL25 (rLjCCL25) only significantly induced the migration of monocytes/macrophages (MO/MΦ) both in vitro and in vivo, but didn't induce that of neutrophils or lymphocytes. Additionally, rLjCCL25 only induced migration of the lipopolysaccharide-stimulated MO/MΦ (M1 type). Knockdown of Japanese sea bass CC chemokine receptor 9 (LjCCR9) expression in MO/MФ by RNA interference inhibited the LjCCL25-induced chemotaxis of resting and M1 type MO/MФ. Moreover, administration of 300 ng/g rLjCCL25 effectively increased the survival of V. harveyi-infected fish and decreased bacterial load. Our study demonstrates that LjCCL25 functions as an MO/MФ chemoattractant via LjCCR9 in Japanese sea bass against V. harveyi.

Topics: Animals; Bass; Chemokines; Chemotaxis; Fish Diseases; Fish Proteins; Immunity, Innate; Ligands; Vibrio; Vibrio Infections

Effect of bamboo vinegar powder (BVP) on growth, immunity, disease resistance, and immune-related gene expressions in juvenile Asian sea bass (barramundi), Lates calcarifer against Vibrio anguillarum was investigated. V. anguillarum infected fish fed by 2g BVP kg

Topics: Acetic Acid; Animals; Anti-Inflammatory Agents; Antioxidants; Bass; Cytokines; Diet; Fish Diseases; Perciformes; Powders; Vibrio; Vibrio Infections

Topics: Animals; Antiviral Agents; Bass; Cytokines; Fish Diseases; Immunologic Factors; Necrosis; Nodaviridae; RNA Virus Infections; Temperature

Viral nervous necrosis (VNN) disease caused by the nervous necrosis virus (NNV) is a major disease, leading to a huge economic loss in aquaculture. Previous GWAS and QTL mapping have identified a major QTL for NNV resistance in linkage group 20 in Asian seabass. However, no causative gene for NNV resistance has been identified. In this study, RNA-seq from brains of Asian seabass fingerlings challenged with NNV at four time points (5, 10, 15 and 20 days post-challenge) identified 1228, 245, 189 and 134 DEGs, respectively. Eight DEGs, including rrm1, were located in the major QTL for NNV resistance. An association study in 445 survived and 608 dead fingerlings after NNV challenge revealed that the SNP in rrm1 were significantly associated with NNV resistance. Therefore, rrm1 was selected for functional analysis, as a candidate gene for NNV resistance. The expression of rrm1 was significantly increased in the gill, liver, spleen and muscle, and was suppressed in the brain, gut and skin after NNV challenge. The rrm1 protein was localized in the nuclear membrane. Over-expression of rrm1 significantly decreased viral RNA and titer in NNV-infected Asian seabass cells, whereas knock-down of rrm1 significantly increased viral RNA and titer in NNV-infected Asian seabass cells. The rrm1 knockout heterozygous zebrafish was more susceptible to NNV infection. Our study suggests that rrm1 is one of the causative genes for NNV resistance and the SNP in the gene may be applied for accelerating genetic improvement for NNV resistance.

Topics: Animals; Bass; Disease Resistance; Fish Diseases; Gene Editing; Nodaviridae; RNA Virus Infections; RNA-Seq; Zebrafish

Toll-like receptors (TLRs) are major pattern recognition receptors (PRRs) that recognize multiple pathogen-associated molecular patterns (PAMPs) through the leucine-rich repeat (LRR) domain and mount effective immune responses. Vibrio parahaemolyticus is the main pathogen that causes vibriosis in aquatic animals, yet the mechanisms of its recognition by innate immune system in teleost fish remain unknown. Here, the results reveal that TLR13 in orange-spotted grouper (Epinephelus coioides) (EcTLR13) recognizes a conserved 23S ribosomal RNA (23S rRNA) sequence in V. parahaemolyticus, and the 13-nucleotide motif near the 23S rRNA ribozyme activation site (VP13) acts as a PAMP. After challenge with RNA and 23S rRNA from V. parahaemolyticus and with the synthetic oligoribonucleotide VP13, the expression of EcTLR13 in grouper spleen cells (GS cells) was significantly increased. EcTLR13-knockdowned GS cells were stimulated with the same stimulants as listed above, the expression of IL-6, IL-12, IL-1β and TNFα was significantly reduced. RNA-protein immunoprecipitation revealed that VP13 could directly bind to EcTLR13. The dual-luciferase reporter assay also showed that EcTLR13 enhanced the fluorescence activity of IFNβ rather than that of NF-κB when the cells were challenged with RNA from V. parahaemolyticus or with synthetic VP13. Our study established the mechanism of fish TLR13-mediated recognition of microbial products during V. parahaemolyticus infection.

Topics: Animals; Bass; Cell Line; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Immunomodulating Agents; Pathogen-Associated Molecular Pattern Molecules; Protein Binding; RNA, Catalytic; RNA, Ribosomal, 23S; Toll-Like Receptors; Vibrio Infections; Vibrio parahaemolyticus

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Topics: Animals; Bass; Biofilms; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gills; Mucus

Non-destructive sampling methods offer practical advantages to detection and monitoring of viral pathogens in economically important farmed fish and broodstock. Here, we investigated whether blood, mucus and fin can be used as non-lethal sample sources for detection of scale drop disease virus (SDDV) in farmed Asian sea bass, Lates calcarifer. Detection of SDDV was performed in parallel from three non-destructive and seven destructive sample types, collected from both clinically sick fish and subclinical fish obtained from an affected farm. The results showed that SDDV was detectable in all 10 sample types with the percentage ranging from 20% to 100%. Blood was the best non-destructive sample source exhibited by the fact that it yielded 100% SDDV-positive tests from both sick (n = 12, 95% CI: 69.9-99.2) and clinically healthy fish (n = 4, 95% CI: 39.6%-97.4%) and is considered a "sterile" sample. This study also revealed concurrent infection of SDDV and two ectoparasites Lernanthropus sp. and Diplectanum sp., in all affected fish (n = 8, 95% CI: 46.7-99.3) during the disease outbreak. These ectoparasites also tested positive for SDDV by PCR, indicating that they were potential sample sources for PCR-based detection of SDDV and possibly other viruses infecting Asian sea bass.

Topics: Animal Scales; Animals; Bass; Copepoda; DNA Virus Infections; Fish Diseases; Iridoviridae; Prevalence; Thailand; Trematoda

Bcl-2-associated athanogene 5 (BAG5) is a kind of molecular chaperone that can bind to the Bcl-2 and modulate cell survival. However, little is known about the functions of fish BAG5. In this study, we characterized a BAG5 homolog from orange-spotted grouper (Epinephelus coioides) gene (Ec-BAG5) and investigated its roles during viral infection. The Ec-BAG5 protein encoded 468 amino acids with four BAG domains, which shared high identities with reported BAG5. The highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte (PBL). And the Ec-BAG5 expression were significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) or Lipopolysaccharide (LPS) stimulation in vitro. Furthermore, Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)). Also, overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor 3 (IRF3), interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 (IFP35), myxovirus resistance gene 1 (Mx1) and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), as well as the activities of NF-κB, ISRE and IFN-1. These data indicate that Ec-BAG5 can affect viral infection through regulating the expression of IFN- and inflammation-related factors, which provide useful information to better understand the immune response against viral infection.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Inflammation; Interferons; Molecular Chaperones; Nodaviridae; RNA Virus Infections; Sequence Alignment; Tissue Distribution; Virus Replication

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that is a major threat to grouper aquaculture. The pathogenesis of SGIV is not well understood so far. Previous studies have revealed that ICP18, an immediate early protein encoded by SGIV ORF086R gene, promotes viral replication by regulating cell proliferation and virus assembly. In the present study, the potential functions of ICP18 were further explored by probing into its interactors using a proximity-dependent BioID method. Since our in-house grouper embryonic cells (a natural host cell of SGIV) could not be efficiently transfected with the plasmid DNA, and the grouper genome data for mass spectrometry-based protein identification is not currently available, we chosen a non-permissive cell (HEK293 T) as a substitute for this study. A total of 112 cellular proteins that potentially bind to ICP18 were identified by mass spectrometry analysis. Homology analysis showed that among these identified proteins, 110 candidate ICP18-interactors had homologous proteins in zebrafish (a host of SGIV), and shared high sequence identity. Further analysis revealed that the identified ICP18-interacting proteins modulate various cellular processes such as cell cycle and cell adhesion. In addition, the interaction between ICP18 and its candidate interactor, i.e., cyclin-dependent kinase1 (CDK1), was confirmed using Co-immunoprecipitation (Co-IP) and Pull-down assays. Collectively, our present data provides additional insight into the biological functions of ICP18 during viral infection, which could help in further unraveling the pathogenesis of SGIV.

Topics: Animals; Bass; Cell Adhesion; Cell Cycle; Cyclin-Dependent Kinases; Fish Diseases; HEK293 Cells; Humans; Iridovirus; Mass Spectrometry; Protein Interaction Domains and Motifs; Singapore; Viral Proteins; Virus Replication

The objective of this study was to investigate safety and efficacy using a low-temperature immunization protocol with NNV in sevenband grouper, Epinephelus septemfasciatus. Further, NNV specific antibody post immunization and intramuscularly challenge was also evaluated. Immunization at low temperature resulted in a low titer virus infection in brain tissues without any clinical symptoms of infection such as sluggish behavior and/or spinning, rotating swimming being observed, and no mortality was observed. Post challenge, NNV titer NNV giving an RPS of 100 %, increased in brain tissues of naïve (non-immunized) sevenband grouper NNV giving an RPS of 100 %, with a cumulative mortality of 100 % at 25 days post-infection. No mortality or disease symptoms NNV giving an RPS of 100 %, as NNV giving and of 100 %, observed in the groups immunized at low temperature with live NNV giving an RPS of 100 %. NNV giving an RPS of 100 %. NNV specific antibody was not detected in live NNV vaccinated sevenband grouper. This is the first study that confirms that field-scale NNV immersion vaccine can protect sevenband grouper against lethal infection with NNV at natural seawater temperature under the gradually increased from 14.3-24.8 °C.

Topics: Animals; Antibodies, Viral; Bass; Cold Temperature; Fish Diseases; Immunization; Nodaviridae; RNA Virus Infections; Viral Vaccines

Asian seabass, Lates calcarifer farming in Southeast Asia, encounters serious disease challenges caused by Streptococcus agalactiae and Streptococcus iniae. However, a vaccine for disease prevention is not yet available. In this study, we investigated the mucosal and systemic antibody (IgM) response kinetics of the Asian seabass following primary immunization with oil-based formalin-killed vaccines (FKVs) prepared from S. agalactiae and S. iniae (monovalent Sa, monovalent Si, and bivalent Sa-Si) and secondary booster with the respective water-based FKVs. The efficacy of vaccines was subsequently evaluated by an experimental challenge. The results revealed similar antibody response kinetics in both systemic and mucosal systems. However, the immune response in the fish vaccinated with the monovalent vaccines was superior to those fish received the bivalent vaccine in terms of specific antibody titer. The fish that received monovalent vaccines required 1-2 weeks to raise a significant level of specific antibody titer in both systemic and mucosal systems while those vaccinated with bivalent vaccine required three weeks. Following booster at day 21, both systemic and mucosal antibody titers in all vaccinated groups had reached the peak at day 28 and gradually declined in the following weeks but remained significantly higher than control until the end of the experiment (day 63). In the challenge test, both monovalent and bivalent vaccines were found to be highly efficacious, with the relative percentage survival (RPS) ranging from 75 to 85%. In summary, this study explored the 63-days antibody response kinetics (both mucosal and systemic systems) of Asian seabass to monovalent and bivalent inactivated vaccines and confirmed that the combination of S. agalactiae and S. iniae in a single injectable vaccine is possible.

Topics: Animals; Antibody Formation; Bass; Fish Diseases; Immunity, Innate; Immunity, Mucosal; Streptococcal Infections; Streptococcal Vaccines; Streptococcus agalactiae; Streptococcus iniae; Vaccines, Combined

Topics: Animals; Bass; Feeding Behavior; Fish Diseases; Leeches; Mouth; Virginia

A new coelozoic myxosporean species, Zschokkella epinepheli n. sp., collected from the gallbladder of the white grouper Epinephelus aeneus (Perciformes: Serranidae) from the bay of Bizerte, Tunisia, is described based on morphological and molecular characteristics. Myxospores and plasmodia were observed floating free in the bile. Mature plasmodia were polysporic and subspherical in shape, measuring 85.0-94.0 μm long and 70.0-82.0 μm wide. Mature myxospores were ovoid in valvular view, measuring 10.0 ± 1.7 (8.0-11.0) μm in length and 7.0 ± 0.3 (6.6-7.5) μm in width. Polar capsules were pyriform and equal in size, measuring 3.0 ± 0.2 (2.8-3.6) μm in length and 2.3 ± 0.3 (1.8-2.7) μm in width. Myxospore valves had 12-14 longitudinal striations. Based on the small subunit rDNA, the new species Z. epinepheli n. sp. differs from all other Zschokkella species for which there is a DNA sequence deposited in GenBank. Phylogenetic analysis revealed that Z. epinepheli n. sp. clustered in the marine subclade of Zschokkella species within the biliary tract IV clade. This is the first report of a Zschokkella species from the gallbladder of an epinephelin fishes.

Topics: Animals; Bass; Bile; DNA, Ribosomal; Fish Diseases; Gallbladder; Myxozoa; Parasitic Diseases, Animal; Phylogeny; Seafood; Tunisia

Five isonitrogenous and isocaloric diets [containing 54, 30, 15, 10, and 5% fishmeal crude-protein (CP), dry matter (DM) basis] were prepared by replacing fishmeal with poultry by-product meal plus soybean meal to feed juvenile largemouth bass (LMB, with an initial mean body weight of 4.9 g) for 8 weeks. All diets contained 54% CP and 13% lipids. There were four tanks of fish per treatment group (15 fish/tank). The fish were fed twice daily with the same feed intake (g/fish) in all the dietary groups. Results indicated that the inclusion of 15% fishmeal protein in the diet is sufficient for LMB growth. However, some of the fish that were fed diets containing ≤ 15% fishmeal CP had black skin syndrome (characterized by skin darkening and retinal degeneration, as well as intestinal and liver atrophies and structural abnormalities). The concentrations of taurine, methionine, threonine and histidine in serum were reduced (P < 0.05) in fish fed the diets containing 5, 10 and 15% fishmeal CP, compared with the 30 and 54% fishmeal CP diets. Interestingly, the concentrations of tyrosine and tryptophan in serum were higher in fish fed diets with ≤ 15% fishmeal CP than those in the 54% fishmeal CP group. These results indicated that 15% fishmeal CP in the diet containing poultry by-product meal and soybean meal was sufficient for the maximum growth and feed efficiency in LMB but inadequate for their intestinal, skin, eye, and liver health. A reduction in dietary methionine and taurine content and the possible presence of antinutritional factors in the fishmeal replacements diets containing high inclusion levels of soybean meal may contribute to black skin syndrome in LMB. We recommend that the diets of juvenile LMB contain 30% fishmeal CP (DM basis).

Topics: Amino Acids; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Bass; Body Composition; Costs and Cost Analysis; Diet; Dietary Proteins; Eating; Fish Diseases; Glycine max; Lipids; Poultry

Azomite is a hydrated calcium sodium aluminosilicat rich in rare earth elements. To investigate the dietary effects of Azomite on growth, intestine microbiota and morphology, immunohematological changes and disease resistance, seven diets with Azomite supplementation of 0 (the control), 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 g/kg (A0, A1, A2, A3, A4, A5, A6), were prepared and fed to largemouth bass, Micropterus salmoides (7.96 ± 0.19) for 60 days. The results revealed that the weight gain (WG) increased first and then decreased with the increasing dietary Azomite, and the A2 group presented the highest WG and lowest feed conversion ratio among all the groups. The supplementation of 2.0 g/kg Azomite significantly increased the intestine protease activity, the crude protein of whole body and protein retention (P < 0.05), and high inclusion of Azomite (6.0 g/kg) significantly reduced the lipid retention (P < 0.05). The amounts of red blood cells in A5, A6 groups, white blood cells in A3, A5, A6 groups and lymphocyte in A2-A6 groups were all significantly higher than those in the control group (P < 0.05). In addition, serum superoxide dismutase and catalase activities in A5, A6 groups, and serum alkaline phosphatase and lysozyme activities in A2-A4 groups showed significantly higher values than the control group (P < 0.05). Intestinal microbiota analysis indicated that the Tenericutes abundance was increased, whereas Proteobacteria abundance was decreased in all Azomite supplemented groups. The villus height in A2-A4 groups, and the villus width in A2 group were significantly higher than those of the control group (P < 0.05). The cumulative mortality was reduced by the addition of 2.0-5.0 g/kg Azomite after challenging with A. hydrophila (P < 0.05). In conclusion, proper addition of Azomite in diets improved the growth, intestine morphology, immune response and disease resistance in largemouth bass, and the optimal inclusion was estimated to be 2.0-3.0 g/kg diet.

Topics: Aluminum Silicates; Animal Feed; Animals; Bacterial Infections; Bass; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Fish Diseases; Gastrointestinal Microbiome; Random Allocation; Trace Elements

A unique strain of Vibrio harveyi is the causative agent of scale drop and muscle necrosis disease (SDMND) in Asian sea bass (Lates calcarifer). This study investigated the protein profiles of SDMND-causing Vibrio harveyi isolates compared to the reference V. harveyi ATCC 14126 strain. A distinct protein band of 33 kDa, namely HP33, found from only V. harveyi SDMND was subjected to analysis by LC-MS/MS and the identified peptide sequences matched to an unknown hypothetical protein. Detection of HP33 coding sequence was investigated at both genomic and transcriptional levels and the results consistently supported the protein analysis. Recombinant HP33 protein was then produced using Escherichia coli system. The rHP33 protein did not cause mortality or visible clinical signs to Asian sea bass. However, the rHP33 protein was able to stimulate antibody response in Asian sea bass as evidenced by Western blotting and agglutination tests. Here, we proposed that rHP33 might be a good protein target for development of subunit vaccine and/or immunostimulant to protect Asian sea bass from SDMND.

Topics: Animal Scales; Animals; Bacterial Proteins; Bass; Fish Diseases; Immunogenicity, Vaccine; Muscular Diseases; Necrosis; Vibrio; Vibrio Infections

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1β, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.

Topics: Animals; Bass; Ciliophora Infections; Fish Diseases; Gene Expression Profiling; Gills; Hymenostomatida; Signal Transduction; Takifugu

Fish parasites can cause diseases in humans and lead to commercial losses in fisheries and aquaculture. The objectives of this study were to analyze E. ongus's parasite fauna regarding food safety and parasite transmission risk between Epinephelus species and test whether E.ongus populations can be distinguished by their parasite community.. We studied the metazoan parasite fauna of 30 white-streaked groupers Epinephelus ongus from the Thousand Islands, Java Sea, Indonesia, and compared the parasite community with specimens from Karimunjawa archipelago, Java Sea, from a former study. We used common fish parasitological methods for fish examination and parasite calculations.. We found 12 metazoan parasite species, establishing five new host and five new locality records, increasing the known parasite fauna of E. ongus by 21%. No anisakid worms infected E. ongus. All but one (trematode Gyliauchen cf. nahaensis) species have been previously reported from Epinephelus. Parasite abundance of E. ongus differed significantly between the two regions.. Due to a certain degree of host specificity to groupers, there is potential risk of parasite transmission from E. ongus into groupers in mariculture or surrounding fishes, which increases (sea) food security related health risks from zoonotic parasites and calls for better monitoring and management plans for E. ongus. The regional separation of the Thousand Islands and Karimunjawa with different food availability and fish ecology causes different parasite abundances, distinguishing two separate E. ongus populations by their parasite fauna.

Topics: Animals; Bass; Fish Diseases; Humans; Indonesia; Islands; Parasites; Trematoda

For the activation of T cells, it is necessary the specific recognition of the peptide by the T cell receptors (TCR) in the surface of antigen-presenting cells (APCs) and additional signals delivered by costimulatory receptors. In fish, knowledge about the presence of these costimulatory signals is limited and functional evidence almost absent. Thus, in this study, we have identified the stimulatory CD28 and the inhibitory cytotoxic T-lymphocyte-associated protein 4 (CTLA4) coreceptors in the European sea bass (Dicentrarchus labrax), and evaluated their transcription. In parallel, the transcription encoding for the T cell markers CD8α and CD4 was also evaluated. Both coreceptors showed the canonical architecture including a signal peptide, an immunoglobulin domain, a transmembrane region and a cytosolic tail. Protein predictions and phylogenetic tree identify them as true mammalian orthologues of CD28 and CTLA4. We found these genes constitutively expressed in all studied organs of European sea bass with high expression in lymphoid organs (thymus, spleen and head-kidney) and liver. The molecular expression pattern of these genes was up-regulated in head-kidney leucocytes stimulated with T mitogens as concanavalin A and phytohemagglutinin (PHA), but not with the B cell mitogen lipopolysaccharide (LPS). Fish challenged with nodavirus (NNV) evidenced a differential and opposing regulation of the cd28 and ctla4 transcription levels in the brain, the target organ for viral replication, and head-kidney. While cd28 transcription tends to decrease over the infection time in both organs the expression of the ctla4 gene tends to increase. Interestingly, the coreceptor expression is highly and significantly correlated to the transcription of the T cell markers. Our results highlight the important role of CD28 and CTLA4 as costimulatory receptors of T cells in European sea bass but further studies are deserved.

Topics: Adaptive Immunity; Amino Acid Sequence; Animals; Bass; CD28 Antigens; CTLA-4 Antigen; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Lymphoid Tissue; Nodaviridae; Perciformes; Phylogeny; RNA Virus Infections; Sequence Alignment; T-Lymphocytes

Antimicrobial peptides, which are crucial effectors of innate immunity, are a promising substitute for antibiotics. The piscidin family is a group of fish-derived antimicrobial peptides that have potent antimicrobial activity and participate in the innate immune response. Here we describe a novel piscidin-like peptide called cerocin from the black sea bass (Centropristis striata), which is a highly valued marine teleost in both commercial and recreational fisheries worldwide. The full-length cDNA of cerocin consists of 567 base pairs, including 5' and 3' untranslated regions of 61 and 209 base pairs, respectively. The active peptide consists of 20 amino acids that form an amphipathic α-helix structure. Cerocin showed highest identity with the cardinalfish (Ostorhinchus fasciatus) piscidin (52%). Phylogenetic tree demonstrated that the cerocin clustered with dicentracin of Liparis tanakae and Perca flavescens. It showed tissue-specific distribution patterns and was predominantly expressed in the gill. After challenge with Vibrio harveyi, C. striata showed time- and tissue-dependent expression of the cerocin gene. Finally, a cerocin peptide was synthesized, and it exerted broad-spectrum antimicrobial activity against a number of bacterial strains, especially Gram-positive pathogens. Analysis of the killing kinetics revealed that the cerocin peptide had a rapid bactericidal effect on the bacteria. Collectively, these data suggest that the piscidin-like cerocin might play a vital role in the immune response of C. striata, and further studies of this gene may provide insight into the innate immune system of this species.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Random Allocation; Sequence Alignment; Vibrio; Vibrio Infections

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Topics: Amino Acids; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Bass; Body Composition; Diet; Dietary Proteins; Dietary Supplements; Eating; Fish Diseases; Lipids; Methionine; Taurine

Recently, the same fish diseases, which have been found in pond farming, have been found in the newly tested largemouth bass (Micropterus salmoides) system. Bacterial septicemia caused by Aeromonas hydrophila occurs frequently in largemouth bass culture leading to significant economic losses. To investigate the role miRNA in the largemouth bass disease resistance, twelve (2 tissues (spleen and head kidney) × 2 experimental groups (infected and control) × three biological replicates) small RNA libraries were constructed and sequenced with miRNA-seq. A total of 26 differentially expressed miRNAs, 8 upregulated and 18 downregulated, were identified in the spleen, and 19 differentially expressed miRNAs, 9 upregulated and 10 downregulated, were identified in head kidney (fold change ≥ 2 or ≤ 0.5 and P ≤ 0.05). The differentially expressed miRNAs with the largest fold change were selected for target gene prediction using GO and KEGG analysis. Six miRNAs in the spleen and 5 miRNAs in the head kidney were selected. Analysis showed that, of all the immune and metabolic pathways, the FoxO signaling pathway was enriched in both the spleen and head kidney groups. Common target genes of the pathway included AMP-activated catalytic subunit alpha 1 (prkaa1), phosphatidylinositol 3-kinase (pik3r3b), serine/threonine-protein kinase (plk2), and forkhead box protein G1 (foxg1a). MiRNAs (such as miR-126-5P, miR-126-3P) are involved in immune response and cell cycle functions as they regulate targeted genes in the FoxO pathway. These results will enhance our understanding of the molecular mechanisms underlying immune responses to bacterial septicemia and facilitate molecular-assisted selection of resistant strains of largemouth bass.

Topics: Aeromonas hydrophila; AMP-Activated Protein Kinases; Animals; Bass; Fish Diseases; Fish Proteins; Forkhead Transcription Factors; Gram-Negative Bacterial Infections; Kidney; MicroRNAs; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Spleen

Black soldier fly larvae meal (BSFLM) has been successfully demonstrated as a promising fish meal (FM) replacer in diets of several fish species including European seabass (Dicentrarchus labrax). However, its impacts on antioxidant capacity, and immune responses of treated fish are still poorly understood. A 60-day feeding trial was carried out to evaluate the effects of partial substitution of FM with different levels of dry BSFLM on the antioxidative status, non-specific immunity, transcriptomic responses, and resistance of European seabass to the challenge with Vibrio alginolyticus. Four isoproteic (45%) and isolipidic diets were formulated by replacing 0.0%, 25%, 35%, and 50% of the dietary FM. Each diet was randomly assigned to four fish groups (in triplicates) (initial mean body weight, 12.1 ± 0.21 g) (20 fish per aquarium) (n = 240). Fish were fed three times daily to the apparent satiation. At the end of the feeding trial, serum antioxidant biomarkers such as malondialdehyde levels, and catalase, superoxide dismutase, and glutathione peroxidase enzyme activities were significantly increased in all BSFLM groups in a dose-dependent manner compared to the control group (P < 0.05). The non-specific immune indices, including phagocytic activity, phagocytic index, serum lysozyme and respiratory burst activities were significantly elevated in BSFLM groups compared to those in the control group (P < 0.05). Significant upregulation of the mRNA expression levels of hepatic heat shock protein 70, interleukin-1beta and interleukin-10 genes were observed in all BSFLM groups compared to the control group (P < 0.05). Additionally, after the challenge with V. alginolyticus, the relative percent of survival was significantly elevated in fish groups fed on diets containing graded levels of BSFLM over the control group (P < 0.05). Conclusively, the present study suggests the potential efficacy of partial replacement of dietary FM protein for up to 50% by BSFLM without negative effects on fish health with possible potentiation of the antioxidative status, and the immune responses of the European seabass.

Topics: Animal Feed; Animals; Antioxidants; Bass; Diet; Dietary Supplements; Diptera; Disease Resistance; Dose-Response Relationship, Drug; Fish Diseases; Immunity, Innate; Larva; Random Allocation; Transcriptome; Vibrio alginolyticus; Vibrio Infections

Cardicola Short, 1953 is the most speciose aporocotylid genus (35 species) and includes marine and estuarine species of fish blood flukes that infect "higher ray-finned fishes" (Euteleostei). Several clades within Cardicola are recovered in phylogenetic analyses of the large subunit ribosomal DNA (28S), but morphological synapomorphies for those nucleotide-based clades remain elusive. The type species, Cardicola cardiocola (Manter, 1947) Short, 1953, has not been recollected in 73 yr and the original description was incomplete; making a genus revision challenging because of the ambiguous systematic position of its type species. Herein, we redescribe C. cardiocola by using the holotype (USNM 1337732) and new specimens collected from the type host, jolthead porgy, Calamus bajonado (Sparidae), from nearby the type locality. It differs from its congeners by the combination of having a body that is 5 times longer than wide, an anterior sucker with concentric rows of spines, 2-6 tegumental body spines per row, an esophageal gland that is 22-43% of the esophageal length, a testis that is 3-5 times longer than wide and that fills the intercecal space, a vitelline duct connecting to the anterior aspect of the oötype, an ascending uterus that lacks any coil, a descending uterus yielding a single coil, an obvious cirrus sac separated by constriction from the seminal vesicle, a tegumental protrusion surrounding the terminal end of cirrus sac, and a male genital pore that is posterior to the remainder of the genitalia. We also describe a new congener infecting the heart of yellowedge grouper, Hyporthodus flavolimbatus (Serranidae), from the Gulf of Mexico. It differs from its congeners by the combination of having an anterior sucker that does not extend beyond the anterior body margin, 2-5 tegumental body spines per row, posterior ceca that are 9 times length of the anterior ceca and that lack any coil, a testis that is 3 times longer than wide and that does not fill the intercecal space, an ovary that is >60% of the body width, a vitelline duct that connects to the anterior aspect of the oötype, a uterus that is >10% of the body width and that extends posterior to all genitalia, and a rounded posterior body margin. It is the first species of Cardicola to be described from a grouper (Serranidae). The 28S and internal transcribed spacer 2 phylogenetic analyses recovered the new species as a distinct lineage within the clade of Cardicola spp.

Topics: Animals; Bass; Bayes Theorem; Fish Diseases; Florida; Gulf of Mexico; Heart; Phylogeny; Prevalence; Trematoda; Trematode Infections

Aquaculture is responsible for more than 50% of global seafood consumption. Bacterial diseases are a major constraint to this sector and associated with misuse of antibiotics, pose serious threats to public health. Fish-symbionts, co-inhabitants of fish pathogens, might be a promising source of natural antimicrobial compounds (NACs) alternative to antibiotics, limiting bacterial diseases occurrence in aquafarms. In particular, sporeforming Bacillus spp. are known for their probiotic potential and production of NACs antagonistic of bacterial pathogens and are abundant in aquaculture fish guts. Harnessing the fish-gut microbial community potential, 172 sporeforming strains producing NACs were isolated from economically important aquaculture fish species, namely European seabass, gilthead seabream, and white seabream. We demonstrated that they possess anti-growth, anti-biofilm, or anti-quorum-sensing activities, to control bacterial infections and 52% of these isolates effectively antagonized important fish pathogens, including Aeromonas hydrophila, A. salmonicida, A. bivalvium, A. veronii, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei. By in vitro quantification of sporeformers' capacity to suppress growth and biofilm formation of fish pathogens, and by assessing their potential to interfere with pathogens communication, we identified three promising candidates to become probiotics or source of bioactive molecules to be used in aquaculture against bacterial aquaculture diseases.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bacillus; Bacterial Infections; Bass; Biofilms; Fish Diseases; Gastrointestinal Microbiome; Probiotics; Quorum Sensing; Sea Bream

Nocardia seriolae is an important pathogenic bacterium that causes nocardiosis in various fish species and leads to economic losses in the fish industry. To develop an effective subunit vaccine against nocardial infection, the truncated resuscitation-promoting factor (tRPF) of N. seriolae was selected and recombinantly produced using the Escherichia coli expression system. Western blotting results indicated that the recombinant protein could be strongly recognised by largemouth bass anti-N. seriolae antibodies. The protective efficacy of tRPF recombinant protein was assessed in combination with the commercial adjuvant Montanide™ ISA 763 A VG. The results showed that emulsified tRPF + ISA significantly induced high serum antibody response and serum lysozyme activity in the vaccinated fish. Quantitative reverse transcription polymerase chain reaction analysis indicated that tRPF + ISA could notably enhance the expression of immune-related genes in both the head kidney and spleen of the vaccinated fish. Finally, vaccinated largemouth bass displayed higher immuno-protection with a relative percent survival of 69.23% compared to the control groups. Taken together, the combination of tRPF + ISA is an ideal vaccine candidate against N. seriolae infection.

Topics: Animals; Bacterial Proteins; Bacterial Vaccines; Bass; Fish Diseases; Immunity; Immunogenicity, Vaccine; Nocardia; Nocardia Infections; Recombinant Proteins; Vaccines, Subunit

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Heat-Shock Proteins; Photobacterium; Vaccines, Synthetic

The response of macrophage aggregates in fish to a variety of environmental stressors has been useful as a biomarker of exposure to habitat degradation. Total volume of macrophage aggregates (MAV) was estimated in the liver and spleen of white perch Morone americana from Chesapeake Bay using stereological approaches. Hepatic and splenic MAV were compared between fish populations from the rural Choptank River (n = 122) and the highly urbanized Severn River (n = 131). Hepatic and splenic MAV increased with fish age, were greater in females from the Severn River only, and were significantly greater in fish from the more polluted Severn River (higher concentrations of polycyclic aromatic hydrocarbons, organochlorine pesticides, and brominated diphenyl ethers). Water temperature and dissolved oxygen had a significant effect on organ volumes, but not on MAV. Age and river were most influential on hepatic and splenic MAV, suggesting that increased MAV in Severn River fish resulted from chronic exposures to higher concentrations of environmental contaminants and other stressors. Hemosiderin was abundant in 97% of spleens and was inversely related to fish condition and positively related to fish age and trematode infections. Minor amounts of hemosiderin were detected in 30% of livers and positively related to concentrations of benzo[a]pyrene metabolite equivalents in the bile. This study demonstrated that hepatic and splenic MAV were useful indicators in fish from the 2 tributaries with different land use characteristics and concentrations of environmental contaminants. More data are needed from additional tributaries with a wider gradient of environmental impacts to validate our results in this species.

Topics: Animals; Bass; Bays; Environmental Monitoring; Female; Fish Diseases; Macrophages; Rivers; Water Pollutants, Chemical

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

Topics: Animals; Apoptosis; Bass; DNA Virus Infections; Fish Diseases; Genes, Viral; Iridovirus; MicroRNAs; NF-kappa B; Transcription Factor AP-1; Virus Replication

The aetiological agents of red sore disease (RSD) reportedly comprise a taxonomically ambiguous stalked ciliate (a species of Epistylis) and Aeromonas hydrophila. The taxonomic identity of each pathogen remains provisional: using supra-specific morphological features for the ciliate and culture-based methods that cannot delineate bacterial strain. On 7 and 9 November 2017 and 28 May 2020, biologists and anglers reported a local epizootic (Hiwassee and Chattahoochee river basins; Georgia) wherein some moribund fish presented RSD-like lesions. The ciliates were assigned to Epistylis by morphology. The ciliate is regarded as Epistylis cf wuhanensis, as nucleotide sequences from its small subunit ribosomal DNA were identical to those of Epistylis wuhanensis. The bacterium was identified as Aeromonas hydrophila by phenotypic markers and nucleotide sequences from the DNA gyrase subunit B; our sequences comprised 3 strains and phylogenetically were recovered sister to strains of Eurasian origin. Histological sections of lesions revealed effacement or partial deterioration of the epithelium covering scales, scale loss, haemorrhaging, necrosis, oedema, and extensive inflammatory infiltrate in the dermis. This is the first nucleotide sequence information for the symbionts implicated in RSD.

Topics: Aeromonas hydrophila; Alabama; Animals; Bass; Ciliophora Infections; Coinfection; Fish Diseases; Georgia; Gram-Negative Bacterial Infections; Lakes; Oligohymenophorea; Perciformes; Sequence Analysis, DNA

The red-spotted grouper, Epinephelus akaara, has been cultured widely in China, and in several countries of Southeast Asia, due to its important economic value. However, in recent years the outbreak of disease caused by red-spotted grouper nervous necrosis virus (RGNNV) has caused mass mortality in the stage of the grouper lifecycle from fry to juvenile, resulting in considerable economic loss in commercial aquaculture. However, the molecular mechanism underlying anti-RGNNV infection in red-spotted grouper has never been fully understood. To identify the anti-RGNNV related markers and candidate genes, we performed a genome-wide association study (GWAS) on a natural population of 100 individuals for a full-genome screen of the red-spotted grouper. In this research, 36,311 single, high quality nucleotide polymorphisms (SNPs) were developed. Two significantly associated SNPs and three suggestively associated SNPs were identified at the genome level. From these identified SNPs, five candidate genes were annotated: EPHA7, Osbpl2, GPC5, CDH4 and Pou3f1. These genes are involved in nervous system development, retinal formation, and lipid metabolism regulation. In combination with studies on the characteristics of NNV infection, it was speculated that in the fry stage of the grouper lifecycle, the immune system is not fully developed. Therefore, improved resistance to RGNNV may come through regulating nervous system development or lipid metabolism related pathways. In addition, the genotypes of SNPs associated with disease-resistant traits were analyzed. The markers and genes obtained in this study may facilitate a marker-assisted selection for red-spotted grouper aiming at disease resistance to RGNNV.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Genome-Wide Association Study; Nodaviridae; Polymorphism, Single Nucleotide; RNA Virus Infections

The polymeric immunoglobulin receptor (pIgR) is an important molecule in the mucosal immunity of teleosts. Previous studies have shown that pIgR can bind and transport polymeric immunoglobulins (pIgs), but few studies have focused on the binding of teleost pIgR to bacteria. In this study, we identified a gene encoding pIgR in largemouth bass (Micropterus salmoides). The pIgR gene contained two Ig-like domains (ILDs), which were homologous to ILD1 and ILD5 of mammalian pIgR. Our results showed that largemouth bass pIgR-ILD could combine with IgM. Moreover, we also found that largemouth bass pIgR-ILD could bind to Aeromonas hydrophila and Micrococcus luteus. Further analysis showed that largemouth bass pIgR-ILD could also combine with lipopolysaccharide (LPS), peptidoglycan (PGN) and various saccharides, and reduced binding to bacteria was observed with LPS and PGN treatment, indicating that largemouth bass pIgR could bind to bacteria to prevent infection and that saccharide binding is an important interaction mechanism between pIgR and bacteria. These results collectively demonstrated that largemouth bass pIgR not only combines with IgM but also binds to bacteria by various saccharides.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Immunity, Mucosal; Immunoglobulin M; Lipopolysaccharides; Micrococcus luteus; Peptidoglycan; Phylogeny; Protein Domains; Receptors, Polymeric Immunoglobulin; Sequence Alignment; Sequence Analysis, DNA

Topics: Animals; Bass; Dinoflagellida; Fish Diseases; Gene Expression; Gills; Head Kidney; Immunity, Innate; Protozoan Infections, Animal; RNA, Messenger

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; L-Amino Acid Oxidase; Recombinant Proteins; Sequence Alignment; Sf9 Cells; Spodoptera; Substrate Specificity; Vibrio parahaemolyticus

Sessiline ciliates live as eco commensals (low numbers) and parasites (high numbers) on different hosts, like mollusks copepods, mysids and fish. Riboscyphidia ecto-protozoan is moderately pathogenic but high numbers of it on the gills can physically prevent gas exchange. The present study aimed to describe the epizoic ciliates Riboscyphidia found on the Red Sea cultured Asian sea bass and obtain more information on the Epidemiology of the parasite with special references to control and histopathological examination of naturally infected sea bass.. The occurrence of epizoic ciliates on the adult Asian Sea bass. About 100 Asian sea bass were collected by the fishing net at a private marine fish farm at Ismailia governorate and transferred to the hydrobiology laboratory at National Research Centre. A parasitological and histopathological study of epizoic sessile ciliate species was done. ANOVA test was used for Statistical analysis.. Riboscyphidia sp. was found and isolated after parasitological examination of investigated adult's Asian sea bass. The prevalence of Riboscyphidiosis was 64%. Sessile ciliates were found on gills, skin and fins. The clinical signs of Riboscyphidiosis were respiratory distress, flashing and off food. Histopathological alterations in naturally infested Asian sea bass were investigated.. The treatment of choice of Riboscyphidiosis was prolonged immersion by Copper citrate with a dose of 0.56 mg mL-1 for 7 days.

Topics: Animals; Antiprotozoal Agents; Aquaculture; Bass; Citrates; Fish Diseases; Indian Ocean; Protozoan Infections, Animal

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.

Topics: Animals; Aquaculture; Bass; Croatia; Disease Outbreaks; Fish Diseases; Piscirickettsia; Piscirickettsiaceae Infections; RNA, Bacterial; RNA, Ribosomal, 16S; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Topics: Animals; Bass; Cytokines; Fish Diseases; Gene Expression Profiling; Head Kidney; Isopoda; Liver; Mediterranean Sea; Parasitic Diseases, Animal

Susceptibility of European sea bass (Dicentrarchus labrax L.) to viral nervous necrosis (VNN) is well-known. Interest towards selective breeding as a tool to enhance genetic resistance in this species has increased sharply due to the major threat represented by VNN for farmed sea bass and limitations concerning specific therapeutical measures. A sea bass experimental population (N = 650) was challenged with nervous necrosis virus (NNV) to investigate genetic variation in VNN mortality. In addition, relationships of this trait with serum cortisol concentration after stress exposure, antibody titer against NNV antigens, and body weight at a fixed age were studied to identify potential indicator traits of VNN resistance.. The estimate of heritability for VNN mortality was moderate and ranged from 0.15 (HPD95%, 95% highest posterior density interval: 0.02, 0.31) to 0.23 (HPD95%: 0.06, 0.47). Heritability estimates for cortisol concentration, antibody titer, and body weight were 0.19 (HPD95%: 0.07, 0.34), 0.36 (HPD95%: 0.16, 0.59) and 0.57 (HPD95%: 0.33, 0.84), respectively. Phenotypic relationships between traits were trivial and not statistically significant, except for the estimated correlation between antibody titer and body weight (0.24). Genetic correlations of mortality with body weight or antibody titer (- 0.39) exhibited a 0.89 probability of being negative. A negligible genetic correlation between mortality and cortisol concentration was detected. Antibody titer was estimated to be positively correlated with body weight (0.49).. Antibody titer against NNV offers the opportunity to use indirect selection to enhance resistance, while the use of cortisol concentration as an indicator trait in breeding programs for VNN resistance is questionable. The estimate of heritability for VNN mortality indicates the feasibility of selective breeding to enhance resistance to NNV and raises attention to the development of genomic prediction tools to simplify testing procedures for selection candidates.

Topics: Animals; Antibodies, Viral; Bass; Body Weight; Disease Resistance; Fish Diseases; Hydrocortisone; RNA Virus Infections

During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome β-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1β, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity; NF-kappa B; Nodaviridae; Phylogeny; Proteasome Endopeptidase Complex; RNA Virus Infections; Sequence Alignment; Signal Transduction

Viral nervous necrosis (VNN), caused by betanodavirus, is a significant viral infection that threatens marine aquaculture. Freshwater and marine fish farms in Turkey are subjected to annual pathogen screenings. In 2016, during the Nervous Necrosis Virus screening program conducted in the Black Sea, betanodavirus was unexpectedly detected using real-time reverse transcription-polymerase chain reaction in apparently healthy sea bass. Phylogenetic analysis of both the RNA1 and RNA2 segments of the virus determined that the betanodavirus detected was red-spotted grouper nervous necrosis virus genotype (RGNNV). Following the initial discovery of betanodavirus in the Black Sea, monitoring studies performed over a 3 yr period have not indicated any additional presence of the virus. The absence of clinical symptoms related to VNN disease in the area's marine fish farms and the surrounding detection zone, and the fact that the virus has not been detected anew in monitoring programmes conducted following the initial detection, indicate that there is no virus circulation in the detection zone.

Topics: Animals; Bass; Black Sea; Fish Diseases; Genotype; Nodaviridae; Phylogeny; RNA Virus Infections; Turkey

Interleukin-8 (IL-8, CXCL8), a pro-inflammatory chemokine secreted by a variety of cell types, plays a critical role in the development of various immune diseases. Interactions between IL-8 and its receptor CXC receptor 1/2 (CXCR1/2) are known to promote chemotaxis and phagocytosis in many immune responses. In this study, we report the molecular characteristics and pharmacological activity of CXCR1 (MsCXCR1) in largemouth bass (Micropterus salmoides) and evaluated the functional involvement of MsCXCR1 in individuals infected with the pathogen Nocardia seriolae. MsCXCR1 was cloned into the pEGFP-N1 plasmid and the subcellular localization of MsCXCR1 on the cell membrane was verified in MsCXCR1-EGFP-expressing HEK293 cells. Following observation of receptor internalization and intracellular signaling detection, we further determined the functional interaction of secreted interleukin-8 (LcIL-8, the ligand for CXCR1 in large yellow croaker) and MsCXCR1 was further determined, and the ERK phosphorylation signal activation mediated by MsCXCR1 was demonstrated. Quantitative real-time PCR assays were conducted to analyze the transcriptional distribution of MsCXCR1 in various tissues of healthy and diseased largemouth bass. These results illustrate the significant elevation of MsCXCR1 expression in the head kidney, spleen and liver of M. salmoides, suggesting that MsCXCR1 was involved in the immune response in N. seriolae-infected largemouth bass and potentially affects the digestive function of this species.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Endocytosis; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fish Diseases; Green Fluorescent Proteins; HEK293 Cells; Humans; Interleukin-8; Nocardia; Nocardia Infections; Phosphorylation; Phylogeny; Receptors, Interleukin-8A; Transcription, Genetic

Mycobacteriosis occurs with high prevalence in the wild striped bass Morone saxatilis of Chesapeake Bay, USA. Etiologic agents of mycobacteriosis in this system are dominated by Mycobacterium pseudoshottsii and Mycobacterium shottsii, both members of the M. ulcerans/M. marinum clade of mycobacteria. Striped bass occupying Chesapeake Bay during summer months where water temperatures regularly approach and occasionally exceed 30°C are thought to be near their thermal maximum, a condition hypothesized to drive high levels of disease and increased natural mortality due to temperature stress. M. shottsii and M. pseudoshottsii, however, do not grow or grow inconsistently at 30°C on artificial medium, potentially countering this hypothesis. In this work, we examine the effects of temperature (20, 25, and 30°C) on progression of experimental infections with M. shottsii and M. pseudoshottsii in striped bass. Rather than exacerbation of disease, increasing temperature resulted in attenuated bacterial density increase in the spleen and reduced pathology in the spleen and mesenteries of M. pseudoshottsii infected fish, and reduced bacterial densities in the spleen of M. shottsii infected fish. These findings indicate that M. pseudoshottsii and M. shottsii infections in Chesapeake Bay striped bass may be limited by the thermal tolerance of these mycobacteria, and that maximal disease progression may in fact occur at lower water temperatures.

Topics: Animals; Bass; Fish Diseases; Mycobacterium; Temperature

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Perciformes; Phylogeny; Ranavirus; Sequence Alignment; TNF Receptor-Associated Factor 5

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry.

Topics: Animals; Antiviral Agents; Aquaculture; Bass; Brain Diseases; Disease Resistance; Fish Diseases; Injections, Intramuscular; Nodaviridae; Peptides; Proteolipids; Retinal Diseases; RNA Virus Infections; Survival Rate

Topics: Animals; Aphanomyces; Bass; Fish Diseases; Infections; Rivers; West Virginia

The orange-spotted grouper (Epinephelus coioides) is an important marine farmed fish in China. It is affected by the bacterial pathogen Vibrio alginolyticus, which causes high mortality and substantial economic losses. We studied the transcriptional changes of the IgZ gene in E. coioides following V. alginolyticus stimulation and investigated the distribution of IgZ in different tissues. The highest expression level of IgZ occurred in the head kidney. When fish were stimulated with live and inactivated V. alginolyticus, the expression levels of IgZ in the head kidney, spleen, intestine, gills and blood cells were significantly upregulated. In an in situ hybridization study, IgZ mRNA-positive cells were detected in the head kidney, spleen and gill, but positive signals were not detected in the liver and intestine. IgZ-labelled cells increased in the head kidney, spleen and gills post-infection with V. alginolyticus for 21 days. The present study provides additional evidence that IgZ is involved in mucosal immune responses and helps explain the role of IgZ in E. coioides defence against V. alginolyticus infection.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Immunoglobulin Heavy Chains; Random Allocation; Vaccination; Vibrio alginolyticus; Vibrio Infections

Nervous necrosis virus (NNV) belongs to the

Topics: Animals; Bass; Capsid Proteins; Cell Nucleus; Fish Diseases; Nodaviridae; Poly(A)-Binding Proteins; Protein Biosynthesis; Protein Transport; RNA Virus Infections; RNA-Dependent RNA Polymerase

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Topics: Animals; Bass; Fish Diseases; Macrophages; Mesencephalon; Microglia; Neurons; Nodaviridae; RNA Virus Infections; RNA-Seq

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; RNA Virus Infections; TNF Receptor-Associated Factor 4; Virus Replication

Topics: Animals; Antiviral Agents; Bacteria; Bass; Cell Line; Cell Membrane; Cell Surface Display Techniques; Cell Survival; Fish Diseases; Fish Proteins; Myxovirus Resistance Proteins; Nodaviridae; Recombinant Proteins; RNA Virus Infections; Salinity; Virus Attachment

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Pseudomonas; Pseudomonas Infections; RNA Interference; RNA-Seq; Transcriptome; Virulence

Loma psittaca, previously described as inhabiting the intestinal mucosa of an anadromous fish, Colomesus pisttacus, from the Amazon Basin, is reported as being found for the first time in a marine fish, the hybrid grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀), from Lingshui city, Hainan Province, China, expanding the geographical distribution and host range of this parasite. Numerous whitish xenomas (0.5-0.7 mm in diameter) of this new isolate of L. psittaca were found distinctly in the muscle layer of the host stomach wall. Electron microscopic observations showed a monokaryotic nucleus in all developmental stages. Round or elongated multinucleate merogonial plasmodia surrounded by numerous mitochondria were observed initially, subsequently transforming into uninucleate sporonts through multiple fissions. Sporonts, each with a large centrally positioned nucleus, further developed into sporoblasts. Each sporoblast mother cell gave rise to two uninucleate sporoblasts by binary fission. Mature spores were ellipsoidal, measuring 4.0 ± 0.3 (3.7-4.3) μm in length and 2.2 ± 0.2 (2.1-2.5) μm in width. Spores possessed a mushroom-like anchoring disk, a bipartite polarplast, isofilar polar filaments arranged in 12-14 turns in one row, and a trilaminar spore wall. The obtained partial SSU rRNA gene sequence of the new isolate was 1330 bp in length and showed 99.4% sequence similarity with an estuary isolate of L. psittaca previously reported in South America. SSU rRNA gene-based phylogenetic analyses demonstrated that the two L. psittaca isolates first clustered together and then formed a dichotomy that included the digestive-tract-infecting Loma species, L. acerinae, with high support values within group I.

Topics: Animals; Bass; Fish Diseases; Loma; Microsporidia; Microsporidiosis; Phylogeny; Stomach

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26 °C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.

Topics: Animals; Bass; Brain; Cell Line; Edwardsiella tarda; Fish Diseases; Salmon; Toxicology

Antimicrobial peptides (AMPs) are considered to be amongst the most powerful tools for the fight against pathogens in fish, since they form part of the innate immune response, which is especially vital in eggs and early larval stages, when the immune system is developing. The fish responsible for a large part of the profits in Mediterranean aquaculture is European sea bass (Dicentrarchus labrax), a species greatly susceptible to nodavirus (NNV), especially in the larval and juvenile stages. In this work, polyclonal antibodies were developed and used to detect and quantify NK-lysin, dicentracin and hepcidin AMPs in European sea bass eggs and during larval development, as well as to evaluate their regulation in juvenile specimens upon NNV infection. Basal and detectable levels of all the AMPs studied were present in eggs, confirming the maternal transfer of peptides, which increased in one or two waves during larval development up to 69 days post-fertilization. After NNV infection, the mRNA of all the AMPs analysed was up-regulated five days after infection in most of the tissues, whilst peptide quantification of all three AMPs decreased in the brain, the target tissue for NNV, but increased in the head-kidney 5 days after infection. Further research should be carried out to ascertain the role of AMPs in fish innate immunity and to understand how NNV evades the immune response to be disseminated.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Fish Proteins; Hepcidins; Immunity, Innate; Nodaviridae; Proteolipids; RNA Virus Infections

Polyunsaturated fatty acids (PUFAs) play important roles in organisms, including the structure and liquidity of cell membranes, anti-oxidation and anti-inflammation. Very little has been done in terms of the effect of PUFAs on cell death, especially on DNA virus. In this study, we demonstrated that the infectious spleen and kidney necrosis virus (ISKNV) can induce host cell death via the apoptotic cell death pathway, which correlated to modulation by PUFAs in grouper fin cell line (GF-1) cells. We screened the PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for the ability of different dosages to prevent cell death in GF-1 cells with ISKNV infection. In the results, each 10 μM of DHA and EPA treatment enhanced host cell viability up to 80% at day 5 post-infection. Then, in Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, DHA- and EPA-treated groups reduced TUNEL positive signals 50% in GF-1 cells with ISKNV infection. Then, through studies of the mechanism of cell death, we found that ISKNV can induce both the Bax/caspase-3 and Fas/caspase-8/tBid death signaling pathways in GF-1 cells, especially at day 5 post-infection. Furthermore, we found that DHA and EPA treatment can either prevent caspase-3 activation on 17-kDa form cleavage or Bid cleaved (15-kDa form) for activation by caspase-8, apparently. On the other hand, the anti-apoptotic gene Bcl-2 was upregulated 0.3-fold and 0.15-fold at day 3 and day 5, respectively, compared to ISKNV-infected and DHA-treated cells; that this did not happen in the EPA-treated group showed that different PUFAs trigger different signals. Finally, ISKNV-infected GF-1 cells treated with either DHA or EPA showed a 5-fold difference in viral titer at day 5. Taken together, these results suggest that optimal PUFA treatment can affect cell death signaling through both the intrinsic and extrinsic death pathways, reducing viral expression and viral titer in GF-1 cells. This finding may provide insight in DNA virus infection and control.

Topics: Animal Fins; Animals; Apoptosis; Bass; Cell Death; Cell Line; DNA Virus Infections; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fish Diseases; Iridoviridae; Signal Transduction

An 8-week feeding trial was conducted to investigate the effects of partial replacement of fish meal by soy protein concentrate (SPC) on the growth performance, immune responses, intestine morphology and relation gene expression of intestinal inflammation for juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) (initial weight 12.5 ± 0.00 g). Eight isonitrogenous and isolipidic diets (48.61% protein and 11.17% lipid) were formulated by replacing 0% (the control), 11%, 22%, 33%, 44%, 55%, 66%, and 77% of fish meal (FM) with SPC, respectively (the eight dietary be named FM, S11, S22, S33, S44, S55, S66, and S77, respectively). With the replacement level increased, the final body weight, weight gain ratio (WGR), specific growth rate (SGR), and survival rate of fish were significantly decreased (P < 0.05) compared with the group FM. By contrast, the feed conversion ratio (FCR) of fish was significantly increased (P < 0.05) when the replacement level up to 44%. Partial FM replacement by SPC (ranging from 11% to 77%) substantially reduced (P < 0.05) the serum total protein, albumin, and total cholesterol contents compared with the group FM. Liver total superoxide dismutase, glutathione peroxidase, catalase activities, and total antioxidant capacity showed the same trend of gradual increase first and then decrease. Their highest values were found in the replacement levels of 55%, 33%, 22%, and 55% and were significantly higher (P < 0.05) than the control group. The lowest malondialdehyde content was observed in group S77 and was significantly lower (P < 0.05) than that of the control group. The complements C3 and C4 contents of fish fed with experimental diets (replacement level ranged from 11% to 66%) were significantly higher (P < 0.05) than the group FM. The liver lysozyme activity of the control group was the lowest and was significantly lower than that of other dietary treatments (P < 0.05). Villus length and muscle thickness in the intestine of fish were significantly lower (P < 0.05) than other groups when the replacement level exceeded 44%. With dietary replacement levels increased, the TLR22, MyD88, p65, pro-inflammatory cytokines (IL-1β, TNF-α, IL-12P40 and INF-γ) and anti-inflammatory cytokines (TGF-β, IL-10, epinecidin, MHCIIβ and hepcidin) mRNA levels in the proximal intestine were significantly up-regulated (P < 0.05). The TLR22, MyD88, p65, pro-inflammatory cytokines (IL-1β, TNF-α, IL-12P40 and INF-γ) and anti-inflamma

Topics: Animal Feed; Animals; Bass; Diet; Dose-Response Relationship, Drug; Fish Diseases; Gastrointestinal Tract; Gene Expression; Immunity, Innate; Inflammation; Intestines; Soybean Proteins

Autophagy is an evolutionarily conserved cellular degradation process that is essential for homeostasis. As a cell steward, autophagy is thought to be a process that may have evolved to combat intracellular pathogens. However, some virus can subvert or utilize autophagy-related membrane structures to increase viral replication. The red-spotted grouper nervous necrosis virus (RGNNV) is a fish pathogen which leads to disastrous viral nervous necrosis in larvae and juvenile groupers and other marine fishes. To better comprehend the pathogenesis and replication mechanism of RGNNV, we investigated the relationship between RGNNV and autophagy. Here, we demonstrated that RGNNV induced autophagy in grouper spleen (GS) cells, as the significant increase in ultrastructural autophagosome-like vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3 (LC3), and the conversion of LC3-I to LC3-II. Additionally, ultraviolet-inactivated RGNNV and the capsid protein also triggered autophagy. Enhancement of autophagy contributed to RGNNV replication, whereas blocked autophagy decreased RGNNV replication. Moreover, impeded fusion of autophagosomes and lysosomes also reduced RGNNV replication, indicating that RGNNV utilized the different steps of autophagy pathway to facilitate viral replication. The further study showed that RGNNV induced autophagy through activating the phosphorylation of eIF2α and inhibiting the phosphorylation of mTOR. These results will assist the search for novel drugs targets and vaccine design against RGNNV from the perspective of downregulating autophagy.

Topics: Animals; Autophagy; Bass; Fish Diseases; Nodaviridae; RNA Virus Infections; Virus Replication

Bacterial pathogen-host interactions are highly dynamic, regulated processes that have been primarily investigated using in vitro assays. The dynamics of bacterial pathogen-host interplay in vivo are poorly understood. Using time-resolved dual RNA-seq in a Pseudomonas plecoglossicida-Epinephelus coioides infection model, we observed that bacterial genes encoding classical virulence factors and host genes involved in immune regulation were dynamically expressed during infection. Using network inferencing, we were able to predict interspecies regulatory networks linking bacterial virulence genes to host immune genes. Together with gene co-expression network analysis of the pathogen, secY was predicted to be a key virulence gene for P. plecoglossicida pathogenicity in the host, fliN was predicted to be a less important virulence gene. The results of bioinformatics prediction were confirmed by animal infection experiments. Our work provides the first paradigm to study dynamic alterations of bacterial pathogen and host interactions based on the elucidation of time-resolved interactive transcriptomes in vivo, and may be developed into a novel and universal method for revealing the true complexity of the bacterial infection process.

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Host-Pathogen Interactions; Immunity, Innate; Pseudomonas; Pseudomonas Infections; RNA-Seq; SEC Translocation Channels; Transcriptome; Virulence; Virulence Factors

This study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) on growth performance, non-specific immunity, expression of some immune-related genes and resistance to Vibrio harveyi in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu). Six isoproteic and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.65, 1.00, 1.35, 1.70, 2.05 and 2.40% of dry matter, respectively), and the 0.65% group was used as control group. Each diet was randomly allocated to triplicate groups of fish in 1000 L fiberglass tank, and each tank was stocked with 40 fish (initial weight 12.06 ± 0.01 g) for 8 weeks. Results showed that feed conversion ratio (FCR), survival rate (SR), hepatosomatic index (HSI) and condition factor (CF) were all not significantly affected by dietary n-3 HUFA levels (P > 0.05). Weight gain (WG) and specific growth rate (SGR) in 1.35% group were significantly higher than those in 2.40% group (P < 0.05). Crude lipid of body in 1.00% group was significantly lower than that in 1.70% and 2.40% groups (P < 0.05). Liver and muscle fatty acid profiles reflected that of diets. Before challenge with Vibrio harveyi, the activity of serum superoxide dismutase (SOD), catalase (CAT) and content of complement 3 (C3) in 1.35% and 1.70% groups significantly higher than those of control group (P < 0.05). After challenge with Vibrio harveyi, serum CAT, glutathione peroxidase (GSH-PX), lysozyme (LZM) and C3 all increased sharply, while SOD showed the opposite trend. Before challenge with Vibrio harveyi, the expression levels of intestine toll-like receptor 22 (TLR22) and myeloid differentiation factor 88 (MyD88) mRNA in 2.40% group were significantly increased, and the expression levels of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) mRNA in 2.05% group were significantly higher than those in 1.00% and 1.35% groups (P < 0.05). In addition, the TLR22 and IL-1β mRNA levels in kidney of 1.70% group were significantly lower than those in control group (P < 0.05). After challenge with Vibrio harveyi, the expression level of MyD88 mRNA in intestine of 1.35% group was significantly higher than that in 1.00% group and from 1.70% to 2.40% groups (P < 0.05), while TNF-α and IL-1β obtained minimum values in 1.70% group. In the kidney, the interleukin 10 (IL10) mRNA expression was significantly higher in 1.70% group than that in other groups, while the IL-1β expressio

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Fish Diseases; Gene Expression; Immunity, Innate; Random Allocation; Vibrio; Vibrio Infections

Heat shock protein 22 (Hsp22) is an important regulatory factor response to various stresses in mammals. In this study, the full length cDNA of Epinephelus coioides Hsp22, which was 1680bp in length, with a 289 bp 5' UTR, a 725 bp 3'UTR, and a 666 bp open reading frame encoding 221 amino acids, was obtained. E. coioides Hsp22 contains a highly conserved α-crystallin domain. E. coioides Hsp22 mRNA was detected in all tissues examined by quantitative real-time PCR, with the highest expression in blood, followed by the spleen, skin, gill, head kidney, muscle, heart, liver, trunk kidney, stomach, pyloric caeca, intestine, brain and thymus. The expression patterns of E. coioides Hsp22 response to infection with Singapore grouper iridovirus (SGIV) and Vribro alginolyticus, the important pathogens of E. coioides, were studied. The expression levels of the gene were up-regulated in the tissues examined. Subcellular localization analysis demonstrated that E. coioides Hsp22 was distributed in both the cytoplasm and nucleus. In addition, E. coioides Hsp22 significantly inhibited the SGIV-induced cell apoptosis. In summary, the E. coioides Hsp22 might play a critical role in pathogenic stimulation.

Topics: Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression; Heat-Shock Proteins; Iridovirus; Vibrio alginolyticus; Vibrio Infections; Virus Diseases

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immune System; Phylogeny; Proto-Oncogene Proteins c-raf; Ranavirus; Sequence Alignment

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. Caspase-1 is a member of inflammatory caspases that play important roles in the innate immune system. However, few studies have been performed in lower vertebrates such as teleosts and implications of extracellular ATP-mediated immune signalling in fish. Here we identified and characterized novel ASC and caspase-1 genes (namely EcASC and EcCaspase-1) from the orange-spotted grouper (Epinephelus coioides). EcASC and EcCaspase-1 encode 204- and 388-aa proteins which shared 55.34% and 72.89% identity with those in Siniperca chuatsi and Perca flavescens, respectively. EcASC contained a PYRIN domain (aa 5-82) and CARD domain (aa 107-201). EcCaspase1 contained a CARD domain (aa 1-88) and a CASc domain (aa 127-376). Both EcASC and EcCaspase-1 were distributed in all tissues tested in the healthy grouper. The expression of EcASC and EcCaspase-1 was significantly upregulated in response to ATP infection. Subcellular localization analysis showed that EcCaspase-1 exhibited a clear distribution in both cytoplasm and nucleus. In contrast, EcASC was observed in the cytoplasm as speck-like structures, which are called "pyroptosomes". EcCaspase-1 co-localized with the spot-like protein (EcASC). Overexpression of EcASC and EcCaspase-1 inhibited NF-κB activation and promoted P53 activation in grouper spleen (GS) cells. Extracellular ATP was an effective signaling molecule that activates the innate immune response, rapidly upregulating the expression of EcASC and EcCaspase1, and enhancing their promotion of proinflammatory cytokine expression in GS cells. Both EcASC and EcCaspase-1 promoted ATP-induced apoptosis. Our results suggested that the interactions of inflammatory EcCaspase-1 with EcASC proteins were associated with extracellular ATP-mediated immune signaling in fish.

Topics: Adenosine Triphosphate; Animals; Bass; CARD Signaling Adaptor Proteins; Caspase 1; Cloning, Molecular; Fish Diseases; Gene Expression Regulation; Immunity, Innate; Phylogeny; Signal Transduction

Lernaeenicus radiatus, a mesoparasitic pennellid copepod, has long been known in the northwest Atlantic with metamorphosed females infecting the muscle of marine fish. The study herein is the first to identify a definitive first host, black sea bass Centropristis striata, for L. radiatus supporting larval development to adults and sexual reproduction in the gills. This finding suggests a two-host life cycle for L. radiatus, with black sea bass as the first host. Heavy infections in the gill were associated with considerable pathology related to a unique and invasive attachment process that penetrated the gill and selectively attached to the gill filament cartilage. The morphology of the developing copepod was highly conserved with that of a related pennellid copepod, Lernaeocera branchialis, though was distinguished by the attachment process, unique pigmentation and other morphologic features described herein. Sequencing the small and large subunits of the ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes demonstrated L. radiatus to share closer identities with Lernaeocera and Haemobaphes spp. pennellid copepods rather than other Lernaeenicus spp. available in GenBank to date. Taxonomy of L. radiatus is discussed in relation to life cycles, tissue tropism, morphology and genetics of other closely related pennellid copepods.

Topics: Animals; Bass; Copepoda; Female; Fish Diseases; Gills; Host-Parasite Interactions; Male; New Jersey; Parasitic Diseases, Animal

Interferon regulatory factors (IRFs) are a family of transcription factors essential to the control of antiviral immune response, cell growth, differentiation and apoptosis. IRF10 was originally found in chicken, which was induced by the v-Rel oncoprotein in lymphoid cell lines and involved in the upregulation of major histocompatibility complex (MHC) class I and guanylate-binding protein. In fish, IRF10 plays negative roles in regulation of the interferon (IFN) response. Here, we identified a splice variant of IRF10, named as EcIRF10-SF in orange spotted grouper, which shares the first three exons with the long form (EcIRF10-LF) and retains part of intron 3, creating a premature termination codon. Furthermore, we observed that the EcIRF10-SF exhibits similar expression pattern compared to its native counterparts. Functional studies demonstrate that the two EcIRF10 isoforms repress DrIFNϕ1 and DrIFNϕ3 promoter activity and negatively regulate fish antiviral gene expression. Subcellular localization analysis shows that the amino acids from 57 to 86 within DBD are required for IRF10 nuclear import. Overall, our description of transcript diversification of IRF10 in the grouper provides a coherent framework to further dissect its roles in immune response.

Topics: Active Transport, Cell Nucleus; Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Immunity, Innate; Interferon Regulatory Factors; Phylogeny; Promoter Regions, Genetic; Protein Isoforms

Vibrio harveyi is the pathogen causing vibriosis in marine-cultured animals, leading to massive deaths in farmed grouper around the world. It is urgent to develop an effective vaccine to prevent vibriosis. In the previous study, we developed a V. harveyi formalin-killed cells vaccine (FKC), and sought an effective adjuvant for enhancing the immune efficacy of vaccine. In this study, we aimed to evaluate the immune responses and protective effect of FKC combined with chitosan oligosaccharide (COS) or Astragalus polysaccharides (APS) in the pearl gentian grouper♀Epinephelus fuscoguttatus × ♂E. lanceolatus. The results indicated the vaccine triggered a remarkably higher expression levels of IL-1β, IL-16, TNF-α, MHC-Iα and IgM in the kidney and spleen of groupers post-vaccination. Antibody titers, lysozyme, catalase, superoxide dismutase and total protein were significantly elevated in the vaccinated fish compared with those in the control. The experimental groupers were challenged intraperitoneally by V. harveyi at 35 d post-vaccination, and the relative percentage of survival (RPS) of group FKC + COS, FKC + APS, COS, APS and FKC were 80%, 72%, 52%, 47% and 55%, respectively. These results demonstrated COS and APS was the potential adjuvants for FKC against V. harveyi in aquaculture.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Aquaculture; Astragalus Plant; Bacterial Vaccines; Bass; Chitosan; Cytokines; Fish Diseases; Formaldehyde; Kidney; Oligosaccharides; Polysaccharides; Spleen; Vaccination; Vaccines, Inactivated; Vibrio; Vibrio Infections

Skin abrasions often occur in farmed fish following handling by labourers, injury by farm facilities, cannibalism and ectoparasites. Vibrio spp. are opportunistic pathogens that can invade host fish through damaged tissues and cause outbreaks of vibriosis. This study describes the effect of skin abrasions on the infectivity of V. harveyi using Asian seabass Lates calcarifer (Bloch, 1790) fingerlings as a case example and compares bacterial load and fish survival following immersion challenge with different doses. In total, 315 fish (6.67 ± 1.8 g) were divided into 3 treatments: skin abrasion followed by immersion infection, immersion infection only and an uninfected, uninjured control. Fish in the infection treatments were divided into 3 subgroups and exposed in triplicate to a 7 d immersion challenge with 106, 107 and 108 CFU ml-1 of live V. harveyi. No mortalities were observed in the control and immersion infection groups. However, fish in the skin abrasion treatment group that were infected with 108 CFU ml-1 of live V. harveyi showed signs of progressing disease throughout the experiment, which resulted in mortalities. Significantly higher bacterial loads (p < 0.05) were recorded in the intestine, liver and gills of the fish in this group. Fish in the skin abrasion treatment that were exposed to 107 and 108 CFU ml-1 of V. harveyi showed 100% mortality by Days 5 and 4, respectively. These findings confirm that skin injuries increase the susceptibility of seabass fingerlings to V. harveyi infection.

Topics: Animals; Bass; Fish Diseases; Perciformes; Vibrio; Vibrio Infections

Grouper (Epinephelus coioides) is an important commercial maricultural fish, which suffers from nervous necrosis virus (NNV) infection. The molecular mechanisms underlying the pathogenesis of the viral infection are not clear. In this study, we combined deep RNA sequencing and label-free mass spectrum for the first time to analyze the transcriptomic and proteomic profiles in infected/dead, infected/survival (persistent), and infection-free (control)orange-spotted groupers in the larval stage. Further analyses showed that the transcriptome and proteome changed dramatically among the three distinct groups, especially differentially-expressed genes in the infected/dead and infected/survival larvae enriched for pathways related to immune response. Notably, the overlapped genes between transcriptomes and proteomes identified that genes related to collagen synthesis and adhesion molecules were enhanced in the persistent (infected/survival) stage, which might contribute to suppressing the acute and lethal immune responses upon NNV infection. These transcriptomic and proteomic datasets enable the investigation of molecular mechanisms underlying NNV infection, thus may help further development of molecular breeding in marine fishery.

Topics: Animals; Bass; Cell Adhesion Molecules; Collagen; Fish Diseases; Fish Proteins; Nodaviridae; Proteome; RNA Virus Infections; Transcriptome

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of EcKLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Iridovirus; Kruppel-Like Factor 6; PPAR delta; Ranavirus

During recent decades, survey studies have documented the widespread presence of oocytes in the testes of male Smallmouth Bass Micropterus dolomieu collected from surface waters throughout the United States. There are few published reports of testicular oocytes (TO) in Smallmouth Bass before the 1990s, so it is difficult to know how long this has been occurring. Consequently, this study was conducted to evaluate the prevalence and severity of TO occurrence in whole fish specimens from two archival collections-the Smithsonian Institution's National Museum of Natural History in Suitland, Maryland, and Cornell University's Museum of Vertebrates in Ithaca, New York. Gonads were excised from 167 preserved male Smallmouth Bass that were originally collected between 1875 and 2004, and routine histologic sections were prepared and examined. The severity of TO was determined using a semiquantitative scoring system. Overall, 52.1% of male Smallmouth Bass were found to have TO. Affected fish had been collected in 11 of the 18 represented states, and TO were found in specimens harvested during decades as early as the 1880s and 1900s. Unfortunately, the small number of samples acquired at the earliest time periods precluded analyses of prevalence and severity trends over time. The results of this study demonstrated that the phenomenon of TO in male Smallmouth Bass is at least a century old and confirmed the widespread nature of this finding throughout the species' historic range. Further research efforts should focus on determining the baseline prevalence of TO in laboratory-reared male Smallmouth Bass that have not been exposed to endocrine active substances or the effects of experimental estrogen exposure on such fish.

Topics: Animals; Bass; Fish Diseases; Male; Oocytes; Prevalence; Testicular Diseases; Testis

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2

Topics: Animals; Aquaculture; Bass; Fish Diseases; Fishes; Flavobacteriaceae; Flavobacteriaceae Infections; Genes, Bacterial; Genome, Bacterial; Microbiota; Perciformes; Phenotype; Phylogeny; RNA, Ribosomal, 16S; Tenacibaculum

Fish NK-lysin (NKL), an orthologous to human granulysin, exerts a dual role as an antimicrobial peptide (AMP) and as a direct executor of T cytotoxic and natural killer cells during the cell-mediated cytotoxic (CMC) response. Although its best-known function is as AMP against bacteria, recent studies point to a special role of NKL in antiviral responses. Nodavirus (NNV) is a spreading threat in Mediterranean aquaculture. In this study, we have identified and compared the expression pattern of European sea bass and gilthead seabream NKL and evaluated its transcription in different tissues and its regulation in head-kidney leucocyte (HKLs) stimulated in vitro with different immunostimulants, under CMC response and upon an in vivo infection with NNV. Our results showed that nkl transcription is highly expressed in spleen, thymus and skin with species-specific differences. Interestingly, the expression pattern in both species was very different upon treatment. While sea bass nkl transcription was increased in HKLs by the T mitogen phytohemagglutinin all the stimulators inhibited it in seabream HKLs. Similar results occurred in NNV-infected fish where the transcription was increased in sea bass tissues and down-regulated in seabream. Curiously, during CMC assays, nkl transcription was significantly increased in seabream HKLs against NNV-infected fish cell lines but this was not observed in sea bass leucocytes. The potential role of NKL as CMC effector molecule or as AMP in fish will be discussed.

Topics: Animals; Antimicrobial Cationic Peptides; Aquaculture; Bass; Cell Line; Fish Diseases; Fish Proteins; Gene Expression; Head Kidney; Immunity, Innate; Nodaviridae; Proteolipids; RNA Virus Infections; Sea Bream

Micropterus Salmoides rhabdovirus (MSRV), as a common aquatic animal virus, can cause lethal and epidemic diseases in the cultivation of largemouth bass. In this study, we reported a kind of immersion single-walled carbon nanotubes-loaded subunit vaccine which composited by glycoprotein (G) of MSRV, and evaluated its protective effect on largemouth bass. The results showed that a stronger immune response including serum antibody levels, enzyme activities (superoxide dismutase, acid phosphatase, alkaline phosphatase and total antioxidant capacity), complement C3 content and immune-related genes (IgM, TGF-β, IL-1β, IL-8, TNF-α, CD4) expression can be induced obviously with single-walled carbon nanotubes-glycoprotein (SWCNTs-G) groups compared with G groups when largemouth bass were vaccinated. After bath immunization with G or SWCNTs-G for 28 days, fish were challenged with a lethal dose of MSRV. The survival rates for control group (PBS), SWCNTs group (40 mg L

Topics: Animals; Antibodies, Viral; Bass; Fish Diseases; Immersion; Immunization; Nanotubes, Carbon; Rhabdoviridae; Rhabdoviridae Infections; Vaccines, Subunit; Viral Vaccines

Nocardia seriolae has become one of the major pathogens affecting the aquaculture industry and causes Nocardiosis, a highly devastating disease of marine and freshwater fish that leads to severe economic losses. Therefore, research efforts towards developing efficacious vaccines to control this disease are of high importance. In this study, the hypoxic response protein 1 (HRP1) cloned into pET32a vector was expressed, and produced in Escherichia coli strain BL21 (DE3). The antigenicity of purified recombinant TRX-tagged HRP (rHRP1) was analysed by western blotting using largemouth bass anti-N. seriolae sera. The results showed that largemouth bass anti-N. seriolae sera could specifically detect a 33 kDa rHRP1 protein. Further, the vaccine efficacy of rHRP1 was evaluated in a largemouth bass fish model by calculating the relative percent survival (RPS). rHRP1 incurred an RPS of 73.33% as compared to the control group. Immunological analysis showed that rHRP1 could produce significantly higher serum concentrations of anti-N. seriolae antibodies and serum lysozyme activity as compared to the control groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that rHRP1 significantly enhanced the expression of immune-related genes, such as IL-12p40, IL-8, IL-1β, TNFα, IFNγ, NKEF, MHCIα, MHCIIα, CD4-1, CD8α, IgM, NF-κβ, STAT3, IRF4, RORα, and CCL20. These results indicate that rHRP1 may be a promising vaccine candidate against nocardiosis.

Topics: Animals; Antigens, Bacterial; Antigens, CD; Bacterial Vaccines; Bass; Cytokines; Fish Diseases; Nocardia; Nocardia Infections; Recombinant Proteins

Largemouth bass ulcerative syndrome virus (LBUSV) is an important virus induce the mortality of largemouth bass (Micropterus Salmoides). In this study, we reported a single-walled carbon nanotubes (SWCNTs) containing LBUSV major capsid protein (MCP) subunit vaccine (SWCNTs-MCP) which was evaluated for its protective effect on largemouth bass by immersion immunization. We found an elevation in serum antibody levels, enzyme activities, complement C3 content and immune-related genes (IgM, TGF-β, IL-1β, IL-8, TNF-α and CD4) expression, in the SWCNTs-MCP immunized groups compared with the pure MCP group. The survival rates for control group, pure MCP protein groups (40 mg L

Topics: Animals; Antibodies, Viral; Bass; DNA Virus Infections; DNA Viruses; Fish Diseases; Immersion; Immunization; Nanotubes, Carbon; Vaccines, Subunit; Vaccines, Synthetic; Viral Vaccines

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKβ-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cytokines; Fish Diseases; Fish Proteins; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; I-kappa B Kinase; Immunity, Innate; NF-kappa B; Pathogen-Associated Molecular Pattern Molecules; Phylogeny; Sequence Alignment; Vibrio Infections; Vibrio parahaemolyticus

Topics: Animals; Anti-Bacterial Agents; Bass; Enrofloxacin; Fish Diseases; Inflammation; Injections, Intramuscular; Time Factors

This study was conducted to examine the combinatory effects of β-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with β-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary β-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g β-glucan per kg feed weight, groups 3-5 received 5 g/kg β-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with β-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary β-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

Topics: Animal Feed; Animals; Bass; beta-Glucans; Chimera; Dietary Supplements; Fish Diseases; Head Kidney; Immunity, Innate; Immunosuppressive Agents; Leukocytes; Oxytetracycline; Phagocytosis; Vibrio alginolyticus; Vibrio Infections

Interferons play an important role in the fish innate immune system against viral infection by inducing the interferon stimulated genes, such as Mx gene. We cloned the MxII gene promoter from orange-spotted grouper and found three MxII gene promoters. All of them contained two interferon stimulated response elements (ISREs), and three dinucleotide repeat sequences located at 5' end of ISREs. Interestingly, there is a polymorphic GT repeat element located upstream of these ISREs. The three MxII gene promoters respectively contained 27, 29, and 31 GT repeats, namely EcMx_27, EcMx_29, and EcMx_31. To determine whether GT repeat element influence the MxII gene expression, the MxII gene promoters were subcloned into promoterless reporter plasmid and transfected into grouper kidney (GK) cells. The results showed that a significant induction by poly(I:C) was detected in GK cells transfected with pEcMx_31 (2.65 folds) whereas there was no induction in GK cells transfected with pEcMx_27 and pEcMx_29. However, the significant induction by nervous necrosis virus (NNV) infection was found in GK cells separately transfected with three reporter plasmids. These results suggest that the GT repeat element plays an important role in modulation of MxII gene expression and the induction by poly(I:C) and NNV may be mediated through different signal transduction pathways.

Topics: Animals; Bass; Fish Diseases; Kidney; Nodaviridae; Poly I-C; Polymorphism, Genetic; Promoter Regions, Genetic; RNA Virus Infections

The transcription factor nuclear factor kappa B (NF-κB) is a critical regulator of immune and inflammatory responses with crucial roles in various pathophysiologic conditions involving cell survival and death. Recent studies in mammals showed that NF-κB was also involved in peroxisome proliferator-activated receptors (PPARs)-mediated immune responses However, the mechanism by which NF-κB regulates PPARδ in teleosts remains unclear. In the present study, we analyzed the potential role of NF-κB/p65 (Ecp65) in the immune response stimulated by various pathogens in the grouper Epinephelus coioides. Ecp65 expression was significantly induced soon after infection with lipopolysaccharide, nervous necrosis virus, poly(I:C), and zymosan A. We also analyzed the promoter to determine the regulatory effect of Ecp65 on PPARδ expression, using progressive EcPPARδ promoter deletion mutations. Among the five truncated mutants, the luciferase reporter activity of the PPARδ-5 promoter region was highest in response to Ecp65, indicating that the core p65-binding region was located in the PPARδ-5 promoter region (+122 bp to +383 bp). Mutation analyses indicated that the luciferase reporter activity of the EcPPARδ promoter was dramatically decreased by mutation of the M3 (+305 bp to +324 bp) and M4 (+346 bp to +365 bp) binding sites, respectively. We further confirmed that Ecp65 bound to the M3 and M4 binding sites in the 5'-untranslated region of EcPPARδ by electrophoretic mobility shift assay. Finally, overexpression of Ecp65 in vitro notably promoted the transcription of EcPPARδ, interferon-related genes, and several inflammatory cytokines. This study demonstrated that Ecp65 plays an important role in modulating the innate immune responses in groupers. These results also further our understanding of the mechanisms involved in the transcriptional regulation of PPARs by p65 in bony fish.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; NF-kappa B; Nodaviridae; Poly I-C; PPAR delta; RNA Virus Infections; Sequence Alignment; Zymosan

Topics: Animals; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Polymerase Chain Reaction; Ranavirus; Sensitivity and Specificity

IκB kinase (IKK) is the core regulator of the nuclear factor-κB (NF-κB) pathway, which is involved in cellular development and proliferation, as well as the inflammatory response. IKKα is an important subunit of the IKK complex. In this study, two IKKαs (EcIKKα-1 and -2) were characterized in E. coioides. Similar to IKKα of other species, EcIKKα-1 and -2 contained a kinase domain, a leucine zipper, a helix-loop-helix domain and a beta NF-κB essential modulator-binding domain. Sequence alignment indicated that EcIKKα-1 and -2 shared high degrees of sequence identity with IKKs from other species (about 63%-96%). EcIKKα-1 and -2 are widely expressed in all tissues, but have different expression profiles in normal groupers. Additionally, EcIKKα-1 and -2 responded rapidly to Cryptocaryon irritans infection at the local infection site (i.e., gill tissue), but there was no significant change in EcIKKα-2 expression. In GS cells, EcIKKα-1 was uniformly distributed in the cytoplasm, while EcIKKα-2 was observed uniformly both in the cytoplasm and nucleus. Both EcIKKα-1 and -2 were found to activate NF-κB, but the luciferase activity of EcIKKα-2 was twice that of EcIKKα-1. In addition, EcIKKα-1 and -2 can regulate the expression of immune-related cytokines (IL-1β, IL-6, IL-8, IL-12 [p35 subunit], and TNF-α). These findings should prove helpful to further elucidate the innate immunity function of IKKα in fish.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Cytokines; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; I-kappa B Kinase; Immunity, Innate; Phylogeny; Sequence Alignment

Effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. Paper lateral flow biosensors (LFBs) have been established as attractive tools for such analytical applications. In the present study a prototype LFB was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. Nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. The LFB was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. Brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the LFB. Formation of a red test line was indicative of nodavirus virions presence. Nodavirus visual detection was completed in short time (30 min). Key factors of the LFB development influencing the assays' detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. Therefore, the proposed LFB assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. The proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. It is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures.

Topics: Animals; Antibodies, Monoclonal; Bass; Biological Assay; Biosensing Techniques; Cell Line; Female; Fish Diseases; Gold; Metal Nanoparticles; Nodaviridae; Particle Size; Rabbits; Reproducibility of Results; Virion

We investigated the antimicrobial properties and the effects of Rheum officinale extract (ROE) on nonspecific immune parameters of orange-spotted grouper (Epinephelus coioides) in vitro and in vivo. The in vitro analysis was conducted by treating grouper primary head kidney leukocytes with various concentrations of ROE. The phagocytic rate of the leukocytes was elevated in a dose-dependent manner from 0.01 to 0.1 mg/ml, but decreased with higher concentrations of ROE (0.5 and 1.0 mg/ml). The production of reactive oxygen species (ROS) was strongly enhanced in a dose-dependent manner by treatment with ROE doses of 0.1-10.0 mg/ml. However, morphological changes (e.g., rounding and shrinkage of cells, chromatin condensation, fragmentation, and appearance of apoptotic bodies) were observed in the leukocytes after incubation with higher concentrations of ROE (1.0 and 10.0 mg/ml). A 28-day feeding trial was performed to assess the impact of dietary administration of ROE on grouper innate immunity parameters. Fish were fed with feed supplemented with 0, 0.1, 1.0, or 5.0 g ROE per kg of feed. The phagocytic activity of the animals' leukocytes was significantly elevated in all ROE-fed groups on day 1 and in groups fed with ROE at 0.1 or 1.0 g/kg on day 14. Production of ROS was substantially increased on day 1 in fish fed with ROE at 1.0 and 5.0 g/kg, but decreased steadily later on. The ability to generate ROS increased steadily until day 7 in fish fed the lowest concentration of ROE (0.1 mg/ml), but decreased thereafter. ROE showed excellent antibacterial activity against six pathogens of aquatic animals: Vibrio parahaemolyticus, V. vulnificus, V. alginolyticus, V. carchariae, Aeromonas hydrophila, and Edwardsiella tarda. The minimum inhibitory and bactericidal concentrations of measured ROE-derived anthraquinones were 10.57-84.53 μg/ml and 10.57-169.05 μg/ml, respectively.

Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bass; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Plant Extracts; Rheum; Vibrio; Vibrio Infections

Tumour necrosis factor receptor-associated factors (TRAFs) are key regulatory proteins in the NF-κB signaling pathways. TRAF2 participates in the activation of both canonical and non-canonical NF-κB pathways, which are crucial for cell inflammation and cell survival. To elucidate its function in teleost fish, TRAF2 homologues of yellow grouper (Epinephelus awoara) and golden pompano (Trachinotus ovatus) have been cloned and characterized in this study. The open reading frame (ORF) of grouper TRAF2 (EaTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.70 kDa. The ORF of golden pompano TRAF2 (ToTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.66 kDa EaTRAF2 and ToTRAF2 share 99.23% and 99.42% identity with orange-spotted grouper (Epinephelus coioides) TRAF2 (EcTRAF2), respectively. Quantitative real-time PCR analysis indicated that the expression of EaTRAF2 was increased in grouper spleen (GS) cells after Red-spotted grouper nervous necrosis virus (RGNNV) infection; while the expression of ToTRAF2 was decreased in golden pompano brain (TOGB) cells after RGNNV infection. Both EaTRAF2 and ToTRAF2 were identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. Both EaTRAF2 and ToTRAF2 enhanced RGNNV replication during viral infection in vitro. Further studies showed that EaTRAF2 and ToTRAF2 overexpression decreased the expression levels of interferon associated cytokines and pro-inflammatory factors. Taken together, these results are important for better understanding of the function of TRAF2 in fish and reveal its involvement in host response to immune challenges in RGNNV.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; RNA Virus Infections; Sequence Alignment; TNF Receptor-Associated Factor 2

Peroxisome proliferative-activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α, IFN-γ, ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; PPAR alpha; Ranavirus; RNA Virus Infections; Sequence Alignment

Grouper is known as a highly economical teleost species in the Asian aquaculture industry; however, intensive culture activities easily cause disease outbreak, especially viral disease. For the prevention of viral outbreaks, interferon (IFN) is among the major defence systems being studied in different species. Fish type I IFNs are known to possess antiviral properties similar to mammalian type I IFNs. In order to stimulate antiviral function, IFN will bind to its cognate receptor, the type I interferon receptor (IFNAR), composed of heterodimeric receptor subunits known as IFNAR1 and IFNΑR2. The binding of type I interferon to receptors assists in the transduction of signals from the external to internal environments of cells to activate biological responses. In order to study the function of IFN, we first need to understand IFN receptors. In this study, we cloned and identified IFNAR1 in orange-spotted grouper (osgIFNAR1) and noted the up-regulated mRNA expression of the receptor and downstream effectors in the head kidney cells with cytokine treatment. The transcriptional expression of osgIFNAR1, which is characterised using polyinosinic-polycytidylic acid (poly[I:C]) and lipopolysaccharide (LPS) treatments, indicated the involvement of osgIFNAR1 in the immune response of grouper. The subcellular localisation of osgIFNAR1 demonstrated scattering across the grouper cell. Viral infection showed the negative feedback regulation of osgIFNAR1 in grouper larvae. Further loss of function of IFNAR1 showed a decreased expression of the virus. This study reported the identification of osgIFNAR1 and characterisation of receptor sensitivity towards immunostimulants, cytokine response, and viral challenge in the interferon pathway of orange-spotted grouper and possible different role of the receptor in viral production. Together, these results provide a frontline report of the potential function of osgIFNAR1 in the innate immunity of teleost.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cytokines; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Nodaviridae; Phylogeny; Poly I-C; Receptor, Interferon alpha-beta; RNA Virus Infections; Sequence Alignment

This study was aimed at characterizing microbiologically Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) produced in two estuarine ecosystems in Andalusia (Spain): the estuary of the river Guadalquivir (La Puebla del Río, Sevilla) (A), and the estuary of the river Guadiana (Ayamonte, Huelva) (B). The collected fish individuals and water were analysed for hygiene indicator microorganisms and pathogens. The statistical analysis of results revealed that microbial counts for the different microbiological parameters were not statistically different for fish type. On the contrary, considering anatomic part, viscera showed significantly higher concentrations for Enterobacteriaceae, total coliforms and for Staphylococcus spp. coagulase +. Furthermore, location A showed in water and fish higher levels for lactic acid bacteria, aerobic mesophilic bacteria, Enterobacteriaceae, total coliforms and Staphylococcus spp. coagulase +. Neither Listeria monocytogenes, nor Salmonella spp. were detected, though Vibrio parahaemolyticus was identified, molecularly, in estuarine water in location B. The predictive analysis demonstrated that the initial microbiological quality could have an impact on product shelf-life, being longer for location B, with better microbiological quality. Results stress the relevance of preventing the microbiological contamination of water in estuary production systems in order to assure the quality and safety of Gilthead sea bream and Sea bass.

Topics: Animals; Aquaculture; Bacteria; Bass; Ecosystem; Enterobacteriaceae; Estuaries; Fish Diseases; Food Storage; Prevalence; Sea Bream; Seafood; Spain; Staphylococcus; Vibrio parahaemolyticus

Vibrio harveyi is regarded as serious pathogen for marine fishes. To evaluate the physiological responses of spotted sea bass (Lateolabrax maculatus) after V. harveyi infection, four biochemical biomarkers including alanine amino transferase (ALT), albumin (ALB), total protein (TP) and glucose (GLU) were measured in serum. Our results showed that V. harveyi infection significantly influenced the concentration of ALT, ALB and GLU. Additionally, five interleukin-17 (IL-17) and five IL-17 receptors (IL-17R) genes were identified in spotted sea bass and their gene structures were characterized. Furthermore, the expression patterns of IL-17 and IL-17R genes were determined by qPCR in liver, intestine, spleen and head kidney after V. harveyi infection. All IL-17 and IL-17R genes exhibited time- and tissue-dependent expressions. Several tested genes were dramatically induced by V. harveyi treatment, particularly IL-17A/F1 in liver and head kidney, IL-17A/F2 in head kidney, IL-17RC in spleen with more than 10-fold increases, which suggested their potential essential roles against bacterial infection.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Head Kidney; Immunity, Innate; Interleukin-17; Organ Specificity; Receptors, Interleukin-17; Signal Transduction; Transcriptome; Vibrio; Vibrio Infections

In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.

Topics: Aldehydes; Animals; Aquaculture; Bass; Biomarkers; Central Nervous System; Fish Diseases; Gene Expression; Gills; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Immunohistochemistry; Immunoprecipitation; Kidney; Liver; Malondialdehyde; Oxidative Stress; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Spleen; Stress, Physiological; Temperature; Tyrosine

Nodavirus, or nervous necrosis virus (NNV), is the causative agent of viral encephalopathy and retinopathy (VER), a severe disease affecting numerous fish species worldwide. European sea bass, a cultured species of great economic importance, is highly susceptible to the disease. To better understand the response of this organism to NNV, we conducted RNA-Seq analysis of the brain and head kidney from experimentally infected and uninfected sea bass juveniles at 24 and 72 hours post-infection (hpi). Contrary to what was expected, we observed modest modulation of immune-related genes in the brain, the target organ of this virus, and some of these genes were even downregulated. However, genes involved in the stress response showed extremely high modulation. Accordingly, the genes encoding the enzymes implicated in the synthesis of cortisol were almost the only overexpressed genes in the head kidney at 24 hpi. This stress response was attenuated after 72 h in both tissues, and a progressive immune response against the virus was mounted. Moreover, experiments were conducted to determine how stress activation could impact NNV replication. Our results show the complex interplay between viral activity, the stress reaction and the immune response.

Topics: Animals; Bass; Brain; Fish Diseases; Fish Proteins; Head Kidney; Immunity, Innate; Nodaviridae; RNA Virus Infections; RNA-Seq; Stress, Physiological

Scale drop disease virus (SDDV) is a megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, we report on a sensitive and specific SYBR Green qPCR assay. The qPCR primers were designed to target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 scale drop disease-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously, using a semi-nested conventional PCR. The higher sensitivity of our SDDV qPCR assay can thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed that SDDV DNA loads varied from 8.0 × 102 to 6.8 × 104 viral DNA copies per 200 ng DNA template among the 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of more than one type of tissue is advisable.

Topics: Animals; Bass; Fish Diseases; Iridoviridae; Real-Time Polymerase Chain Reaction

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.

Topics: Animals; Bass; Brain; Fish Diseases; Gills; Immunity; Nodaviridae; RNA Virus Infections

Topics: Animals; Bass; Capsid Proteins; Fish Diseases; Fish Proteins; Nodaviridae; Quasispecies; RNA Virus Infections; Tumor Necrosis Factor-alpha

Grouper is an important food fish due to its commercial value. A major production constraint in culturing grouper is that the fish can be fatally affected by leeches and diseases. Leeches are an ectoparasite of grouper, which can cause significant loss in fish number, and thus controlling their prevalence is indispensable as a tool of aquaculture management. In order to understand the prevalence and infestation intensity of a piscicolid leech Zeylanicobdella arugamensis, field observation of hybrid grouper was conducted in two aquaculture farms in Brunei Darussalam. The prevalence rates were 100% for the two farms throughout the study period, while the infestation intensity levels were significantly different between the two farms. The farm with the higher salinity ranging from 26 to 28 psu was found to have a significantly higher number of leeches (505 ± 271 leeches fish

Topics: Animals; Bass; Brunei; Fish Diseases; Leeches; Prevalence

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population.

Topics: Algeria; Animals; Bass; Brain Diseases; Endemic Diseases; Fish Diseases; Mediterranean Sea; Prevalence; Retinal Diseases

Non-native parasites are often translocated into novel areas together with their natural hosts, but the parasite diversity is frequently lower compared to the host's native range.. This paper provides morphological and molecular characterisation for non-native monogenean parasite Onchocleidus principalis (Ancyrocephalidae) co-introduced with its fish host to Portugal, Europe, including new details on the species' vaginal morphology and metrics.. Two host species, the largemouth bass Micropterus salmoides and pumpkinseed sunfish Lepomis gibbosus, were sampled from two reservoirs (Landeira, Coruche) in the Atlantic Sea drainage. Morphometric analysis and sequencing of 28S rDNA were performed.. Presence of O. principalis was confirmed in all individuals of largemouth bass. Accidental findings on pumpkinseed sunfish at both sampling sites represent new host records for this parasite species. The morphometric description of O. principalis showed high similarity to the species in its native range. No intraspecific sequence variation of partial 28S rDNA was observed between specimens from the two sampling sites. Presence of another North-American ancyrocephalid parasite Onchocleidus dispar was confirmed in pumpkinseed sunfish at both sites, and in largemouth bass from the Coruche Reservoir, this representing the first record of O. dispar on largemouth bass in Europe.. Onchocleidus principalis has been confirmed to parasitise both centrarchid fish species introduced to Europe. Compared to other studies, the species collected in Portugal exhibits pronounced vaginal armament.

Topics: Animals; Bass; DNA, Ribosomal; Europe; Female; Fish Diseases; Humans; Perciformes; Trematoda

Understanding the functions of genes related to disease resistance and identifying polymorphisms in these genes are essential in molecular breeding for disease resistance. Viral nervous necrosis (VNN) is one of the major diseases in the Asian seabass, Lates calcarifer. Our previous works on QTL mapping, GWAS and cell-line transcriptome analysis of the Asian seabass after NNV challenge revealed that the gene GAB3 might be a candidate gene for VNN resistance. In this study, we cloned and characterized GAB3, and identified SNPs in the gene of the Asian seabass. The cDNA of the gene was 2165 bp, containing an ORF of 1674 bp encoding 557 amino acids. The gene consisted of 10 exons and nine introns. It was ubiquitously expressed in normal fish. An analysis of the association between two SNPs in the second intron and NNV resistance in 1035 fish descended from 43 families revealed that the two SNPs were significantly associated with VNN resistance. After NNV infection, the expression of GAB3 was significantly increased in the brain, spleen, muscle and gut, and was suppressed in the liver. The GAB3 protein was localized in the nucleus. Overexpression of GAB3 with specific GAB3-pcDNA was positively correlated to increased viral RNA and titer in NNV-infected Asian seabass cells. Our study provides new evidence to support that GAB3 may be an important gene related to NNV resistance. In addition, the SNPs provide DNA markers for the selection of candidate genes resistance to NNV at the juvenile stage of Asian seabass.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; GRB2 Adaptor Protein; Immunity, Innate; Nodaviridae; Phylogeny; RNA Virus Infections

Interferon-induced transmembrane proteins (IFITMs) have been identified as important host restriction factors in mammals for the control of infection by multiple viruses. However, the antiviral functions of IFITMs against fish viruses remain largely uncertain. In this study, the IFITM3 homolog from orange spotted grouper (EcIFITM3) was cloned and its roles in grouper virus infection were investigated. The full-length cDNA of EcIFITM3 was 737 bp, which was composed of a 16 bp 5'-UTR, a 274 bp 3'-UTR, and a 447 bp ORF. EcIFITM3 encodes a 148-amino-acid polypeptide, which contains five domains, i.e., the N-terminal domain (aa 1-65), TM1 (aa 66-90), the cytoplasmic domain (aa 91-110), TM2 (aa 111-140), and the C-terminal domain (aa 141-148), and shares 78% and 47% identity with IFITM3 of gilthead seabream (Sparus aurata) and human (Homo sapiens), respectively. EcIFITM3 mRNA was detected in 12 tissues of healthy groupers, with the highest expression levels in the head kidney. Additionally, the in vitro mRNA levels of EcIFITM3 were significantly upregulated by infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV), or treatment with polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS). Subcellular localization analysis showed that EcIFITM3 was mainly distributed in the cell membrane of grouper cells. In vitro, the ectopic expression of EcIFITM3 inhibited SGIV and RGNNV infection, as demonstrated by the reduced severity of the cytopathic effect, decreased virus production, and low levels of viral mRNA and proteins. Consistently, knockdown of EcIFITM3 by small interfering RNAs (siRNAs) enhanced SGIV and RGNNV replication. EcIFITM3 overexpression and knockdown experiments both suggested that EcIFITM3 inhibits the infection of SGIV and RGNNV by restricting virus entry.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Membrane Proteins; Nodaviridae; Phylogeny; Poly I-C; Ranavirus; RNA Virus Infections; Sequence Alignment

Lates calcarifer herpes virus (LCHV) is a novel virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per μl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.

Topics: Animals; Asia, Southeastern; Bass; Benzothiazoles; Diamines; Fish Diseases; Fisheries; Herpesviridae; Quinolines; Real-Time Polymerase Chain Reaction

The TRAF family member-associated nuclear factor (NF)-κB activator (TANK) was first identified as a TRAF-binding protein with both stimulatory and inhibitory properties in host innate immune activation. To elucidate the roles of TANK in teleosts, we cloned and characterized the TANK homologue of orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcTANK consists of 1026 nucleotides encoding a 342 amino acid protein with a predicted molecular mass of 38.24 kDa. EcTANK shares 89.47% and 88.89% identity with Larimichthys crocea TANK and Lates calcarifer TANK, respectively. EcTANK was distributed in all 11 examined tissues. The expression of EcTANK in the spleen increased after infection with Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV). EcTANK was mainly located in the cytoplasm of grouper spleen cells. EcTANK enhanced SGIV and RGNNV replication during viral infection in vitro. Overexpression EcTANK decreased the expression levels of interferon-associated cytokines and pro-inflammatory factors, and enhanced activation of NF-κB. Taken together, these results suggest that EcTANK may play an important role in antiviral innate immune activation in grouper.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; Sequence Alignment

Vibrio alginolyticus is posting an increasing threat to survival of grouper. Classical complement cascade can trigger initiation of immunity, while complement 9 (C9) is a major complement molecule involved in final step of membrane attack complex (MAC) formation. In this study, full-length EcC9 contained an ORF sequence of 1779 bp, encoding a polypeptide of 592 amino acids. A high-level expression of EcC9 mRNA was observed in liver. Following vibrio challenge, increased expression levels of EcC1q, EcBf/C2, EcC4, EcC6, EcC7 and EcC9 mRNA were detected in liver and kidney. These results implied that elevated expression level of classical complement pathway (CCP) and terminal complement components (TCCs) may assess toxicological effect of V. alginolyticus.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Complement C9; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Sequence Alignment; Vibrio alginolyticus

Ubiquitin-specific protease 14 (USP14), one of the USP family members which belong to deubiquitinating enzymes (DUBs), plays a key role in maintaining cellular protein homeostasis by trimming ubiquitin chains from their substrates. However, the roles of USP14 in response to virus infection still remains largely unknown. In the current study, a USP14 homolog from orange spotted grouper (EcUSP14) was cloned and its roles in innate immune response were investigated. EcUSP14 was composed of 1479 base pairs encoding a 492-amino acid (aa) polypeptide. Sequence analysis indicated that EcUSP14 shared 96.14% and 81.30% identity to USP14 of bicolor damselfish (Stegastes partitus) and humans (homo sapiens), respectively. EcUSP14 contains conserved ubiquitin-like (UBL) domain (aa 3-76) and peptidase-C19A domain (aa 106-481). In response to Singapore grouper iridovirus (SGIV) infection in vitro, EcUSP14 was significantly up-regulated. Subcellular localization showed that EcUSP14 was predominantly localized in the cytoplasm of grouper spleen (GS) cells and mostly co-localized with the viral assembly sites after SGIV infection. The ectopic expression of EcUSP14 significantly promoted the replication of SGIV, as demonstrated by the accelerated progression of severity of cytopathic effect (CPE), the increased viral gene transcription and viral protein synthesis during infection. Consistently, treatment with IU1, a USP14 specific inhibitor, significantly inhibited the replication of SGIV, suggesting that USP14 function as a pro-viral factor during SGIV replication. Further analysis showed that EcUSP14 overexpression decreased the promoter activities of interferon (IFN)-1, IFN-3, IFN-stimulated response element (ISRE), and nuclear factor of kappa B (NF-κB). Furthermore, the ectopic expression of EcUSP14 decreased the activities of IFN-1 promoter evoked by TANK-binding kinase (TBK)-1 and melanoma differentiation-associated protein (MDA)-5, but not stimulator of interferon genes (STING). Thus, we speculated that EcUSP14 facilitated virus replication by negatively regulating the IFN response. Taken together, our results firstly demonstrated that fish USP14 functioned as a pro-viral factor by negatively regulating interferon response against virus infection.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Ranavirus; Sequence Alignment; Ubiquitin Thiolesterase

The marine leech, Zeylanicobdella arugamensis, is a major threat to aquaculture in grouper-producing countries including Indonesia. This study aimed at investigating prevalence, intensity and histopathology of the ectoparasite in humpback and hybrid groupers cultured in different rearing systems. A total of 260 groupers (60 humpback groupers and 200 hybrid groupers) were used for samples. The marine leech was observed on skin, fins, gills and mouth, followed by histopathological assay on the skin tissue. The results showed that prevalence of the leech in both groupers was higher when they were cultured in the floating net cages compared with the hatchery, p < .05. Furthermore, humpback grouper had a higher prevalence than hybrid grouper when they were cultured in a similar system, p < .05. Meanwhile, there was no significant difference in intensity between the two groupers, p > .05. Within the hybrid groupers, the highest prevalence was obtained from hybrid groupers reared in the earthen ponds. Histopathological studies showed that the infected groupers exhibited inflammation, congestion and erosion of the epidermis layer. Hybrid grouper had more severe histopathological lesions in the skin tissues. These results suggested that species and type of aquaculture system had significantly determined the prevalence, intensity and severity of lesion in Z. arugamensis infestation.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Indonesia; Leeches; Prevalence

Topics: Animals; Bass; Cichlids; Disease Susceptibility; Fish Diseases; Francisella; Gram-Negative Bacterial Infections; Granuloma; Head Kidney; Injections, Intraperitoneal; Spleen

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut

Topics: Animals; Bass; Brain; Fish Diseases; Gene Expression Profiling; Head Kidney; Nodaviridae; RNA Virus Infections; Transcriptome; Virulence

Nervous necrosis virus (NNV) infection in susceptible grouper larvae has been reported to cause high mortalities, leading to great economic losses in aquaculture industry. Although the effects of NNV vaccines on grouper have been broadly investigated, vaccination strategies have not been fully established. To this end, we introduced the parsimonious epidemiological models that explored the assessment of key epidemiological parameters and how they changed when vaccinations showed the effects. We showed that the models capture the published cumulative mortality data accurately. We estimated a basic reproduction number R

Topics: Animals; Aquaculture; Basic Reproduction Number; Bass; Fish Diseases; Larva; Models, Theoretical; Nodaviridae; RNA Virus Infections; Taiwan; Vaccination

Largemouth bass virus (LMBV) is the causative agent of a disease causing high mortality rates in largemouth bass during summer. However, there is little information available about the development of vaccines for LMBV disease. Hence, a DNA vaccine, named pCDNA3.1(+)-MCP-Flag, was constructed by inserting the cloned LMBV major capsid protein (MCP) gene into the pCDNA3.1(+)-Flag plasmid. The expression of the recombinant plasmid was confirmed by Western blot (WB) and RT-PCR. The WB result revealed that the MCP protein produced a band of approximately 53 kDa, consistent with the expected result. The RT-PCR results also confirmed that MCP was transcribed in the EPC cells transfected with the recombinant plasmid. The largemouth bass in the DNA vaccine group were immunized with the pCDNA3.1(+)-MCP-Flag plasmid by pectoral fin base injection, and the relative percent survival (RPS) of fish challenged with LMBV was 63%. The relative immunological analyses were as follows. Compared with the PBS and pCDNA3.1(+) groups, the DNA vaccine group showed significantly upregulated expression of IL-1β, IL-8, TNF-α and Mx in the spleen, head kidney and liver. All largemouth bass immunized with the DNA vaccine produced a high titre of LMBV-specific neutralizing antibody during the immunization period. The titre was 1:375 ± 40 and peaked at 14 days post-vaccination. The expression of the recombinant plasmid was analysed in the tissues of the DNA vaccine group by RT-PCR. The recombinant plasmid was expressed in the spleen, head kidney and liver, and MCP protein was successfully expressed after vaccination. In conclusion, the recombinant plasmid expressing LMBV MCP induced significant immune responses in largemouth bass, and might represent a potential LMBV vaccine candidate for largemouth bass.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Ranavirus; Vaccines, DNA; Viral Vaccines

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-β, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Topics: Animals; Bass; Capsid Proteins; DNA Virus Infections; Fish Diseases; Iridoviridae; Nanotubes, Carbon; Vaccination; Vaccines, Subunit; Viral Vaccines

Epinephelus coioides is an important economic culture marine fish and is susceptible to various pathogenic diseases. Increasingly evidences showed that miRNAs participated in the regulation of the cell proliferation, differentiation and immune response. MiR-122 has been reported to play an essential role in immune response by triggering an inflammatory reaction. However, the function of miR-122 in response to bacterial infection is unclear in Epinephelus coioides. Herein, we report that miR-122 is involved in response to Aeromonas hydrophila infection of grouper spleen cells (GS). IL-15, IL-6 and IL-1β are inhibited in overexpression miR-122 GS cells, while induced in silence miR-122 GS cells. In addition, IL-15 is predicted to be the target gene of miR-122, which is further confirmed by LUC. Taken together, we propose that miR-122 regulates the immune response to bacterial infection by triggering IL-15.

Topics: Aeromonas hydrophila; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Interleukin-15; MicroRNAs; Phylogeny; Sequence Alignment; Spleen

The present study was designed to determine the modulatory effects of arginine and citrulline dietary supplementation on the immune condition and inflammatory response of European seabass, Dicentrarchus labrax. Four diets were manufactured: a control diet (CTRL) was formulated to meet the indispensable amino acids profile established for seabass. Based on this formulation, three other diets were supplemented with l-arginine at two different levels (0.5% and 1%, ARG1 and ARG2, respectively) and l-citrulline at 0.5% (CIT). Fish were fed these diets for 2 or 4 weeks under controlled conditions. At the end of 4 weeks, fish from all dietary treatments were intraperitoneally-injected with Photobacterium damselae piscicida and sampled after 4, 24 our 48 h. Immune status was characterized by a lymphocyte time-dependent decrease regardless of dietary treatment, whereas peroxidase values dropped in time in fish fed ARG1 and ARG2 and was lower at 4 weeks in fish fed ARG1 than in fish fed CTRL. Up-regulation of several genes was more evident in ARG1-and CIT-fed fish, though pro-inflammatory cytokines were down-regulated by CIT dietary treatment. Following immune stimulation, seabass fed ARG1 showed a decrease in neutrophils and monocytes circulating numbers. On the other hand, expression of 17 selected immune and inflammatory responses genes was barely affected by dietary treatments. Based on the analyzed parameters, results suggest an active role of dietary arginine/citrulline supplementation in modulating immune defences that seem to translate into a suppressed immune repertoire, mostly at the cell response level. The observed changes due to citrulline dietary supplementation were in part similar to those caused by arginine, suggesting that citrulline might have been used by macrophages as an arginine precursor and then engaged in similar immune-impairment leading mechanisms.

Topics: Animal Feed; Animals; Arginine; Bass; Citrulline; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Inflammation; Photobacterium; Random Allocation

Groupers are popular aquaculture species in South-East Asia, but their cultivation is affected by infectious disease outbreaks. Mucosa-associated lymphoid tissues provide a first-line defence against pathogens; however, few studies are available relating to cellular or proteomic responses of mucosal immunity in grouper. Skin, gill and intestine were sampled from brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) at 4 and 96 hr post-infection (hpi) and 7 days post-infection (dpi) following intraperitoneal infection with Vibrio harveyi, and stained with haematoxylin/eosin and Alcian Blue/periodic acid-Schiff. Skin mucus was analysed by 2D-gel electrophoresis, and proteins modulated by the bacterial infection identified. In the infected fish, significant increases in sacciform cells in skin and increased levels of nucleoside diphosphate kinase in mucus were detected at 4 hpi. At 96 hpi, goblet cells containing acidic mucins significantly increased in the intestine, while those containing mixed mucins increased in skin and gills of infected fish. Proteasome subunit alpha type-I and extracellular Cu/Zn superoxide dismutase levels also increased in mucus. Rodlet and mast cells did not appear to respond to the infection. Mucosal tissues of grouper appeared actively involved in response to Vibrio infection. This information may help future research on improving grouper health, production and vaccine development.

Topics: Animals; Bass; Fish Diseases; Goblet Cells; Immunity, Mucosal; Mucous Membrane; Mucus; Proteome; Vibrio; Vibrio Infections

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bass; Cells, Cultured; Cytokines; DNA Virus Infections; Fish Diseases; Fish Proteins; Host-Pathogen Interactions; Immunity, Innate; Iridovirus; Nodaviridae; Receptor-Interacting Protein Serine-Threonine Kinases; RNA Virus Infections; Signal Transduction

Palmitic acid is the most common saturated fatty acid in animals, plants, and microorganisms. Studies highlighted that palmitic acid plays a significant role in diverse cellular processes and viral infections. Accumulation of palmitic acid was observed in fish cells (grouper spleen, GS) infected with Singapore grouper iridovirus (SGIV). The fluctuated content levels after viral infection suggested that palmitic acid was functional in virus-cell interactions. In order to investigate the roles of palmitic acid in SGIV infection, the effects of palmitic acid on SGIV induced cytopathic effect, expression levels of viral genes, viral proteins, as well as virus production were evaluated. The infection and replication of SGIV were increased after exogenous addition of palmitic acid but suppressed after knockdown of fatty acid synthase (FASN), of which the primary function was to catalyze palmitate synthesis. Besides, the promotion of virus replication was associated with the down-regulating of interferon-related molecules, and the reduction of IFN1 and ISRE promotor activities by palmitic acid. We also discovered that palmitic acid restricted TBK1, but not MDA5-induced interferon immune responses. On the other hand, palmitic acid decreased autophagy flux in GS cells via suppressing autophagic degradation, and subsequently enhanced viral replication. Together, our findings indicate that palmitic acid is not only a negative regulator of TBK1-IRF3/7 pathway, but also a suppressor of autophagic flux. Finally, palmitic acid promotes the replication of SGIV in fish cells.

Topics: Animals; Autophagy; Bass; Cell Line; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Host-Pathogen Interactions; Interferon Regulatory Factor-3; Interferon Regulatory Factor-7; Iridovirus; Palmitic Acid; Protein Serine-Threonine Kinases; Signal Transduction; Virus Replication

Peptidoglycan recognition proteins (PGRPs), which are structurally conserved innate immune molecules in invertebrate and vertebrate animals, play the important roles in regulation of innate immune responses. In this paper, three PGRP genes of spotted sea bass, Lateolabrax maculatus, were cloned, designated as Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2, respectively. Sequence analysis showed that the deduced amino acid sequences of Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2 proteins contained respectively 468, 482 and 167 amino acid residues, and had the typical structural features of PGRPs, i.e. conserved PGRP domain and Zn

Topics: Animals; Bass; Carrier Proteins; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Fish Proteins; Lipopolysaccharides; Poly I-C; Staphylococcus aureus; Vibrio

Topics: Animals; Bass; Female; Fish Diseases; Ovarian Neoplasms; Sex Cord-Gonadal Stromal Tumors

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

Topics: Animals; Bass; Brain; Brain Diseases; Esophagus; Eye; Fish Diseases; Gills; Nodaviridae; Real-Time Polymerase Chain Reaction; Retinal Diseases; RNA Virus Infections; Skin

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5-7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.

Topics: Animals; Antigens, Protozoan; Bass; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Hymenostomatida; Immunity, Humoral; Immunity, Mucosal; Major Histocompatibility Complex

The emergence of diseases has caused much health and economic damage. Viral Nervous Necrosis (VNN) is considered as one of the most important threats to aquatic ecosystems. VNN can cause severe mortality and economic loss in fish farms. The high water temperatures in southern Iran and the observed incidences of fish mortality in the Persian Gulf led to the hypothesis of the possible emergence of VNN. Therefore, this study aimed to monitor two species of fish susceptible to VNN using PCR, and Nested PCR methods and comparing the sensitivity of these methods to the identification of Betanodavirus infection in apparently healthy and symptomatic fish. About 850 Grouper (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) fish of the Persian Gulf were collected randomly and examined. Molecular methods were used to identify NNV in visibly healthy and symptomatic fish of the Persian Gulf of Iran. The results of the PCR showed no positive cases, but the Nested PCR revealed some positive results. Then, the phylogenetic analysis of the virus sequence was performed. The nucleotide sequence of Nested PCR products revealed a 98-100% homology with Red Spotted Grouper Viral Nervous Necrosis (RGNNV). This is the first report on VNN tracing and detection as well as phylogenetic analysis of the virus from the Persian Gulf of Iran. Therefore, considering the importance of emerging viral diseases and the irreparable damage they cause, continuous monitoring and epidemiological studies of VNN were recommended by authorized organizations.

Topics: Animals; Bass; Ecosystem; Fish Diseases; Indian Ocean; Iran; Nodaviridae; Phylogeny; RNA Virus Infections

To reduce the largemouth bass ulcer syndrome (LBUSV) aquatic economic losses, it must take effective preventive measures and coping strategies should be urgently investigated. In this research, the effects of a functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in largemouth bass (Micropterus Salmoides) against LBUSV were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to largemouth bass against LBUSV. We found more than 10 times increase in serum antibody levels, enzyme activities and immune-related genes (IL-6, IL-8, IFN-γ, IgM and TNF-α) expression, in the SWCNTs-pcDNA-MCP immunized groups compared with PBS group and the pure SWCNTs group. The survival rates for control group (PBS), pure SWCNTs groups (40 mg L

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Immunity, Innate; Immunization; Iridoviridae; Nanotubes, Carbon; Vaccination; Vaccines, DNA; Viral Vaccines

This study was carried out to investigate the effects of dietary vitamin A (VA) on growth performance, antioxidant capacity, digestion, intestinal immune response, and mRNA expression of intestinal tight junction proteins for juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Six isonitrogenous and isolipidic experimental diets were formulated to obtain VA levels (317, 1136, 2038, 4142, 7715, 15204 IU/kg diet, respectively). The triplicate groups of fish (average weight of 9.01 ± 0.27 g) were fed twice daily (8:00 and 16:00) for 7 weeks. Based on the broken-line analysis model of WG and LYZ activity, the dietary VA requirement of hybrid grouper were estimated to be 2688.58 and 4096.36 IU/kg diet. The results showed that VA deficiency or excess could reduce Weight gain, specific growth rate, and protein efficiency ratio, and increase feed conversion ratio and hepatosomatic index (P < 0.05). In addition, VA deficiency could reduce the serum activities of acid phosphatase (ACP), superoxide dismutase, and total antioxidant capacity and increase the malondialdehyde content (P < 0.05). VA deficiency also could reduce intestinal activities of ACP, alkaline phosphatase, lysozyme, complement 3, complement 4 contents, and activities of alpha-amylase, lipase, and trypsin (P < 0.05). Meanwhile, VA deficiency could reduce villus height in proximal intestine (PI) and mid intestine (MI), as well as muscle thickness in PI and distal intestine (DI) (P < 0.05). Moreover, VA deficiency could down-regulated antimicrobial peptides (β-defensin, Hepcidin [not in MI and DI], Epinecidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1 [not in DI]), tight junction proteins (occluding and claudin3) mRNA levels in the PI, MI and DI, and up-regulated pro-inflammatory cytokines (tumor necrosis factor α [not in MI] and interleukin 1β [not in MI]), signaling molecules c-Rel and p65 (P < 0.05). Collectively, VA deficiency could reduce growth performance because of a negative effect on intestinal health by depressing digestive abilities, intestinal morphology, immunity and tight junction function in the intestine.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Intestines; Random Allocation; Tight Junction Proteins; Vitamin A Deficiency

The ectoparasite protozoan

Topics: Animals; Bass; Dinoflagellida; Fish Diseases; Gills; High-Throughput Nucleotide Sequencing; Protozoan Infections, Animal; Protozoan Proteins; Transcriptome; Virulence Factors

Recent surveys of white perch Morone americana from Chesapeake Bay, USA, revealed a high prevalence of hepatic and biliary lesions, including neoplasia, and bile duct parasites. Here, we describe lesions in the liver and gallbladder and evaluate for statistical associations among lesions, parasites, and biomarkers of chemical exposure in fish from 2 tributaries of Chesapeake Bay. Fish were collected from an estuarine site in the Choptank River (n = 122, ages 3-11), a tributary with extensive agriculture within the watershed, and the Severn River (n = 131, ages 2-16), a tributary with extensive urban development. Passive integrative samplers were deployed at the fish collection site and an upstream, non-tidal site in each river for 30 d. Intrahepatic biliary lesions observed in fish from both rivers included neoplasia (23.3%), dysplasia (16.2%), hyperplasia (46.6%), cholangitis (24.9%), and dilated ducts containing plasmodia of Myxidium sp. (24.9%). Hepatocellular lesions included foci of hepatocellular alteration (FHA, 15.8%) and neoplasia in 4 Severn River fish (2.3%). Age of fish and Myxidium sp. infections were significant risk factors for proliferative and neoplastic biliary lesions, age alone was a risk factor for FHA, and Goussia bayae infections were associated with cholangitis and cholecystitis. Lesion prevalence was higher in fish from the Severn River, which contained higher concentrations of PAHs, organochlorine pesticides, and brominated diphenyl ethers. Metabolite biomarkers indicated higher PAH exposures in Severn River fish. This study suggests Myxidium sp. as a promoter of bile duct tumors, but more data are needed to evaluate the biological effects of environmental contaminants in this species.

Topics: Animals; Bass; Bile Ducts; Environmental Monitoring; Fish Diseases; Neoplasms; Parasites; Prevalence; Water Pollutants, Chemical

Nocardia seriolae is the causative agent of nocardiosis in both marine and freshwater fish. Here, we report on multiple outbreaks of nocardiosis associated with elevated mortality (23-35%) in farmed largemouth bass in Sichuan, China, from 2017 to 2018. A total of 9 strains isolated from diseased largemouth bass were identified as N. seriolae by phenotypic characterization, 16S rRNA and hsp65 gene sequence analysis. The clinical signs of infected largemouth bass included hemorrhage, skin ulcers and prominent tubercles varying in size in the gill, liver, spleen and kidney. Experimental infection indicated that these isolates were the pathogens responsible for the mortalities. In vitro antibacterial activities of 12 antibiotics against N. seriolae isolates were determined as minimum inhibitory concentrations. Histopathological observation of diseased fish infected with N. seriolae showed necrotizing granulomatous hepatitis, nephritis, splenitis, epithelial hypertrophy and hyperplasia with degenerative changes of the epithelium in the gill. Large quantities of bacterial aggregates were found in the necrotic area of the granuloma by Lillie-Twort Gram stain and immunocytochemistry. Our findings indicated that N. seriolae is a serious threat to the largemouth bass Micropterus salmoides industry in Southwest China.

Topics: Animals; Bass; China; Fish Diseases; Nocardia; Phylogeny; RNA, Ribosomal, 16S

Bacterial fish diseases constitute a major problem in aquaculture, it was found in the environment and under stressors cause severe economic losses to fish. This work aimed to investigate the bacterial causes and suitable treatments of mass mortality in some cultured marine fish farms in Damietta governorate.. The study was performed on 5 farms suffered from mass mortality. Total of 100 diseased fish (10 sea bass and 10 sea bream/farm) and 20 water samples were randomly collected from these farms. Bacteriological examinations were carried out followed by in vitro sensitivity tests. Treatment trial was performed using the most effective antibacterial agent on isolated bacteria.. From fish and water samples Pseudomonas spp., Aeromonas spp. and Vibrio spp. were isolated with the rat of (16, 10%), (22, 10%) and (28, 10%) respectively. These results were confirmed biochemically. Some virulence genes of isolated bacteria were detected using PCR; meanwhile, enrofloxacin reduced significantly the mortality rates in examined farms.. It could be concluded that, Pseudomonas spp., Aeromonas spp. and Vibrio spp. are the main bacterial species causing mass mortality in marine fish farms. These bacteria were highly sensitive to enrofloxacin in vitro and in vivo.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Bass; Enrofloxacin; Fish Diseases; Fisheries; Microbial Sensitivity Tests; Polymerase Chain Reaction; Sea Bream; Virulence

In Egypt, Nile tilapia represents the main cultured type due to its economical price, palatability and easy culturing. This study was aimed to elucidate the pathogenicity of V. alginolyticus isolated from diseased sea bass and experimentally infected healthy Nile tilapia fish.. Healthy Nile tilapia fish were injected I/P with V. alginolyticus isolated from diseased sea bass. Symptoms and mortality rates of infected Nile tilapia fish were recorded during the experimental period. Re-isolation of V. alginolyticus was done from infected tilapia fish by bacteriological methods. For confirmation the pathogenicity of Vibrio isolated either from marine fish or tilapia fish, PCR test was done using tdh and bla gens. Liver and kidney function tests with histopathological examinations of some organs were performed. Treatment trial was done according to the antibiotic sensitivity test.. The isolated Vibrio is highly pathogenic to Nile tilapia fish causing deterioration in all parameters which finished by severe mortalities. Treatment with florfenicol, enrofloxacin, or oxytetracycline reduced the mortality rate and improved liver and kidney function parameters of infected Nile tilapia fish.. V. alginolyticus can infect both marine and fresh water fish inducing a high mortality rate. Treatment of infected fish with florfenicol, enrofloxacin, or oxytetracycline reduces the mortality rate.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Cichlids; Enrofloxacin; Fish Diseases; Oxytetracycline; Thiamphenicol; Vibrio alginolyticus; Vibrio Infections

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Nectins; Nodaviridae; RNA Virus Infections

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process used to maintain cell survival and homeostasis. A series of autophagy-related genes (Atgs) are involved in the autophagic pathway. In mammals, a growing number of studies have attributed functions to some Atgs that are distinct from their classical role in autophagosome biogenesis, such as resistance to pathogens. However, little is known about the functions of fish Atgs. In this study, we cloned and characterized an atg12 homolog from orange spotted grouper (Epinephelus coioides) (Ecatg12). Ecatg12 encodes a 117 amino acid protein that shares 94.0% and 76.8% identity with gourami (Anabas_testudineus) and humans (Homo sapiens), respectively. The transcription level of Ecatg12 was lower in cells infected with Singapore grouper iridovirus (SGIV) than in non-infected cells. Fluorescence microscopy revealed that EcAtg12 localized in the cytoplasm and nucleus in grouper spleen cells. Overexpression of EcAtg12 significantly increased the replication of SGIV, as evidenced by increased severity of the cytopathic effect, transcription levels of viral genes, levels of viral proteins, and progeny virus yield. Further studies showed that EcAtg12 overexpression decreased the expression levels of interferon (IFN) related molecules and pro-inflammatory factors and inhibited the promoter activity of IFN-3, interferon-stimulated response element, and nuclear factor-κB. Together, these results demonstrate that EcAtg12 plays crucial roles in SGIV replication by downregulating antiviral immune responses.

Topics: Amino Acid Sequence; Animals; Autophagy-Related Protein 12; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Ranavirus; Sequence Alignment

Topics: Animal Feed; Animals; Bacillus; Bass; Diet; Disease Resistance; Fish Diseases; Probiotics; Random Allocation; Vibrio; Vibrio Infections

Fish rhabdoviruses are a family of viruses responsible for large-scale fish die-offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT-PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT-PCR. Electron microscopy showed the presence of bullet-shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole-genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen-free largemouth bass juveniles. VHSVLB2018-infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%-93.3% by 7 days post-infection. VHSVLB2018 was re-isolated from dead fish and confirmed by RT-PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Novirhabdovirus; Phylogeny; Rhabdoviridae Infections; Sequence Alignment; Viral Proteins

The male and subgravid female of Philometra serranellicabrillae Janiszewska, 1949 (Philometridae) collected from the gonads of Serranus cabrilla (Linnaeus) (Serranidae) off Tunisia are described for the first time based on light and scanning electron microscopical studies. The male of this nematode can be separated from other congeneric gonad-infecting nominal species in the structure and shape of the gubernaculum (e.g. absence of a dorsal protuberance and a median smooth field). The shape of the male posterior region is unique in that it bears a pair of big circular papillae posterior to the cloacal opening, which is also present in other Philometra spp. from serranids, i.e. P. indica Moravec & Manoharan, 2014, P. inexpectata Moravec, Chaabane, Justine & Neifar, 2016 and P. jordanoi (López-Neyra, 1951) Yamaguti, 1961. Moreover, P. serranellicabrillae differs from its congeners in other fish families from the Mediterranean Sea, in the length of spicules and gubernaculum.

Topics: Animals; Bass; Dracunculoidea; Female; Fish Diseases; Gonads; Male; Microscopy, Electron, Scanning; Species Specificity; Tunisia

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Breeding; DNA Shuffling; Fish Diseases; Fish Proteins; Fisheries; Gene Expression Profiling; High-Throughput Screening Assays; Whole Genome Sequencing

Peroxisome proliferator-activated receptor δ (PPAR-δ), also called PPAR-β or PPAR-β/δ, is a member of the peroxisome proliferator-activated receptor (PPAR) family, which belongs to the nuclear steroid receptor superfamily. Activated PPARs participate in the regulation of lipid and glucose metabolism and also affect cellular proliferation, differentiation, and apoptosis, and the immune responses. To investigate the roles of PPAR-δ in Singapore grouper iridovirus (SGIV) infection, we cloned and characterized the gene encoding a PPAR-δ homologue from the orange-spotted grouper, Epinephelus coioides (EcPPAR-δ). EcPPAR-δ encodes a 514-amino-acid polypeptide, with 95.29% and 74.76% homologue to the Seriola dumerili and human proteins, respectively. EcPPAR-δ contains a typical DNA-binding domain and a ligand-binding domain. Its expression was induced by SGIV infection in vitro. A subcellular localization analysis showed that EcPPAR-δ localizes throughout the cytoplasm and nucleus, with a diffuse intracellular expression pattern. SGIV replication was reduced by EcPPAR-δ overexpression, which was evident in the reduced severity of the cytopathic effect, reduced viral gene transcription, and the reduced expression of the viral capsid protein. The replication of SGIV increased with the knockdown of EcPPAR-δ. The overexpression and silencing of EcPPAR-δ in grouper spleen cells showed that EcPPAR-δ plays a positive role in the regulation of the interferon signaling pathway, but has an anti-inflammatory effect on the inflammatory response. The anti-inflammatory effect of EcPPAR-δ may be related to its function in maintaining cell homeostasis. Because the interferon signaling pathway plays an important role in antiviral immune responses, we speculate that the activation of the interferon signaling pathway by EcPPAR-δ overexpression underlies its inhibitory effect on SGIV replication. Together, our data greatly extend our understanding of the roles of the EcPPAR-δ family members in the pathogenesis of fish viruses.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; PPAR delta; Ranavirus; Sequence Alignment

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), one of the interferon stimulated genes (ISGs), is strongly induced by type I interferon (IFN), double-stranded RNAs and virus infection. To investigate the actions of fish IFIT1 in response to virus infection, we cloned an IFIT1 homolog from orange spotted grouper (EcIFIT1) and clarified its function in this study. The full-length cDNA of EcIFIT1 is 1839 bp, which is composed of 436 amino acid (aa) residues, with 77.8% and 22.8% identity to IFIT1 homolog of yellow perch (Perca flavescens) and humans (homo sapiens), respectively. Sequence alignment analysis showed that EcIFIT1 contained three tetratricopeptide repeats (TPRs). Tissue distribution analysis indicated that EcIFIT1 was abundant in intestine, spleen, liver, and heart. Moreover, EcIFIT1 was significantly up-regulated by Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infection, and polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) treatment in vitro. Under fluorescence microscopy, EcIFIT1 was found to localize throughout the cytoplasm in transfected cells. EcIFIT1 overexpression significantly suppressed the replication of SGIV and RGNNV, demonstrated by decreasing the cytopathic effect (CPE) severity, viral gene transcription and the virus titers. Further studies showed that the ectopic expression of EcIFIT1 increased the transcription level of IFN related molecules, including IFN regulatory factor (IRF) 3, IRF7, IFN stimulated gene (ISG) 15 and myxovirus resistance gene (MX) I. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcIFIT1. In addition, flow cytometry analysis suggested that EcIFIT1 overexpression affected cell cycle progression by mediating S/G2 transition. Taken together, our results indicated that EcIFIT1 might exert antiviral function against fish virus by up-regulating interferon response or affecting cell cycle.

Topics: Amino Acid Sequence; Animals; Bass; Carrier Proteins; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Nodaviridae; Phylogeny; Poly I-C; Ranavirus; RNA Virus Infections; Sequence Alignment

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.

Topics: Amino Acid Sequence; Animals; Autophagy-Related Proteins; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; Sequence Alignment

An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus × ♂ Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24 h, and the effects increased at 48 h postinfection. The transmission electron microscopy results showed that viral particles that were about 200 nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1 h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1 h. The analyses showed that SGIV-Gx was stable for 1 h at 4°C and 25°C but was inactivated after 1 h at 40, 50, and 60°C.

Topics: Animals; Bass; China; DNA Virus Infections; Fish Diseases; Microscopy, Electron, Transmission; Ranavirus; Spleen

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

Topics: Adaptive Immunity; Amino Acid Sequence; Animals; Base Sequence; Bass; Beclin-1; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Sequence Alignment

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

Topics: Animals; Bass; Cell Size; Dietary Supplements; Fish Diseases; Fish Proteins; Functional Food; Galactose; Gene Expression Profiling; Gene Expression Regulation, Developmental; Intestinal Mucosa; Mannans; Oligosaccharides; Software; Vibrio

We report the complete genome sequencing of the first fish peribunyavirus determined using a next-generation sequencing approach. The virus was isolated during a routine health assessment of wild largemouth bass (Micropterus salmoides) in Wisconsin in April of 2009. Further research is needed to determine the epidemiology and pathogenicity of the largemouth bass bunyavirus.

Topics: Animals; Bass; Fish Diseases; Genome, Viral; High-Throughput Nucleotide Sequencing; Orthobunyavirus

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ± 0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recomm

Topics: Aeromonas hydrophila; Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Yeast, Dried

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were

Topics: Adjuvants, Immunologic; Animals; Bass; Disease Resistance; Fish Diseases; Immunity, Innate; Lactococcus lactis; Prebiotics; Probiotics; Stramenopiles; Vibrio; Vibrio Infections

European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.

Topics: Amino Acid Substitution; Animals; Bass; Capsid Proteins; Fish Diseases; Nodaviridae; RNA Virus Infections; Virulence; Virus Replication

Tumor necrosis factor (TNF) receptor type 1-associated DEATH domain protein (TRADD) is a TNFR1-associated signal transducer and an essential component of the TNFR1 complex that is involved in activating both apoptotic and nuclear factor (NF)-κB pathways as an adaptor. It also is required for TNFR-1-initiated neuronal apoptosis following

Topics: Animals; Apoptosis; Bass; Cells, Cultured; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Immunity, Innate; Iridovirus; Sequence Analysis, DNA; TNF Receptor-Associated Death Domain Protein; Virus Replication

Macrophage aggregates (MAs) are focal accumulations of pigmented macrophages in the spleen and other tissues of fish. A central role of MAs is the clearance and destruction of degenerating cells and recycling of some cellular components. Macrophage aggregates also respond to chemical contaminants and infectious agents and may play a role in the adaptive immune response. Tissue damage or physiological stress can result in increased MA accumulation. As a result, MAs may be sensitive biomarkers of environmental stress in fish. Abundance of MAs in tissues has been reported in a variety of ways-most commonly as density, mean size, and relative area-but the utility of these estimates has not been compared. In this study, four different types of splenic MA abundance estimates (abundance score, density, relative area, and total volume) were compared in two fish populations (Striped Bass Morone saxatilis and White Perch M. americana) with a wide range in ages. Stereological estimates of total volume indicated an increase in MA abundance with spleen volume, which generally corresponded to fish age, and with splenic infections (mycobacteria or trematode parasites). Abundance scores were generally limited in the ability to detect changes in MA abundance by these factors, whereas density estimates were greatly influenced by changes in spleen volume. In some instances, densities declined while the total volume of MAs and spleen volume increased. Experimentally induced acute stress resulted in a decrease in spleen volume and an increase in MA density, although the total volume of MAs remained unchanged. Relative area estimates accounted for the size and number of MAs but not for changes in organ volume. Total volume is an absolute measure of MA abundance irrespective of changes in organ volume or patterns of accumulation and may provide an improved means of quantifying MAs in the spleens of fish.

Topics: Animals; Bass; Female; Fish Diseases; Immunologic Techniques; Macrophages; Male; Spleen; Splenic Diseases; Stress, Physiological

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.

Topics: Animals; Bass; DNA, Ribosomal Spacer; Electron Transport Complex IV; Fish Diseases; Fisheries; Nova Scotia; Nucleic Acid Conformation; Oncorhynchus mykiss; Phylogeny; RNA, Ribosomal, 18S; Salmo salar; Saprolegnia; Trout

The interactions between host and pathogen is exceedingly complex, which involves alterations at multiple molecular layers. However, research to simultaneously monitor the alterations of transcriptome and proteome between a bacterial pathogen and aquatic animal host through integrated dual RNA-seq and dual iTRAQ of tissue during infection is currently lacking. The important role of a diguanylate cyclase gene (L321_RS15240) in pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides was suggested by previous dual RNA-seq of our lab. Then L321_RS15240-RNAi strains of P. plecoglossicida were constructed with pCM130/tac, and the mutant with the best silencing effect was selected for follow-up study. The RNAi of L321_RS15240 resulted in a significant decrease in bacterial virulence of P. plecoglossicida. The E. coioides spleens infected by wild type strain or L321_RS15240-RNAi strain of P. plecoglossicida were subjected to dual RNA-seq and dual iTRAQ, respectively. The results showed that: RNAi of L321_RS15240 led to 1)alterations of host transcriptome associated with complement and coagulation cascades, ribosome, arginine and proline metabolism, and oxidative phosphorylation; 2)high expression of host proteins which related to phagosome and metabolism responses (metabolism of glutathione, amino sugar and nucleotide sugar); 3)the highly differentially expression of host lncRNAs and miRNAs. The differentially expressed proteins and mRNAs of pathogen were different after infection, but the functions of these proteins and mRNAs were mainly related to metabolism and virulence. This study provides a new insight to comprehensively understand the gene functions of pathogens and hosts at multiple molecular layers during in vivo infection.

Topics: Animals; Bass; Escherichia coli Proteins; Fish Diseases; Gene Expression Profiling; Host-Pathogen Interactions; Immunity, Innate; Phosphorus-Oxygen Lyases; Pseudomonas; Pseudomonas Infections; RNA Interference; RNA-Seq; Transcriptome; Virulence

Granzymes (Gzm) are serine proteases, contained into the secretory granules of cytotoxic cells, responsible for the cell-mediated cytotoxicity (CMC) against tumor cells and intracellular pathogens such as virus and bacteria. In fish, they have received little attention to their existence, classification or functional characterization. Therefore, we aimed to identify and evaluate their functional and transcriptomic relevance in the innate CMC activity of two relevant teleost fish species, gilthead seabream and European sea bass. Afterwards, we wanted to focus on their regulation upon nodavirus (NNV) infection, a virus that causes great mortalities to sea bass specimens while seabream is resistant. In this study, we have identified genes encoding GzmA and GzmB in both seabream and sea bass, as well as GzmM in seabream, which showed good phylogenetic relation to their mammalian orthologs. In addition, we found enzymatic activity related to tryptase (GzmA and/or GzmK), aspartase (GzmB), metase (GzmM), or chymase (GzmH) in resting head-kidney leucocytes (HKLs), with the following order of activity: GzmA/K ~ GzmM >> GzmH >>> GzmB. In addition, during innate CMC assays consisting on HKLs exposed to either mock- or NNV-infected target cells, though all the granzyme transcripts were increased only the tryptase activity did. Thus, our data suggest a high functional activity of GzmA/K in the innate CMC and a marginal one for GzmB. Moreover, GzmB activity was detected into target cells during the CMC assays. However, the percentage of target cells with GzmB activity after the CMC assays was about 10-fold lower than the death target cells, demonstrating that GzmB is not the main inductor of cell death. Moreover, in

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Granzymes; Immunity, Innate; Nodaviridae; RNA Virus Infections; Sea Bream

The mucosal surfaces of fish harbour microbial communities that can act as the first-line of defense against pathogens. Infectious diseases are one of the main constraints to aquaculture growth leading to huge economic losses. Despite their negative impacts on microbial diversity and overall fish health, antibiotics are still the method of choice to treat many such diseases. Here, we use 16 rRNA V4 metataxonomics to study over a 6 week period the dynamics of the gill and skin microbiomes of farmed seabass before, during and after a natural disease outbreak and subsequent antibiotic treatment with oxytetracycline. Photobacterium damselae was identified as the most probable causative agent of disease. Both infection and antibiotic treatment caused significant, although asymmetrical, changes in the microbiome composition of the gills and skin. The most dramatic changes in microbial taxonomic abundance occurred between healthy and diseased fish. Disease led to a decrease in the bacterial core diversity in the skin, whereas in the gills there was both an increase and a shift in core diversity. Oxytetracycline caused a decrease in core diversity in the gill and an increase in the skin. Severe loss of core diversity in fish mucosae demonstrates the disruptive impact of disease and antibiotic treatment on the microbial communities of healthy fish.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Disease Outbreaks; Fish Diseases; Gills; Gram-Negative Bacterial Infections; Microbiota; Oxytetracycline; Photobacterium; Skin

Amyloodinium ocellatum infects the gills and skin of both marine and brackish water fishes. The aim of the present study was to examine pathogenesis, prevalence, trials for treatment and histopathological alterations of Amyloodinosis in naturally infested Asian Seabass Barramundi Lates calcarifer and Hamour Epinephelus polyphekadion in Ismailia Governorate, Egypt.. A total number of 1447 Red Sea cultured Seabass (Lates calcarifer) broadstock and a total number of 53 Red Sea cultured Hamour, Epinephelus polyphekadion broadstock were collected and subjected for the study. Fishes showed symptoms of sudden death and respiratory distress besides Amyloodiniosis on gills and skin. All fishes were treated with various treatment protocols while gills of naturally infected fishes were examined histopathologically.. The clinical signs of infested fishes were flashing, surfacing, off food and respiratory distress. The intensity of infestation of Amyloodiniosis was more sever in Asian Seabass than Epinephelus polyphekadion while treatment of choice was copper sulphate (prolonged bath), freshwater bath and formalin consequently.. Treatment of choice for Amyloodinium ocellatum infestation in Asian Seabass was copper sulphate (prolonged bath) followed by freshwater bath then formalin.

Topics: Alveolata; Animals; Bass; Copper Sulfate; Egypt; Fish Diseases; Fishes; Formaldehyde; Fresh Water; Gills; Perciformes; Skin

Topics: Animals; Bass; Escherichia coli Proteins; Fish Diseases; HSP70 Heat-Shock Proteins; Immunologic Factors; Larva; Recombinant Proteins; Vibrio Infections

Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described. Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro. In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV. Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus. These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.

Topics: Animals; Bass; Cross Protection; Fish Diseases; Injections, Intraperitoneal; Nodaviridae; RNA Virus Infections; Vaccination

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.

Topics: Animals; Bass; Dinoflagellida; Disease Outbreaks; Fish Diseases; Fish Proteins; Gene Expression; Gills; Head Kidney; Protozoan Infections, Animal

The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cathepsin C; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Hemorrhagic Disease Virus, Epizootic; Immunity, Innate; Phylogeny; Reoviridae Infections; Sequence Alignment

Disease outbreaks and mortalities caused by largemouth bass virus (LMBV) in largemouth bass ( Micropterus salmoides) have been reported in the US. Blood and mucus samples tested by PCR to assess the presence of LMBV in largemouth bass in northeastern Mexico were negative, and further monitoring is needed.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Iridoviridae; Mexico

In mammals, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial intracellular adaptor protein, which performs a vital role in numerous signaling pathways that activate NF-κB, MAPKs, and IRFs. In the present study, three TRAF2 sequences were identified from the orange-spotted grouper (Epinephelus coioides), and named EcTRAF2-1, EcTRAF2-2, and EcTRAF2-3. These sequences contained conserved structure features that were similar to those of mammals. EcTRAF2-1 shared relatively low sequence identity with the other two EcTRAF2s. In healthy E. coioides, EcTRAF2s were widely expressed in all tissues tested, but with distinct expression profiles. After infection with Cryptocaryon irritans, EcTRAF2s was markedly upregulated in the gill and head kidney at most time points, implying that EcTRAF2s may be involved in host defense against C. irritans infection. In HEK293T cells, EcTRAF2s were scattered in the cytoplasm. EcTRAF2-1 and EcTRAF2-2 increased the activity of NF-κB, while EcTRAF2-3 reduced NF-κB activation mediated by EcTRAF2-1 implying that EcTRAF2-3 might be a negative regulator of EcTRAF2-1.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; HEK293 Cells; Humans; Immunity, Innate; Phylogeny; Random Allocation; TNF Receptor-Associated Factor 2

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2-1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density-gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral-like particles (~100-180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%-99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow-out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.

Topics: Animals; Aquaculture; Bass; DNA Virus Infections; Fish Diseases; Iridoviridae; Microscopy, Electron, Transmission; Polymerase Chain Reaction; Thailand

MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases, which act as an integration point by transferring extracellular signals to the nucleus. To investigate the function of MEK in teleost fish, we cloned MEK1 and MEK2 cDNA sequences from the orange-spotted grouper (Epinephelus coioides). EcMEK1 and EcMEK2 shared 80% amino acid identity with each other. EcMEK1 had 89-99% amino acid identity with teleosts or mammals, whereas EcMEK2 shared 85-97% amino acid identity. The exon structures of the grouper MEK1/2 genes were conserved with zebrafish and human MEK1/2. Tissue distribution analysis showed that EcMEK1 and EcMEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs. Both EcMEK1 and EcMEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells. Overexpression of EcMEK1 or EcMEK2 activated Activator protein 1 dependent luciferase. The phosphorylation levels of EcMEK1/2 and EcERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment. The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Phylogeny; Sequence Alignment

Probiotics are widely used for the improvement of animals' growth and health. However, few marine aquatic probiotics are applied and licensed in China. In this study, a Bacillus spp. strain was isolated from the Hulong grouper gastrointestinal tract, which was identified as a new strain of Bacillus subtilis and was named as 7k. B. subtilis 7k showed desirable capability of sporulation and resistance to heat, simulated gastric juice and simulated duodenum juice, indicating its potential as probiotics. Seven antimicrobial chemicals were found in the secretion of the B. subtilis 7k. B. subtilis 7k addition in diet promoted the growth rate of Hulong groupers. Moreover, B. subtilis 7k can inhibit infection by iridovirus, making B. subtilis 7k a suitable kind of probiotic for maintaining fishes' health. Our results also revealed that B. subtilis 7k induced non-specific immune response in Hulong grouper under virus infection. Hulong grouper fed by diets containing B. subtilis 7k at 10

Topics: Aeromonas hydrophila; Animal Feed; Animals; Bacillus subtilis; Bass; Diet; DNA Virus Infections; Fish Diseases; Gastrointestinal Tract; Hybridization, Genetic; Immunity, Innate; Micrococcus; Probiotics; Ranavirus; Staphylococcus aureus; Vibrio

Nervous necrosis virus (NNV) causes viral nervous necrosis (VNN), a disease that leads to almost 100% mortality among larvae and juvenile fish, severely affecting the aquaculture industry. VNN vaccines based on inactivated viruses or virus-like particles (VLPs) are unsuitable for fish fry with immature adaptive immune systems. Here, we applied an anti-NNV strategy based on affinity peptides (AFPs). Three phage display peptide libraries were screened against RBS, the VLP of orange-spotted grouper nervous necrosis virus (OGNNV). From the positive clones, a dodecapeptide with the highest binding capacity (BC) to RBS was selected. This AFP agglutinated or disrupted virion particles, inhibiting RBS entry into sea bass (SB) cells. To enhance BC and solubility, we amended the AFP sequence as "LHWDFQSWVPLL" and named as 12C. One to three copies of 12C in tandem were prokaryotically expressed with a maltose binding protein (MBP) linked by a flexible peptide. Of the recombinant proteins expressed, MBP-triple-12C (MBP-T12C) exhibited the highest BC, efficiently blocked RBS entry, and strongly inhibited OGNNV infection at viral entry. Moreover, MBP-T12C bound the VLPs of all NNV serotypes, displaying broad-spectrum anti-NNV ability, and recognized only OGNNV and mud crab virus, demonstrating binding specificity. Therefore, these anti-NNV AFPs specifically bound NNV, aggregating or disrupting the viral particles, to reduce the contact probability between the virus and cell surface, subsequently inhibiting viral infection. Our results not only provided a candidate of anti-NNV AFP, but a framework for the development of antiviral AFP.

Topics: Animals; Antiviral Agents; Bass; Fish Diseases; Fish Proteins; Nodaviridae; Peptides; Recombinant Proteins; RNA Virus Infections; Virus Internalization

Vibrio alginolyticus is well-known as an opportunistic Gram-negative pathogen, which endangers the development of global aquaculture as well as human health. In this study, a ΔacfA mutant strain and complementation of the ΔacfA mutant (C-acfA) were constructed. The ΔacfA mutant was tested in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the ΔacfA mutant caused a high antibody titer and a significant reduction in the ability to colonize the intestine of pearl gentian grouper. Grouper vaccinated with ΔacfA mutant were more tolerant of the infection by virulent V. alginolyticus HY9901 without inducing clinical symptoms and obvious pathological changes. The relative percent survival value of pearl gentian grouper vaccinated with ΔacfA mutant intraperitoneal injection reached 81.1% after challenging with V. alginolyticus HY9901. The specific antibody titers immunized with ΔacfA was significantly higher than that in the PBS group. The antibody titer of ΔacfA group displayed the tendency of rising up from the first to fourth week and declining from fifth to eighth week and reached the peak at the fourth week. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, TNF-α, IL-16, IgM, CD8α and MHC-Iα, was up-regulated after vaccination, indicating that the ΔacfA can induce effective and durable immune response in pearl gentian grouper and it may be an effective attenuated live vaccine candidate for the prevention of infections by V. alginolyticus.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Vaccination; Vaccines, Attenuated; Vibrio alginolyticus; Vibrio Infections

Virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (viperin), is an antiviral protein, induced by interferon (IFN), poly(I:C) and viral infection to exert antiviral function. To investigate the roles of viperin during fish virus infection, a viperin homolog from orange spotted grouper (Epinephelus coioides) (Ecviperin) was cloned and characterized in this study. Ecviperin encoded a 361-aa protein which shared 87% and 69% identity with Siniperca undulata and Homo sapiens, respectively. Amino acid alignment analysis showed that Ecviperin contained a conserved radical-SAM domain (aa73-281). Phylogenetic analysis indicated that Ecviperin showed the nearest relationship with S. undulata. In healthy grouper, Ecviperin was distributed in all tissues, and the expression of Ecviperin was the highest in kidney and spleen. In vitro, the mRNA expression of Ecviperin was significantly up-regulated in response to Singaporean grouper iridovirus (SGIV) infection. Subcellular localization analysis showed that Ecviperin was distributed in the cytoplasm and co-localized with endoplasmic reticulum (ER). The ectopic expression of Ecviperin significantly inhibited the replication of SGIV. Furthermore, overexpression of Ecviperin positively regulated the interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), myxovirus resistance gene I (MXI), interferon-induced 35-kDa protein (IFP35), and TNF receptor-associated factor 6 (TRAF6). In addition, the expression of pro-inflammation cytokines was differently regulated by Ecviperin overexpression. Furthermore, reporter gene analysis showed that the overexpression of Ecviperin enhanced the activity of nuclear factor of kappa B (NF-κB), IFN-1 and interferon-stimulated response element (ISRE) promoter, suggesting that Ecviperin might restrict SGIV replication by the positive regulation of interferon and inflammatory response. Taken together, our results demonstrated that Ecviperin encoded an ER-localized protein, and exerted antiviral function against fish DNA virus by up-regulating interferon and pro-inflammatory response.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Interferon Regulatory Factors; Interferons; Iridovirus; Phylogeny; Sequence Alignment

Macrophage migration inhibitory factor (MIF) is a cytokine playing critical roles in inflammatory and immune responses. However, its functions have not been well studied in fish. In this study, we identified a MIF molecule from Japanese sea bass (Lateolabrax japonicus; LjMIF). Multiple sequence alignment showed that LjMIF has the typical structural features of MIFs. Phylogenetic tree analysis revealed that LjMIF is most closely related to the yellowfin tuna (Thunnus albacares), large yellow croaker (Larimichthys crocea), and red drum (Sciaenops ocellatus) homologs. Constitutive mRNA expression of LjMIF was detected in all tested tissues, with the highest level in the liver. Upon Vibro harveyi infection, LjMIF transcripts were altered in the tested tissues, including the liver, spleen, and head kidney. Subsequently, we prepared recombinant LjMIF (rLjMIF) and the corresponding antibody (anti-LjMIF). The in vitro study showed that rLjMIF inhibited the trafficking of Japanese sea bass monocytes/macrophages (MO/MΦ) and lymphocytes, but not of neutrophils, while anti-LjMIF had the opposite effect. rLjMIF also enhanced phagocytosis and intracellular killing of V. harveyi by MO/MΦ, while anti-LjMIF only inhibited phagocytosis by MO/MΦ. The in vivo study showed that rLjMIF aggravated the course of V. harveyi infection in Japanese sea bass, but anti-LjMIF increased the survival rate of the fish and decreased the bacterial burden. In conclusion, our observation revealed that LjMIF is closely involved in the immune responses of Japanese sea bass for combating V. harveyi infection.

Topics: Animals; Antibodies; Bass; Fish Diseases; Fish Proteins; Leukocytes; Macrophage Migration-Inhibitory Factors; Phagocytosis; Phylogeny; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Vibrio; Vibrio Infections

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Iridovirus; Nodaviridae; RNA Virus Infections; SUMO-1 Protein; Ubiquitins

This study investigated the environmental factors associated with the presence of Vibrionaceae in economically important cage-cultured tropical marine fishes: the Asian Seabass Lates calcarifer, snapper Lutjanus sp., and hybrid grouper Epinephelus sp. Fish sampling was conducted at monthly intervals between December 2016 and August 2017. The body weight and length of individual fish were measured, and the skin, eye, liver, and kidney were sampled for bacterial isolation and identification. Water physicochemical parameters during the sampling activities were determined, and the enumeration of total Vibrionaceae count was also conducted from water and sediment samples. Nine species of Vibrio were identified, including V. alginolyticus, V. diabolicus, V. harveyi, V. campbellii, V. parahaemolyticus, V. rotiferianus, V. furnissii, V. fluvialis, and V. vulnificus. Photobacterium damselae subsp. damselae was also identified. A total of 73% of the isolated Vibrio belonged to the Harveyi clade, followed by the Vulnificus clade (5.5%) and Cholera clade (0.6%). Highest occurrence of Vibrio spp. and P. damselae subsp. damselae was found in hybrid grouper (72%), followed by Asian Seabass (48%) and snapper (36%). The associations of Vibrio spp. and P. damselae subsp. damselae with the host fish were not species specific. However, fish mortality and fish size showed strong associations with the presence of some Vibrio spp. On average, 60% of the infected cultured fish exhibited at least one clinical sign. Nevertheless, inconsistent associations were observed between the pathogens and water quality. The yearlong occurrence and abundance of Vibrionaceae in the environmental components indicate that they might serve as reservoirs of these pathogens.

Topics: Animals; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Perciformes; Photobacterium; Vibrio; Vibrio Infections

Vibrio parahaemolyticus is the major pathogen of vibriosis in aquatic animals and causes inflammation that may be related to tissue damage. Here, we have established a V. parahaemolyticus flagellin stimulation model using grouper spleen (GS) cell line. Purified V. parahaemolyticus flagellin was used to stimulate GS cells. Our results showed that the mRNA levels of orange-spotted grouper (Epinephelus coioides) toll-like receptor 5M (EcTLR5M), EcTLR5S and downstream cytokines, such as interferon-γ2 (IFN-γ2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were all significantly increased after stimulation with V. parahaemolyticus flagellin in GS cells. Gene silencing of the EcTLR5M and EcTLR5S in GS cells by using small interfering RNA resulted in suppression of the V. parahaemolyticus flagellin-induced cytokines expression. We further demonstrated that activation of both mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were required for cytokines expression. We observed that the phosphorylation of NF-κB inhibitor-α (IκBα), extracellular signal-regulated kinase (ERK) and p38 were induced following treatment with flagellin. Additionally, most of p65, a NF-κB subunit, was found to translocate to the nucleus after 60 min stimulation. Overall, our results suggest that V. parahaemolyticus flagellin influences cytokines expression, such as IFN-γ2, IL-6 and TNF-α, via EcTLR5s recognition and MAPKs/NF-κB signaling pathway activation in GS cells.

Topics: Animals; Bass; Cytokines; Fish Diseases; Fish Proteins; Flagellin; Gene Expression; Signal Transduction; Toll-Like Receptor 5; Vibrio Infections; Vibrio parahaemolyticus

The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4 days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V. campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.

Topics: 4-Butyrolactone; Animals; Bass; Fish Diseases; Homoserine; Lactones; Larva; Mutation; Quorum Sensing; Vibrio; Virulence

Phosphorylation of eukaryotic initiation factor 2 alpha subunit (eIF2α) occurs under a variety of conditions, including viral infection. Heme-regulated inhibitor (HRI) is an eIF2α kinase that modifies this phosphorylation. In this study, a HRI homologue (EcHRI) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EcHRI encodes a 664-amino acid polypeptide that shares a high degree of similarity with HRIs from other species. Quantitative real-time polymerase chain reaction analysis indicated that EcHRI was distributed in all examined tissues. Expression of EcHRI in the spleen of E. coioides was up-regulated when challenged with the synthetic analog of double-stranded RNA (dsRNA) of polyinosine-polycytidylic acid (poly I:C). EcHRI was significantly increased in red-spotted grouper nervous necrosis virus (RGNNV) infected cells. EcHRI was abundantly distributed in the nucleus of grouper spleen (GS) cells. Overexpression of EcHRI inhibited the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. Furthermore, our results showed that EcHRI overexpression significantly increased the expression of interferon (IFN)-related cytokines and enhanced activation of IFN-β, interferon-sensitive response element (ISRE), and nuclear factor κB (NF-κB). Taken together, these results suggest that EcHRI is involved in the fish immune response to virus challenge.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Protein Serine-Threonine Kinases; RNA Virus Infections; Sequence Alignment

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a key adapter molecule that participates in numerous signaling pathways. The function of TRAF5 in fish is largely unknown. In the present study, a TRAF5 cDNA sequence (EcTRAF5) was identified in grouper (Epinephelus coioides). Similar to its mammalian counterpart, EcTRAF5 contained an N-terminal RING finger domain, a zinc finger domain, a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. The EcTRAF5 protein shared relatively low sequence identity with that of other species, but clustered with TRAF5 sequences from other fish. Real-time PCR analysis revealed that EcTRAF5 mRNA was broadly expressed in numerous tissues, with relatively high expression in skin, hindgut, and head kidney. Additionally, the expression of EcTRAF5 was up-regulated in gills and head kidney after infection with Cryptocaryon irritans. Intracellular localization analysis demonstrated that the full-length EcTRAF5 protein was uniformly distributed in the cytoplasm; while a deletion mutant of the coiled-coil domain of EcTRAF5 was observed uniformly distributed in the cytoplasm and the nucleus. After exogenous expression in HEK293T cells, TRAF5 significantly activated NF-κB. The deletion of the EcTRAF5 RING domain or of the zinc finger domain dramatically impaired its ability to activate NF-κB, implying that the RING domain and the zinc finger domain are required for EcTRAF5 signaling.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Sequence Alignment; Signal Transduction; TNF Receptor-Associated Factor 5

Topics: Animals; Anti-Bacterial Agents; Bass; California; Disease Outbreaks; Edwardsiella; Enterobacteriaceae Infections; Fish Diseases; Kidney; Microbial Sensitivity Tests; Sepsis; Splenomegaly

In March and April 2016, 150 white perch ( Morone americana) were collected from various localities in Chesapeake Bay and examined for coccidia. A previously undescribed species of coccidia was observed in the hepatic bile ducts and gallbladder of all white perch (100%) examined. We describe this species using morphological characteristics, histology, and gene sequences of the small-subunit ribosomal DNA ( rDNA), large-subunit rDNA, and mitochondrial genes cytochrome oxidase 1 ( COI), cytochrome oxidase b ( Cytb), and cytochrome oxidase 3 ( COIII). Oocysts of Goussia bayae n. sp. were subspherical with a single-layered smooth wall and measured (length [L] × width [W]) 26.2 × 21.8 μm, with a L/W ratio of 1.2. A micropyle was present but a micropyle cap, polar granules, and oocyst residuum were absent. Each oocyst contained 4 sporocysts that were ellipsoidal and measured (L × W) 12.6 × 7.8 μm, with a L/W ratio of 1.6. A pair of sporozoites was present, but sporocysts lacked a Stieda body and residuua. Meronts and gamonts were epicellular in biliary epithelial cells and oocysts were coelozoic in hepatic and common bile ducts and gallbladder. This is the first report of Goussia spp. from white perch and the first mitochondrial DNA sequence reported from a Goussia species. Phylogenetic analysis indicates basal placement of G. bayae to Eimeriidae, Choleoeimeria, and Sarcocystidae.

Topics: Animals; Bass; Bays; Bile Ducts, Intrahepatic; Coccidiosis; DNA, Ribosomal; Eimeriidae; Electron Transport Complex IV; Female; Fish Diseases; Gallbladder; Male; Maryland; Mitochondria; Oocysts; Phylogeny; Rivers; Virginia

Chemokine receptors are a superfamily of seven-transmembrane domain G-coupled receptors and have important roles in immune surveillance, inflammation, and development. In previous studies, a series of CXCRs in grouper (Epinephelus coioides) was identified; however, the function of CXCR in viral infection has not been studied. To better understand the effect of the CXCR family on the fish immune response, full-length CXCR1a was cloned, and its immune response to Singapore grouper iridovirus (SGIV) was investigated. Grouper CXCR1a shared a seven-transmembrane (7-TM) region and a G protein-coupled receptor (GPCR) family 1 that contained a triaa stretch (DRY motif). Phylogenetic analysis indicated that CXCR1a showed the nearest relationship to Takifugu rubripes, followed by other fish, bird and mammal species. Fluorescence microscopy revealed that CXCR1a was expressed predominantly in the cytoplasm. Overexpression of CXCR1a in grouper cells significantly inhibited the replication of SGIV, demonstrating that CXCR1a delayed the occurrence of cytopathic effects (CPE) induced by SGIV infection and inhibited viral gene transcription. Furthermore, our results also showed that CXCR1a overexpression significantly increased the expression of interferon-related cytokines and activated ISRE and IFN promoter activities. Taken together, the results demonstrated that CXCR1a might have an antiviral function against SGIV infection.

Topics: Animals; Bass; Cytokines; DNA Virus Infections; Fish Diseases; Microscopy, Fluorescence; Phylogeny; Ranavirus; Receptors, Chemokine; Virus Replication

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.

Topics: Animals; Apoptosis; Base Sequence; Bass; Cell Line; Cloning, Molecular; Cystatin C; Fish Diseases; Fish Proteins; Iridovirus; Spleen; Up-Regulation

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune r

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Regulatory Factors; Interferons; Iridovirus; Phylogeny; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Spleen; Tripartite Motif Proteins; Zebrafish

Viral necrosis virus (NNV) or nodavirus causes fish viral encephalopathy and retinopathy worldwide. In some cases, mortalities in aquaculture industry can reach up to 100%, some species being especially sensitive as is the case of European sea bass (Dicentrarchus labrax), one of the main cultured species in the Mediterranean, with the consequent economical loses. Development of new vaccines against NNV is in the spotlight though few researches have focused in European sea bass. In this study we have generated a recombinant NNV (rNNV) vaccine produced in Escherichia coli expressing the capsid protein and administered it to European sea bass juveniles by two different routes (intraperitoneal and oral). The last being considered non-stressful and desired for fish farming of small fish, which in fact are the most affected by NNV. Oral vaccine was composed of feed pellets containing the recombinant whole bacteria, and injected vaccine was composed of recombinant bacteria previously lysed. Our results revealed production of specific anti-NNV IgM following the two vaccination procedures, levels that were further increased in orally-vaccinated group after challenge with NNV. Genes related to interferon (IFN), T-cell and immunoglobulin markers were scarcely regulated in head-kidney (HK), gut or brain. Vaccination by either route elicited a relative survival response of 100% after NNV challenge. To our knowledge, this is the first report of a recombinant vaccine followed by no purification steps which resulted in a complete protection in European sea bass when challenged with NNV.

Topics: Administration, Oral; Animals; Antibodies, Viral; Aquaculture; Bass; Escherichia coli; Fish Diseases; Immunity, Humoral; Injections, Intraperitoneal; Nodaviridae; RNA Virus Infections; Vaccination; Vaccines, Synthetic; Viral Vaccines

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (

Topics: Animals; Antiviral Agents; Bass; Cytokines; Endoplasmic Reticulum; Fish Diseases; Fish Proteins; Immunity; Inflammation; Interferon-gamma; Iridovirus; Perciformes; Poly I-C; Signal Transduction; Steroid Hydroxylases; Transcription, Genetic; Virus Internalization; Virus Replication

Transducin β-like 1 X-linked receptor 1 (TBLR1) was identified as an important component of nuclear receptor corepressor (N-CoR) complex, and functionally participated in regulation of transcriptional activation. However, the potential roles of TBLR1 in innate immune response still remain uncertain. In the present work, a novel TBLR1 from orange-spotted grouper, Epinephelus coioides (named as EcTBLR1) was cloned and its effect on fish virus infection was characterized. The full length open reading frame (ORF) of EcTBLR1 was 1548 bp and encoded a putative 515-aa polypeptide, which shared 99% and 95% identity with its homologue from large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Quantitative PCR (qPCR) analysis revealed a ubiquitous expression of EcTBLR1 in different tissues with remarkable expression in brain, spleen and head-kidney. Subcellular location analysis showed that EcTBLR1 was mainly located in cytoplasm of grouper spleen cells, and partly translocated into nucleus after infection with red spotted grouper nervous necrosis virus (RGNNV). Moreover, RGNNV infection suppressed the protein synthesis of EcTBLR1 in grouper cells. Using RNA interference (RNAi) technology, we found that effective knock-down of EcTBLR1 significantly suppressed the transcription of RGNNV capsid protein (Cp) and RNA-dependent RNA polymerase (RdRp) genes, which implied the crucial role of EcTBLR1 in RGNNV infection. Consistently, overexpression of EcTBLR1 in vitro significantly inhibited IFN promoter activity, as well as the transcription of IFN-related downstream effectors, including interferon stimulated gene 15 (ISG15) and interferon regulatory factor 3 (IRF3). Together, our results for the first time demonstrated that fish TBLR1 might exert critical roles during fish RNA virus replication by negatively regulating interferon response.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Interferons; Nodaviridae; Phylogeny; Repressor Proteins; RNA Virus Infections; Sequence Alignment

Vibrio alginolyticus is an opportunistic and halophilic Gram-negative pathogen in limiting the development of aquatic industry and affecting human health. SODs are oxidative enzymes that play a critical role in oxidative defense. In this study, an in-frame deleted mutant strain (ΔsodB) was constructed by allelic exchange mutagenesis to investigate physiological role of sodB in pathogenicity of V. alginolyticus. The results exhibited that ΔsodB showed no differences in growth compared with wild-type strain HY9901 (WT), but led to increasing in biofilm formation, ECPase activity and sensitivity to hydrogen peroxide, decreasing in swarming motility, adherence to CIK cells, SOD activity and virulence. In addition, ΔsodB induced a high antibody titer and provided a valid protection with a relative percent survival value of 86.5% without inducing clinical symptoms after challenging with WT. These results suggest that sodB is important for normal physiological function, oxidation resistance and virulence in V. alginolyticus, and ΔsodB may be considered as an effective live attenuated vaccine against V. alginolyticus.

Topics: Animals; Antioxidants; Bacterial Proteins; Bacterial Vaccines; Bass; Fish Diseases; Mutagenesis; Stress, Physiological; Superoxide Dismutase; Vaccines, Attenuated; Vibrio alginolyticus; Vibrio Infections; Virulence; Virulence Factors

Back Bay is an oligohaline, coastal bay in southeast Virginia, USA. Since 2004, leeches have been observed in the oral cavities of largemouth bass (Micropterus salmoides) in this body of water. Leeches (Myzobdella lugubris) have previously been documented in the oral cavities of largemouth bass in the Currituck Sound, which is confluent with Back Bay on its southern border. Supplemental stocking of largemouth bass in Back Bay since 2009 has resulted in an increasing population; however, concern exists that leech infestation may be negatively affecting health of larger fish, which are still less abundant than expected. Despite the wide distribution of this leech, there is little available literature regarding its health impacts on hosts. In this study, we examine potential impacts of oral leech infestations on stress markers and haematological parameters of largemouth bass in Back Bay. No significant changes in plasma glucose or cortisol were observed between leech-infested and uninfested fish, and haematological parameters were not significantly different between the groups. Further, there was no evidence of systemic infections associated with leech infestation.

Topics: Animals; Bass; Ectoparasitic Infestations; Fish Diseases; Leeches; Mouth; Prevalence; Virginia

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; CD59 Antigens; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Sequence Alignment; Vibrio alginolyticus; Vibrio Infections

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Topics: Animals; Bacterial Proteins; Bacterial Vaccines; Bass; Fish Diseases; Pseudomonas; Pseudomonas Infections; Vaccination; Vaccines, Attenuated

Megalocytiviruses, particularly red seabream iridovirus, infect a broad range of fish including both freshwater and marine species. Although a limited number of infectious spleen and kidney necrosis virus (ISKNV) strains have been reported in association with mortality events in marine aquaculture species, the potential host range for ISKNV strains, particularly of those that have been detected in ornamental fish, has not been well characterised. There have also been few reports on the susceptibility of euryhaline fish species that could potentially transmit megalocytiviruses between freshwater and marine environments. We found that the euryhaline Australian native percichthyid fish, Australian bass Macquaria novemaculeata, is susceptible experimentally to ISKNV (strain DGIV-10), obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. Australian bass developed clinical disease following direct inoculation and also following cohabitation with infected fish, and were able to transmit DGIV-10 to naïve Murray cod Maccullochella peelii. This study demonstrated the potential for a euryhaline species to become infected with, and transmit, the megalocytivirus ISKNV between fish populations.

Topics: Animals; Australia; Bass; Disease Vectors; DNA Virus Infections; Fish Diseases; Iridoviridae; Phylogeny

Streptococcus agalactiae (Group B Streptococcus, GBS) is emerging as a genetically diverse species infecting farmed and wild fish, including commercially and culturally important groupers. To better understand how S. agalactiae are pathogenic in fish, we investigated interactions between isolates from fish and terrestrial hosts and the cellular immune system of Queensland grouper Epinephelus lanceolatus using flow cytometry. Adherent head-kidney leucocytes (HKL) from Queensland grouper displayed two main cell populations with distinct forward and side scatter by flow cytometry. The population of smaller and less complex cells (P1) was composed of monocytes, lymphocytes and thrombocytes, while the population of primarily larger and more complex cells (P2) comprised predominantly of macrophages and neutrophils. The cells in P2 had higher phagocytic index and capacity when incubated with fluorescent latex beads. HKL were activated by phorbol myristate acetate (PMA) but were unresponsive to lipopolysaccharide (LPS) and peptidoglycan (PTG), suggesting the absence of specific receptors on the surface of these cells for these ligands or a requirement for intermediates. In in vitro phagocytosis assays, all fish isolates of GBS activated a respiratory burst in P2 indicated by significant production of intracellular reactive oxygen species (ROS). Similarly, dog and cat isolates of different serotype and sequence type also induced ROS production in grouper HKL. However, human, crocodile and bovine isolates of GBS did not elicit significant ROS in HKL although they coincided with the highest phagocytic index. This suggests that these strains are capable of quenching ROS production. Terrestrial isolates significantly increased mortality of Queensland grouper leucocytes in vitro, aligned with a more diverse repertoire of cellular toxins in these strains. Opsonisation of a marine strain and terrestrial strain of GBS with antiserum raised against the marine strain resulted in an increase in ROS production by HKL in both cases although there was low antigenic cross reactivity between the two strains by flow cytometry, reflecting their diverse serotypes (Ib vs III). However, pre-incubation of either strain with normal serum from grouper also increased ROS production of HKL suggesting other opsonins may be involved. Based on these results it appears that piscine and terrestrial GBS isolates have contrasting strategies when interacting with the cellular immune system of Quee

Topics: Animals; Bass; Fish Diseases; Flow Cytometry; Head Kidney; Leukocytes; Streptococcal Infections; Streptococcus agalactiae

A 10-week growth trial was conducted to investigate the effects of replacing dietary fishmeal with plant proteins on nutrition metabolism, immunity, inflammation and apoptosis responses in liver tissues of Japanese seabass, Lateolabrax japonicas (initial body weight = 10.42 ± 0.01 g). Two isonitrogenous and isoenergetic diets were formulated. A basal diet containing 54% fishmeal (FM), whereas another diet was prepared by totally replacing FM with a plant protein blend (PP) composed with soybean protein concentrate and cottonseed protein concentrate. Although essential amino acids, fatty acids, and available phosphorus had been balanced according to the FM diet profile, the significantly lower growth performance, metabolic disorder, and fatty liver symptom were observed in the PP group. Compared with the FM group, fish in the PP group showed significantly lower plasma free EAA level and PPV. Glucose metabolism disorder was expressed as the uncontrollable fasting glycolysis and pyruvate aerobic oxidation at postprandial 24 h with significantly up-regulated GK, PK and PDH genes expression, which potentially over-produced acetyl-CoA as the substrate for protein and lipid synthesis. Significantly reduced plasma GLU, but increased GC level, along with very significantly reduced liver GLY storage could be observed in the PP group. Plasma TG and hepatic NEFA contents were significantly decreased, but the hepatic TC content was very significantly increased in the PP group, in addition, hepatocyte vacuolation appeared. The significantly up-regulated cholesterol synthesis gene (HMGCR) expression but down-regulated bile acid synthesis gene (CYP7A1) expression could be the main reason for the fatty liver induced by cholesterol accumulation. The reduced plasma IgM content accompanied by the up-regulated mRNA levels of pro-inflammatory cytokines (TNFα and IL1β) and activated apoptosis signals of liver tissues were found in the PP group. The hyperthyroidism (higher plasma T3 and T4) and the accelerated energy metabolism rate decreased the growth performance in the PP group. The activated p65NF-kB may promote the hepatocytes apoptosis via the extrinsic pathway (caspase8/caspase3). Simultaneously, a "self-saving" response could be observed that activated cAMP promoted the lipolysis/β-oxidation process and up-regulated gene expression of anti-inflammatory cytokine IL10 via promoting CREB expression, further inhibited the over-phosphorylation of JNK protein, which might impe

Topics: Animal Feed; Animals; Basal Metabolism; Bass; Diet; Fatty Liver; Fish Diseases; Liver; Metabolic Diseases; Plant Proteins, Dietary; Signal Transduction

The aim of this study was to evaluate the effect of trypanosomes on cultured largemouth bass (Micropterus salmoides) and describe the taxonomic identification of the parasite. The effects of the parasite on M. salmoides were examined based on clinical symptoms, hemograms, histopathology, and serum biochemistry. Diseased fish showed typical clinical symptoms of trypanosomiasis, which included lethargy, anorexia, and histopathological lesions in the liver, head kidney, and spleen. The serum of diseased fish had significantly lower concentrations of glucose, triglyceride, and low-density lipoprotein, and significantly higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities. The morphology of the trypanosomes was also analyzed using light microscopy, and their 18S rDNA sequence was analyzed to establish genetic relationships with other known strains. We found that the trypomastigote form of the trypanosomes from M. salmoides was similar to those isolated from Pelteobagrus fulvidraco. The trypanosomes had a slender and narrow body with a relatively long free flagellum, not well-developed undulating membrane, and an oval kinetoplast located near the subterminal posterior end of the body. The 18S rDNA sequences of the trypanosome from M. salmoides had the highest similarity (99.8%) with that of P. fulvidraco, suggesting they are identical species. Based on the differences in morphological characteristics and 18S rDNA sequence compared to trypanosomes isolated from other freshwater fish, it is considered as a new species and we propose the name Trypanosoma micropteri n. sp.

Topics: Animals; Bass; Catfishes; China; DNA, Ribosomal; Fish Diseases; Fresh Water; Phylogeny; Trypanosoma; Trypanosomiasis

In recent years, with the rapid development of marine farming activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing heavy economic losses. Singapore grouper iridovirus (SGIV) is one of the most important viruses causing disease in fish. In the present study, we isolated and identified a virus from diseased groupers by coculturing the affected tissue cells with grouper spleen cells. The genome of the isolated virus shared 99.83% nucleotide sequence homology with those of SGIV reference strains in the GenBank database. The virus clustered with SGIV on an evolutionary tree constructed based on "major capsid protein" (MCP) amino acid sequences, so it was designated 'Singapore grouper iridovirus Hainan' (SGIV-HN). To evaluate the pathogenic potential of SGIV-HN in fish, orange-spotted groupers were infected by intraperitoneal injection with the virus. Infected groupers began to die from the fourth day after infection, and survivors tended to be stable by the eighth day. The death rate was 83.33%. In a mock-infected control group, only two fish died, and the mortality rate was 6.67%. Dissection showed that the fish had enlarged spleens with hemorrhage, and enlarged cells were visible with Giemsa staining. This is the first report of isolation of SGIV from naturally infected fish in China, and we show that SGIV-HN is highly infectious, causing massive deaths in groupers.

Topics: Amino Acid Sequence; Animals; Bass; Capsid Proteins; Cells, Cultured; China; Fish Diseases; Fishes; Genome, Viral; Iridovirus; Spleen

N-Carbamylglutamate (NCG), an analogue of N-acetylglutamate (NAG), can promote the synthesis of endogenous Arginine (Arg) in mammals, but not well studied in fish. This study was conducted to investigate the capacity of Arg endogenous synthesis by NCG, and the effects of various dietary NCG doses on growth performance, hepatic health and underlying nutrient regulation metabolism on ERK1/2-mTOR-S6K1 signaling pathway in Japanese seabass (Lateolabrax japonicus). Four experimental diets were prepared with NCG supplement levels of 0 (N0), 360 (N360), 720 (N720) and 3600 (N3600) mg/kg, in which N360 was at the maximum recommended level authorized by MOA, China in fish feed, and the N720 and N3600 levels were 2 and 10-fold of N360, respectively. Each diet was fed to 6 replicates with 30 Japanese seabass (initial body weight, IBW = 11.67 ± 0.02 g) in each tank. The results showed that the dietary NCG supplementation had no significant effects on the SGR and morphometric parameters of Japanese seabass, but 360-720 mg/kg NCG inclusion promoted PPV, while the 10-fold (3600 mg/kg) overdose of NCG had remarkably negative effects with significantly reduced feed efficiency, PPV and LPV. We found that Japanese seabass can utilize 360-720 mg/kg NCG to synthesis Arg to improve the amino acid metabolism by increasing plasma Arg and up-regulating intestinal ASL gene expression. Increased plasma GST and decreased MDA indicated the improved antioxidant response. Dietary NCG inclusion decreased plasma IgM and down-regulated the mRNA levels of inflammation (TNF-α and IL8), apoptosis (caspase family) and fibrosis (TGF-β1) related genes in the liver. The immunofluorescence examination revealed significantly decreased hepatic apoptosis and necrosis signals in the NCG groups. The ameliorated liver function and histological structure were closely related to the improved lipid metabolism parameters with decreased plasma VLDL and hepatic TG and NEFA accumulation, down-regulated fatty acid and cholesterol synthesis and simultaneously increased lipolysis gene mRNA levels, which regulated by inhibiting phosphorylation of ERK1/2-mTOR-S6K1 signaling pathway. Consuming 3600 mg/kg of dietary NCG is not safe for Japanese seabass culturing with the significantly increased FCR and decreased protein and lipid retention, and reduced plasma ALB. Accordingly, the observed efficacy and safety level of dietary NCG in the diet of Japanese seabass is 720 mg/kg.

Topics: Animal Feed; Animals; Apoptosis; Arginine; Bass; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Fish Proteins; Glutamates; Hepatocytes; Liver Diseases; Metabolic Diseases; Nutrients; Random Allocation; Signal Transduction

MicroRNAs (miRNAs) are a kind of small non-coding RNAs that have been reported to play a vital role in mediating host-pathogen interactions. High-throughput sequencing technology was applied to identify and illuminate mRNAs and miRNAs from grouper infected with Vibrio alginolyticus. The KEGG pathway enrichment analysis showed that the most significate DEGs are associated with Toll-like receptor signaling pathway and NOD-like receptor signaling pathway. We obtained 374 known miRNAs and 116 novel miRNAs. During them, there are 31 up-regulated miRNAs and 93 down-regulated miRNAs. miRNA-mRNA GO and KEGG analysis show that there are 90 miRNAs associated with the immune system. The target genes of immune-related miRNAs (miR-142, miR-146, miR-150, miR-155, miR-203, miR-205, miR-24, miR-31) and genes (CD80, IL-2, AMPK, PI3K) in Epinephelus coioddes were predicted and validated. This study provides an opportunity to further understanding the molecular mechanisms especially the immune system of miRNA regulation in Epinephelus coioddes host-pathogen interactions.

Topics: Animals; Bass; Fish Diseases; Gene Expression Regulation; Immunity, Innate; MicroRNAs; RNA, Messenger; Vibrio alginolyticus; Vibrio Infections

DEAD (Asp-Glu-Ala-Asp)-box polypeptide 41 (DDX41) is a member of the DEXDc family of helicases, that has recently been identified to be a crucial intracellular DNA sensor that triggers multiple signaling molecules to activate the type I interferon response. However, the precise function of DDX41 in fish during a viral infection remains unknown. In the present study, the DDX41 homolog from orange spotted grouper, Epinephelus coioides (EcDDX41), was cloned and its potential role in the immune response to a fish viral infection were investigated. EcDDX41 encodes a putative protein of 614 amino acid residues that contained two conserved domains: 1) DEADc domain; and 2) HELICc domain. The sequence analysis indicated that EcDDX41 shared 99%, 94%, and 86% identity with Asian seabass (Lates calcarifer), zebrafish (Danio rerio), and humans (Homo sapiens), respectively. EcDDX41 mRNA was present in all of the detected tissues, with the highest level of expression in the gills. The level of EcDDX41 expression was up-regulated following infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) in grouper spleen (GS) cell cultures, suggesting that EcDDX41 may be involved in fish virus infection. Furthermore, EcDDX41 overexpression in GS cells significantly inhibited SGIV and RGNNV replication. EcDDX41 overexpression significantly increased the expression of antiviral and inflammatory cytokine genes, including interferon regulatory factor genes (e.g., IRF1, IRF2, IRF3, and IRF7), interferon induced genes (e.g., ISG15, ISG56, IFP35, Viperin, and MXI), and pro-inflammatory cytokine genes (e.g., TNFα, IL-1β, and IL-8). Moreover, EcDDX41 positively regulated the mitochondrial antiviral-signaling protein (MAVS) and TANK-binding kinase 1 (TBK1)-induced interferon immune response, but did mediate IRF3 activation (MITA) to evoke an interferon immune response in unstimulated cells. Together, our results provide novel insight into the role of fish DDX41 in the antiviral innate immune response.

Topics: Amino Acid Sequence; Animals; Bass; DEAD-box RNA Helicases; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; Sequence Alignment

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV.

Topics: Animals; Bass; Fish Diseases; Gene Expression Profiling; Iridovirus; Open Reading Frames; Proteome; Proteomics; Tandem Mass Spectrometry; Viral Structural Proteins

Excessive lipid accumulation and chemical abuse can induce fatty liver diseases in fish, but the underlying mechanism and therapies are unknown. The present study aims to evaluate the effects of Xiaochaihu Decoction (XCHD) on the growth performance, lipid metabolism and antioxidant function of hybrid grouper in vitro and in vivo, and provide evidence as to whether it can be potentially used as a medicine for liver diseases in aquaculture. In vitro, steatosis model of hybrid grouper primary hepatocytes were incubated for 48 h in control or lipid emulsion (LE)-containing medium with or without 24 h post-treatment with XCHD. XCHD treatment reversed the LE-induced intracellular lipid accumulation, cell viability and hepatocytes morphological structure. In vivo, a total of 300 hybrid grouper with an average initial weight of 25.43 ± 0.18 g were fed diets containing five graded levels of XCHD at 150-1200 mg/kg diet for 8 weeks. After that, a challenge trial was conducted by injection of D-GalN/LPS to induce liver injury. As a result, dietary supplementation with 150-300 mg/kg XCHD diets can significant improve growth performance and feed utilization (P < 0.05). Dietary XCHD down-regulated the expression of lipogenic-related genes (G6PD, DGAT2 and ME1) and up-regulated lipolysis-related genes (ATGL, PPARα and LPL) expression in the liver of hybrid grouper. Livers challenged with D-GalN/LPS exhibited extensive areas of vacuolization with the disappearance of nuclei and the loss of hepatic architecture. These pathological alterations were ameliorated by XCHD treatment. XCHD significantly down-regulated the D-GalN/LPS induced apoptosis-related genes caspase-3, caspase-9 and p53 mRNA expression and up-regulated the antioxidant-related genes CAT and MnSOD mRNA expression in dose dependent manner, respectively. XCHD potently reduced hepatic lipid accumulation and enhanced antioxidant capability in hybrid grouper and may be a potential fish-feed additive to prevent fatty liver diseases onset and progression.

Topics: Animal Feed; Animals; Antioxidants; Apoptosis; Bass; Diet; Dietary Supplements; Drugs, Chinese Herbal; Fatty Liver; Female; Fish Diseases; Galactosamine; Lipid Metabolism; Lipopolysaccharides; Liver; Male

Topics: Animal Feed; Animals; Bass; Blood Bactericidal Activity; Blood Cell Count; Body Weight; Complement Pathway, Alternative; Disease Resistance; Dose-Response Relationship, Drug; Erythrocyte Indices; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Hemoglobins; Hydrocortisone; Immunity, Humoral; Inflammation; Muramidase; Neuroimmunomodulation; Nutritional Requirements; Peroxidases; Photobacterium; Tryptophan

The coccidium Goussia bayae infects the gallbladder and bile ducts of white perch Morone americana from Chesapeake Bay, USA. Seasonal changes in coccidian infections were analyzed from bile specimens of 1588 fish from the Choptank River during 2016-2018 using wet mount preparations with a Sedgwick-Rafter counting chamber. Histopathology of the gallbladder and liver was analyzed from a subset (n = 480) of these fish. Maximum parasite prevalence (100%) and intensities in the gallbladder occurred during the fish spawning season in March and April. Asynchronous coccidian development and prevalence of infections in fish increased gradually during autumn and winter, but coccidian intensity increased sharply 2-4 wk prior to the onset of fish spawning activity and decreased after spawning activity concluded. Sporulation was internal, and the gallbladder was the primary reservoir for oocysts. Two previously undescribed species of coccidia were observed in the intestine. Lesions in the gallbladder were rare and included cholecystitis and epithelial necrosis. Intrahepatic bile duct lesions were more common and included distension, cholangitis, epithelial erosion and necrosis, cholestasis, hyperplasia, and neoplasia. Cholangitis and necrosis of intrahepatic bile ducts were significantly associated with coccidial infections, while plasmodia of a myxosporean (spore morphology consistent with the genera Myxidium and Zschokella) were significantly associated with bile duct hyperplasia. Biliary neoplasia included cholangiomas (5% prevalence) and cholangiocarcinomas (1% prevalence). No association was detected between G. bayae and biliary neoplasms, but an association may exist between these lesions and the myxosporean plasmodia.

Topics: Animals; Bass; Bays; Fish Diseases; Seasons

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. The expression of an ABC transporter gene-L321_23611 of P. plecoglossicida at 18 °C was found significant higher than those at 28 °C by RNA-seq and qRT-PCR. RNAi significantly reduced the content of L321_23611 mRNA in P. plecoglossicida with a maximal decrease of 89.2%. Compared with the wild type strain, the infection of L321_23611-RNAi strain resulted in the reduction in mortality and the onset time delay of a kind of marine teleosts, Epinephelus coioides. The results of dual RNA-seq showed that the RNAi of L321_23611 resulted in a significant change in both pathogen and host transcriptome in the spleens of infected E. coioides. The result of GO and KEGG analysis from dual RNA-seq data showed both host genes of chemokine signaling pathway, coagulation and complement system, hematopoietic cell lineage pathway as well as hemoglobin complex GO term and pathogenic genes of bacterial-type flagellum-dependent cell mortality GO term and flagellar assembly, biosynthesis of amino acids and lysine biosynthesis systems pathways were mainly affected by L321_23611 gene of P. plecoglossicida. The results indicated that: 1. ABC transporter gene-L321_23611 was a virulent gene of P. plecoglossicida. 2. Both the activation of the host immune pathways and depression of pathogenic virulence-related pathways facilitated E. coioides to remove L321_23611-RNAi strain than the wild type strain of P. plecoglossicida.

Topics: Animals; ATP-Binding Cassette Transporters; Bass; Fish Diseases; Fish Proteins; Host-Pathogen Interactions; Immunity, Innate; Pseudomonas; Pseudomonas Infections; Sequence Analysis, RNA

Topics: Animals; Bacterial Proteins; Bass; Fish Diseases; Host-Pathogen Interactions; HSP90 Heat-Shock Proteins; Pseudomonas; Pseudomonas Infections; RNA-Seq; Spleen; Virulence

Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods.. In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2'-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses.. The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days' post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family.. These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

Topics: Animals; Aquaculture; Bass; Cell Line; Fish Diseases; Genome, Viral; High-Throughput Nucleotide Sequencing; Infectious bursal disease virus; Microscopy, Electron, Transmission; Phylogeny; Polymerase Chain Reaction; Singapore

Apolipoproteins (Apos), which are the protein components of plasma lipoproteins, play important roles in lipid transport in vertebrates. It has been demonstrated that in teleosts, several Apos display antimicrobial activity and play crucial roles in innate immunity. Despite their importance, apo genes have not been systematically characterized in many aquaculture fish species. In our study, a complete set of 23 apo genes was identified and annotated from spotted sea bass (Lateolabrax maculatus). Phylogenetic and homology analyses provided evidence for their annotation and evolutionary relationships. To investigate their potential roles in the immune response, the expression patterns of 23 apo genes were determined in the liver and intestine by qRT-PCR after Vibrio harveyi infection. After infection, a total of 20 differentially expressed apo genes were observed, and their expression profiles varied among the genes and tissues. 5 apo genes (apoA1, apoA4a.1, apoC2, apoF and apoO) were dramatically induced or suppressed (log2 fold change >4, P < 0.05), suggesting their involvement in the immune response of spotted sea bass. Our study provides a valuable foundation for future studies aimed at uncovering the specific roles of each apo gene during bacterial infection in spotted sea bass and other teleost species.

Topics: Animals; Apolipoproteins; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Multigene Family; Phylogeny; Transcriptome; Vibrio; Vibrio Infections

Cyclophilin A (CypA) is a ubiquitously expressed cellular protein and involves in diverse pathological conditions, including infection and inflammation. CypA acts as a key factor in the replication of several viruses. However, little is known about the role of CypA in the replication of the red-spotted grouper nervous necrosis virus (RGNNV). In the present report, grouper CypA (GF-CypA) was cloned from the grouper fin cell line (GF-1) derived from orange-spotted grouper (Epinephelus coioides). Sequence analysis found that GF-CypA open reading frame (ORF) of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4 kDa. The deduced amino acid sequence shared highly conserved regions with CypA of other animal species, showing that GF-CypA is a new member of Cyclophilin A family. We observed that GF-CypA was up-regulated in the GF-1 cells infected with RGNNV. Additionally, overexpression of CypA could significantly inhibit the replication of RGNNV in GF-1 cells. By contrast, when the GF-CypA was knock-downed by siRNA in GF-1 cells, the replication of RGNNV was enhanced. Furthermore, the expressions of pro-inflammatory factors, such as TNF-2, TNF-α, IL-1b, and ISG-15, were increased in GF-CypA transfected GF-1 cells challenged with RGNNV, indicating that GF-CypA might be involved in the regulation of the host pro-inflammatory factors. Altogether, we conclude that GF-CypA plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1 cells during RGNNV infection. These results will shed new light on the function of CypA in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cyclophilin A; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; RNA Virus Infections; Sequence Alignment; Virus Replication

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.

Topics: Animals; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Photobacterium; Sea Bream

This is the first description of a betanodavirus infection in the dusky grouper Epinephelus marginatus within the marine protected areas (MPAs) of the Balearic Islands. Histopathology techniques were employed to describe neurological lesions in infected fish. Abnormal swimming, mortality, and neurological lesions were detected in all analysed grouper individuals. Virus particles were observed by means of transmission electron microscopy. Reverse transcription of RNA1 and RNA2 followed by cDNA amplification and sequencing allowed viral classification. Phylogenetic analysis showed the isolates from wild E. marginatus of the Balearic Islands MPAs to be closely related to Dicentrarchus labrax and Mullus barbatus strains from Cyprus and Italy. Although vertical transmission from infected spawners has been described as the major route for nodavirus infection, we point out in this work that horizontal transmission among sub-clinical fishes after migration or commercial import for aquaculture production could play a major role in the spreading of the disease in MPAs.

Topics: Animals; Bass; Brain Diseases; Fish Diseases; Italy; Nodaviridae; Phylogeny; RNA Virus Infections; Spain

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; MAP Kinase Kinase 6; Phylogeny; Ranavirus; Sequence Alignment; Vibrio alginolyticus; Vibrio Infections

Singapore grouper iridovirus (SGIV) is the main grouper-infecting virus in southern China that causes serious economic losses. However, there is no effective way to control this viral disease. In this study, SGIV ORF19R (SGIV-19R) encoding a viral membrane protein was constructed into pcDNA3.1-HA and then was used to evaluate the immune protective effects in grouper Epinephelus coioides. Subcellular localization showed that SGIV-19R distributed in the cytoplasm and co-localization analysis indicated the protein partially co-localized with the endoplasmic reticulum (ER). RT-PCR and Western blot analyses confirmed the expression of the vaccine plasmids in grouper muscle tissues. Moreover, the transcription levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), myxovirus resistance 1 (Mx1) and immunoglobulin M (IgM) genes were significantly up-regulated in the spleen, liver and kidney of vaccinated groupers. SGIV challenge experiments showed the relative percent survival (RPS) was significantly enhanced in fish with 49.9% at the DNA dose of 45 μg pcDNA3.1-19R, while 75.0% RPS when using 90 μg pcDNA3.1-19R. Meanwhile, vaccination with pcDNA3.1-19R significantly reduced the virus replication, evidenced by a low viral load in the spleen of survivals groupers after SGIV challenge. These results imply that pcDNA3.1-19R could induce protective immunity in grouper, and might be a potential vaccine candidate for controlling SGIV disease.

Topics: Adaptive Immunity; Animals; Bass; DNA Virus Infections; Fish Diseases; Immunity, Innate; Injections, Intramuscular; Iridovirus; Ranavirus; Random Allocation; Vaccines, DNA; Viral Matrix Proteins; Viral Vaccines

Macrophage expressed gene 1 (Mpeg1) is a molecule that can form pores and destroy the cell membrane of invading pathogens. In this study, we identified two Mpeg1 isoforms from the orange-spotted grouper (Epinephelus coioides) and named them EcMpeg1a and EcMpeg1b. Predicted proteins of the two EcMpeg1s contained a signal peptide, a conserved membrane attack complex/perforin (MACPF) domain, a transmembrane segment, and an intracellular region. Sequence alignment demonstrated that two EcMpeg1 proteins share a high sequence identity with that of other teleosts. Tissue distribution analysis showed that EcMpeg1s were expressed in all tissues tested in healthy grouper, with the highest expression in the head kidney and spleen. After infection with the ciliate parasite Cryptocaryon irritans, expression of the two EcMpeg1s was significantly upregulated in the spleen and gills. Furthermore, the recombinant EcMpeg1a showed antiparasitic and antibacterial activity against Gram-negative and -positive bacteria, whereas EcMpeg1b had an inhibitory effect only against Gram-positive bacteria. These results indicated that EcMpeg1s play an important role in the host response against invading pathogens.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Immunity, Innate; Membrane Proteins; Phylogeny; Sequence Alignment

Accumulated evidence suggests that some of the tripartite motif (TRIM) -family proteins function as critical regulators of carcinogenesis, immunity, and antiviral functions. TRIM44 is an atypical TRIM family protein that lacks the entire RING domain and has been demonstrated to play a crucial role in cancer and viral infection. To our knowledge, the role of TRIM44 in fish still remains largely unknown. Here, we cloned and characterized a novel TRIM44-like gene from orange spotted grouper (EcTRIM44L). Sequence analysis indicated that EcTRIM44L encoded a 393 amino acid peptide, which shared 81.44% and 51.02% identity with large yellow croaker (Larimichthys crocea) and zebrafish (Danio rerio), respectively. However, EcTRIM44L only exhibited 24.69% identity with the TRIM44 protein of humans (Homo sapiens). Moreover, EcTRIM44L contained two conserved domains, including a B-Box domain and a coiled-coil domain, but not a RING domain. Using fluorescence microscopy, we observed green fluorescence in the cytoplasm of the EcTRIM44L-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcTRIM44L transcription was significantly up-regulated in red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM44L might be involved in fish virus infections. The in vitro overexpression of EcTRIM44L significantly enhanced RGNNV replication, as demonstrated by the accelerated cytopathic effect (CPE) progression induced by RGNNV, as well as the increased expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcTRIM44L significantly decreased the level of interferon (IFN) related signaling molecules and pro-inflammatory cytokine expression, suggesting that EcTRIM44L affected virus replication by negatively regulating the IFN response. In addition, the melanoma differentiation-associated protein 5 (MDA5) and mitochondrial antiviral-signaling protein (MAVS), but not mediator of IRF3 activation (MITA)-evoked IFN response was negatively regulated by EcTRIM44L. Together, for the first time, our results indicate that EcTRIM44L negatively regulates the interferon response against grouper RNA virus infection.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; RNA Virus Infections; Sequence Alignment; Tripartite Motif Proteins

Toll-like receptor (TLR) genes are the earliest reported pathogen recognition receptors (PRRs) and have been extensively studied. These genes play pivotal roles in the innate immune defense against pathogen invasion. In this study, a total of 16 tlr genes were identified and characterized in spotted sea bass (Lateolabrax maculatus). The tlr genes of spotted sea bass were classified into five subfamilies (tlr1-subfamily, tlr3-subfamily, tlr5-subfamily, tlr7-subfamily, and tlr11-subfamily) according to the phylogenetic analysis, and their annotations were confirmed by a syntenic analysis. The protein domain analysis indicated that most tlr genes had the following three major TLR protein domains: a leucine-rich repeat (LRR) domain, a transmembrane region (TM) and a Toll/interleukin-1 receptor (TIR) domain. The tlr genes in spotted sea bass were distributed in 11 of 24 chromosomes. The mRNA expression levels of 16 tlr genes in response to Vibrio harveyi infection were quantified in the head kidney. Most genes were downregulated following V. harveyi infection, while only 5 tlr genes, including tlr1-1, tlr1-2, tlr2-2, tlr5, and tlr7, were significantly upregulated. Collectively, these results help elucidate the crucial roles of tlr genes in the immune response of spotted sea bass and may supply valuable genomic resources for future studies investigating fish disease management.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Genome; Immunity, Innate; Toll-Like Receptors; Vibrio; Vibrio Infections

Epinephelus moara is an economically important fish in Southeast Asian countries but is suffering from nervous necrosis virus (NNV) infection. A deeper understanding of the host-NNV interaction mechanisms makes sense for disease control, however, at present, the pathogenesis of natural NNV infection and the resistance mechanism in host remains poorly understood. In this study, asymptomatic and diseased E. moara with clinical symptoms of viral nervous necrosis (VNN) from a grouper farm were both detected with a positive RT-PCR signal of NNV, then transcriptome sequencing of their immune tissues (liver, spleen and kidney) were performed for comparation analysis. The de novo assemblies yielded 53,789 unigenes which had a length varied from 201 to 19,675 bp and a N50 length of 2115 bp, and 29,451 unigenes were functionally annotated, with 83, 250 and 5632 unigenes being differentially expressed in liver, spleen and kidney respectively. KEGG pathway enrichment analysis of the DEGs showed many DEGs were enriched in immune related pathways. Although the expression of class I major histocompatibility complex (MHC) was significantly higher in three immune tissues of the diseased grouper, many immune related genes, including humoral immune molecules (such as antibodies), the cellular mediated cytotoxic molecules (such as perforin) and some adhesion related genes were down regulated in the diseased grouper. Our results provided many unigenes that might play important roles in NNV resistance for further research. Furthermore, a total of 8666 unigenes containing 11,623 simple sequence repeats (SSRs) were identified, which provided useful information for screening molecular markers associated with NNV resistance in E. moara.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Nodaviridae; RNA Virus Infections; Transcriptome

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Myeloid Differentiation Factor 88; Phylogeny; Sequence Alignment

MDA5 is the pivotal member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and is reported to play a crucial role in type I IFN-mediated responses against pathogen-associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full-length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N-terminal CARD domains, a DExDc domain, a HELICc domain and a C-terminal RIG-1_C-RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time-points following challenge with Gram-negative bacterium, Vibrio alginolyticus, and Gram-positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development.

Topics: Animals; Bass; Cell Line; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression Regulation; Humans; Immunity, Innate; Immunity, Maternally-Acquired; Interferon-Induced Helicase, IFIH1; Pathogen-Associated Molecular Pattern Molecules; Poly I-C; Protein Domains; Staphylococcal Infections; Staphylococcus aureus; Vibrio alginolyticus; Vibrio Infections

MicroRNA-146a (miR-146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR-146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR-146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR-146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR-REPORT Luciferase system, we found that the 3' untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR-146a. Further studies showed that its downstream target gene pro-inflammatory cytokines, including TNF-α, IL-8 and IL-1β, were all significantly decreased in miR-146a mimic transfected cells, but increased in miR-146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR-146a exerted crucial roles in RGNNV infection through TRAF6-mediated inflammatory response.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; MicroRNAs; Nodaviridae; RNA Virus Infections; TNF Receptor-Associated Factor 6; Virus Replication

Mitochondrial antiviral signaling protein (MAVS), also known as IPS-1, VISA, and Cardif, has been well studied for its crucial roles in the mammalian interferon immune response. To better understand the actions of MAVS in fish immune response, a MAVS homolog from orange spotted grouper (Epinephelus coioides) (EcMAVS) was cloned and characterized in this study. EcMAVS encoded a 563-amino acid peptide which showed 64% and 20% identity to rock bream (Oplegnathus fasciatus) and human (Homo sapiens), respectively. Sequence alignment analysis showed that EcMAVS shared a conserved CARD domain at N terminal, a central proline-rich region and a TM domain at C terminal. Phylogenetic analysis indicated that EcMAVS showed the nearest relationship to rock bream, followed by other fishes, birds and mammals. In healthy grouper, the transcript of EcMAVS was predominantly detected in gill, intestine and skin. In vitro, the expression level of EcMAVS was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) infection, but only slightly increased at the late stage of Singapore grouper iridovirus (SGIV) infection, suggested the EcMAVS might exert various roles in response to different viruses. Subcellular localization analysis showed that the fluorescence in EcMAVS transfected cells primarily co-localized with mitochondria. Overexpression of EcMAVS in grouper cells significantly inhibited the replication of RGNNV, demonstrated by the delay of CPE progression and the decrease of viral gene transcription. Differently, the replication of SGIV was almost not affected by the ectopic expression of EcMAVS. Furthermore, our results also showed that EcMAVS overexpression significantly increased the expression of interferon related cytokines, and activated both IRF3- and IRF7-mediated interferon promoter activities. Taken together, our results demonstrated that grouper MAVS exerted antiviral function against nodavirus infection via up-regulating the interferon immune response.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Interferons; Nodaviridae; Organ Specificity; RNA Virus Infections; Virus Replication

Mitochondrial antiviral signaling protein (MAVS), an innate immune signaling adapter coordinates the signals received from two independent cytosolic pathogen recognition receptors (RIG-1 and MDA5) to induce antiviral genes. In the present study the MAVS gene of Lates calcarifer (LcMAVS) was cloned and characterized. The complete cDNA sequence of LcMAVS was 3160 bp and encodes a poly peptide of 577 amino acids. Structural analysis of LcMAVS revealed an N-terminal CARD-like domain, central proline-rich domain and a C-terminal transmembrane domain. Phylogenetic analysis indicated that LcMAVS exhibited the closest relationship to P. olivaceous MAVS. LcMAVS was ubiquitously expressed in all tested tissues of healthy fish viz., brain, gill, heart, liver, spleen, kidney and intestine, with highest transcript level in spleen. The mRNA transcript level of LcMAVS in different developmental stages showed constitutive expression in all the stages tested suggesting the maternal transfer of the gene. Significant up regulation in MAVS expression was observed post nervous necrosis virus (NNV) challenge in vivo in all the selected tissues. Further, time course analysis showed that LcMAVS transcripts significantly increased in the brain and spleen tissues after NNV infection. These findings provide useful information for further elucidating the function of LcMAVS in antiviral innate immune response against NNV in Asian seabass.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression Profiling; Immunity, Innate; Mitochondria; Nodaviridae; Phylogeny; RNA Virus Infections; Signal Transduction; Spleen; Zebrafish Proteins

Cryptocaryon irritans is an important protozoan ciliate, which has led to heavy economic losses in marine aquaculture. Previous studies have indicated that C. irritans infection could induce the migration of neutrophils to infection sites. Myeloperoxidase (MPO) mainly exists in the cytoplasmic granules of the neutrophil and performs its function by a unique enzymatic capacity to produce hypohalous acid and other toxic oxidants. To determine the involvement of MPO and neutrophils against C. irritans infection in the host, we amplified MPO cDNA (EcMPO) from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcMPO encodes a putative polypeptide of 770 amino acids and has typical structural characteristics of mammalian MPO, including a signal peptide, a propeptide, a light chain, a heavy chain, and a peroxidase domain. Bioinformatics analysis has demonstrated that the most important functional sites in mammalian MPO were also conserved in grouper and other piscine MPO, implying the functional conservation of this protein during evolution. A rabbit anti-MPO recombinant protein polyclonal antibody was produced, which could recognize the native MPO protein. The expression of EcMPO was higher in the lympho-hematopoietic organs, such as head kidney, trunk kidney, spleen, but lower in muscle, heart, and brain. After infection with C. irritans, the EcMPO transcript was significantly up-regulated at specific time points in the infection sites (skin and gill) and systemic immune organs (head kidney and spleen); The number of EcMPO positive cells first increased and then decreased in the gill, but was still higher than the control after 7 days. These results demonstrated that EcMPO and its positive cells may be involved in anti-C. irritans infection in the grouper, which is attributed to the innate immune mechanisms of the host against parasite infection.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Peroxidase; Phylogeny; Sequence Alignment

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south-east and north-east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR

Topics: Animals; Anti-Bacterial Agents; Bass; China; DNA, Bacterial; Drug Resistance, Bacterial; Fish Diseases; Necrosis; Phylogeny; Prevalence; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Vibrio; Vibrio Infections

Two marine antimicrobial peptides (AMPs), PC-hepc from large yellow croaker (Pseudosciaena crocea) and scygonadin from mud crab (Scylla serrata), are potently active against specific bacteria and thus they could be used as substitutes for antibiotics in aquaculture. However, how to utilize the AMPs feasibly for marine cultured animals has been so far confused. In our study, a 510 bp of the Scy-hepc sequence was cloned into pMDC85 expression vector, which was then electroporated into Chlorella sp., and thus a transgenic Chlorella, in which the Scy-hepc gene was effectively expressed, was developed. The Scy-hepc fusion protein was successfully expressed in Chlorella sp. and it showed obvious bactericidal activity. In addition, the in vivo efficacy of the transgenic Chlorella was evaluated using Sparus macrocephalus and the hybrid Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂). Results showed that the survival rate of S. macrocephalus fed with transgenic Chlorella (80 ± 10% after 72 h) was significantly higher than that of fish fed with the same dosage of wild-type Chlorella (33.33 ± 11.55% after 72 h). Similarly, results showed that the survival rate of the hybrid grouper fed with transgenic Chlorella (55 ± 5% after 36 h) was much higher than that of fish fed with the same dosage of wild-type Chlorella (25 ± 5% after 36 h). Therefore, in vitro and in vivo results indicated that the constructed transgenic Chlorella with the marine AMPs Scy-hepc could exert effective protection for fish against the Aeromonas hydrophila infection, providing an encouraging prospect for the expected use of transgenic Chlorella in aquaculture in future.

Topics: Aeromonas hydrophila; Animals; Bass; Brachyura; Chlorella; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Hepcidins; Organisms, Genetically Modified; Perciformes

The tiger grouper, Epinephelus fuscoguttatus, is an economically important fish in Southeast Asia but has been plagued by several diseases. Spatholobus suberectus (S), Phellodendron amurense (P), and Eclipta prostrate (E) are three commonly used Chinese medicinal herbs. Although previous pharmacological and clinical studies indicated that S, P, and E possess a variety of beneficial functions in mammals, little is known about their functions in farmed fish and the underlying molecular mechanism of their actions. Challenge tests in this study showed that after 14 days of diet supplement, all these herbs could effectively enhance the disease resistance of E. fuscoguttatus against Vibrio harveyi. However, the non-specific immune parameters of the herb-supplemented groups were not significantly different from the control group. To further explore the molecular mechanism of herbal immune-regulating effects on E. fuscoguttatus, transcriptome sequencing and RNA-Seq technique were applied on E. fuscoguttatus kidney. De novo transcriptome assembly of E. fuscoguttatus kidney yield 80,014 unigenes, among which, 44,901 (56.12%) were annotated with at least one of the public databases (Nr, Nt, Swiss-Prot, KEGG, COG, GO). Among these, 22,738, 11,700 and 27,457 unigenes were assigned to 57, 25 and 258 categories of GO, COG and KEGG databases, respectively. Using Solexa/Illumina's DGE platform, a total of 231, 186 and 144 putative differentially expressed genes (DEGs) were detected in P, E and S group compared with the control group. GO analysis indicated that in P and E, down-regulated DEGs were dominant in almost every GO term; whereas in S, up-regulated DEGs were more dominant. KEGG pathway analysis revealed that putative DEGs in all three herb groups were obviously enriched in the pathways related to infective diseases and immune system. We also identified a number of immune relative genes and pathways (TLR5, IL8 and MAPK pathway, for instance) associated with P, E and S's regulatory effects on E. fuscoguttatus. This study will enrich the E. fuscoguttatus transcriptome database, contribute to a better understanding of the molecular mechanisms associated with the immunoregulatory activities of Chinese medicinal herbs on teleost and provide valuable information on the prevention of grouper Vibrio diseases using Chinese medicinal herbs.

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Eclipta; Fabaceae; Fish Diseases; Head Kidney; Immunity, Innate; Phellodendron; Plant Extracts; Plants, Medicinal; Random Allocation; Transcriptome; Vibrio; Vibrio Infections

TANK-binding kinase-1 (TBK1) has been well studied in mammals because of its importance in type I interferon induction in antiviral immunity. However, the roles of fish TBK1 in virus infection still remained largely uncertain. In the current study, a TBK1 homolog from orange-spotted grouper (Epinephelus coioides) (EcTBK1) was cloned and its roles in fish viral infections were investigated. Sequence analysis showed that EcTBK1 encoded a 723-amino acid peptide which shared 98% and 73% identity to large yellow croaker (Larimichthys crocea) and human (homo sapiens), respectively. Multiple sequence alignments indicated that EcTBK1 contained conserved domains, including N-terminal kinase domain (KD), the middle ubiquitin-like domain (ULD) and C-terminal coiled-coil (CC) domains. The tissue distribution profiles demonstrated that EcTBK1 gene was constitutively expressed in all examined tissues, with predominant expression in intestine. Temporal expression analysis in vitro showed that the expression levels of EcTBK1 were significantly up-regulated in response to both red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection, suggested that EcTBK1 might exert crucial roles in fish virus infection. Subcellular localization indicated that EcTBK1 expression was primarily in the cytoplasm in GS cells. The ectopic expression of EcTBK1 significantly inhibited both SGIV and RGNNV replication. Furthermore, EcTBK1 overexpression significantly increased the expression levels of interferon related cytokines and pro-inflammatory factors. In addition, the overexpression of EcTBK1 increased the IRF3- and IRF7-regulated interferon promoter ISRE and IFN activity, and the regulatory effect on interferon immune response were dependent on its kinase domain. Together, we speculated that grouper TBK1 exerted antiviral activity against iridovirus and nodavirus via regulating the interferon immune and inflammatory response.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Protein Serine-Threonine Kinases; Ranavirus; Sequence Alignment

Photobacterium damselae species are one of the most devastating bacterial pathogens in mariculture worldwide. Some species of Photobacterium are pathogenic for marine animals and human. They are the causative agents of photobacteriosis, formerly known as pasteurellosis. A total of (202) marine fishes of three different species were represented as: seabass (Dicentrarchus labrax), seabream (Sparus aurata) and gray mullet (Mugil capitus) randomly collected from Lake Temsah at Ismailia governorate along the parallel Pelagic road to the lake in the governorate from August 2015 to July 2016. The clinical picture and gross lesions of the diseased fishes were recorded. Isolation and identification of suspected bacteria using traditional and molecular methods. Samples from affected organs were collected for studying the histopathological alterations of these pathogens. Fifty one fishes were found to be infected with Photobacterium damselae subsp. Piscicida. Seabass (Dicentrarchus labrax) was the most infected fish species (23), followed by seabream (Sparus aurata) (18) finally gray mullet (Mugil capitus) was (10). 91fishes were found to be infected with P. damselae subsp. damselae, seabass (Dicentrarchus labrax) was the most infected fish sp. (36), followed by seabream (Sparus aurata) (32), then gray mullet (Mugil capitus) (23). The results indicated that, the total prevalence of P. damselae subsp. piscicida in all examined species (25.24%), the highest seasonal prevalence was recorded in summer season (37.09%) followed by autumn (26%) then spring (20.37%) and winter (11.11%). On the other hand, the total prevalence of P. damselae subsp. damselae in all examined species (45.04%), the highest seasonal prevalence was recorded in summer season (67.74%) followed by autumn (52%) then spring (29.62%) and winter (19.44%). Molecular diagnosis with conventional PCR used to confirm the traditional isolation was applied by using specific primers of two genes (polycapsular saccharide gene and urease C gene). The histopathological studies of naturally infected marine fishes showed severe inflammatory reactions in different organs with accumulation of melanomacrophages and necrosis. The results confirm that P. damselae subspecies damsalea is the most prevalent pathogen between marine fishes, and seabass (Dicentrarchus labrax) was the highly affected marine fishes in this study.

Topics: Animals; Anti-Bacterial Agents; Bacterial Capsules; Bacteriological Techniques; Base Sequence; Bass; DNA Primers; DNA, Bacterial; Fish Diseases; Fishes; Genes, Bacterial; Lakes; Microbial Sensitivity Tests; Pasteurella Infections; Pathology, Molecular; Phenotype; Photobacterium; Polymerase Chain Reaction; Sea Bream; Seasons; Urease

A suspected new enteric Sphaerospora species was believed to be directly associated with the mass mortality of yearling groupers of Epinephelus spp. in South China. The epizootic generally emerged from late September to late April of the following year. The infection prevalence and mortality rate were significantly negatively correlated with fish size. Clinical signs included anorexia, cachexia and extrusion of white pulp-like substance from anus after gentle pressure on the abdomen. Upon necropsy, severe intestinal oedema, thin and transparent intestinal wall, swollen spleen, kidney and gall bladder could be observed. Wet preparation of the infected samples showed large amount of typical disporous plasmodia of the genus Sphaerospora, but no mature spores were observed. Epidemiological investigation showed that this parasite exclusively infected Epinephelus groupers. Histopathologically, this species mainly infected the epithelium of intestine and kidney tubules and caused severe epithelia sloughing and the collapse of intestinal villus. Interestingly, this enteric myxosporidiosis did not cause severe emaciation of infected fish for mass mortality usually emerged within 2-3 days after appearance of clinical signs. The species was most genetically related to Sphaerospora fugu (89% sequence identity) and phylogenetically positioned within marine Sphaerospora lineage. This is the first report of enteric sphaerosporosis of groupers.

Topics: Animals; Bass; China; Disease Outbreaks; Fish Diseases; Myxozoa; Parasitic Diseases, Animal; Phylogeny; Sequence Analysis, DNA

Mitogen-activated protein kinases (MAPKs), a group of serine-threonine protein kinases, play a crucial role in immunoreaction response to extra environmental stresses. In this study, two novel MAPKs, Ec-ERK1 and Ec-ERK2, were identified from Epinephelus coioides. Both Ec-ERK1 and Ec-ERK2 sequences contain a highly conserved Thr-Glu-Tyr (TEY) motif, an HRD domain, and an ATP binding loop containing GXGXXG. An analysis of phylogenetic relationships demonstrated that ERK amino acid sequences were conserved between different species indicating that the functions may be similar. Ec-ERK1 and Ec-ERK2 mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of these two genes in E. coioides were also detected against Cryptocaryon irritans infection, which is capable of killing large numbers of fish in a short time and has a serious impact on aquaculture. The expression was up-regulated in most of the tissues examined, with the highest expressions of Ec-ERK1 (3.9 times) occurring in the head kidney and Ec-ERK2 (3.5 times) occurring in the spleen. There was no significant correlation between the expression of Ec-ERK1/Ec-ERK2 and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-ERK1/ERK2 were conserved, Ec-ERK1/ERK2 showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly varied post C. irritans infection, suggesting Ec-ERK1/ERK2 may play important roles in these tissues during pathogen-caused inflammation.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; MAP Kinase Signaling System; Phylogeny; Sequence Alignment

Toll-like receptors (TLRs) are one of the most important innate immune receptors, which recognize various pathogen-associated molecular patterns and activate the downstream immune response. Mouse TLR13 has been found to recognize a highly conserved sequence from bacterial or viral RNA and activate the myeloid differentiation primary response gene 88-dependent signaling response. The function of teleost tlr13 is still not fully understood, especially its relationship with bacterial RNA. In our study, we identified and characterized a tlr13 from Epinephelus coioides (orange-spotted grouper). The full-length cDNA of Eco. tlr13 contained a 2844 bp open reading frame, encoding 947 amino acids. The polypeptide was constitutive of a signal peptide, 13 leucine-rich repeats domains, a C-terminal leucine-rich repeats, a transmembrane domain and a conserved Toll/interleukin (IL)-1 receptor domain, indicating that Eco. Tlr13 exhibited a typical TLR structure. Multiple alignments showed that the Toll/IL-1 receptor domain of Eco. Tlr13 was identical with other homologues, and the phylogenetic tree suggested that Eco. Tlr13 was clustered with other TLR13s and had the closest relationship with predicted Lates calcarifer (sea bass) Tlr13. Subcellular localization analysis revealed that Eco. Tlr13 colocalized with the endoplasmic reticulum and early endosome. Moreover, Eco. tlr13 was broadly observed in all tested tissues with the relatively high expressions in the brain and immune-related tissues. After challenged with 19-mer Staphylococcus aureus 23S ribosomal RNA-derived oligoribonucleotide (ORN Sa19), the expression of Eco. tlr13 was significantly up-regulated in grouper spleen cells. Also, the luciferase assay further revealed that with the overexpression of Eco. Tlr13 in human embryonic kidney 293T cells, ORN Sa19 activated the promoter activity of interferon-β in a dose-dependent pattern. These results indicate that Eco. tlr13 may involve in the recognition of bacterial RNA.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Sequence Alignment; Toll-Like Receptors

A new brain-cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L-15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2-mercaptoethanol (2-ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18-30° C, with the maximum growth between 24 and 30° C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. After being transfected with pEGFP-N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real-time PCR (qrt-PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.

Topics: Animals; Bass; Brain; Cell Line; Fish Diseases; Glutamate-Ammonia Ligase; Iridoviridae; Karyotype; Kelp; Nodaviridae; Primary Cell Culture; Transfection

As the major viral pathogen of grouper aquaculture, Singapore grouper iridovirus (SGIV) has caused great economic losses in China and Southeast Asia. In the previous study, we have generated highly specific ssDNA aptamers against SGIV-infected grouper spleen cells (GS) by Systematic Evolution of Ligands by Exponential Enrichment technology (SELEX), in which Q2 had the highest binding affinity of 16.43 nM. In this study, we would try to identify the specific sequences in the aptamer Q2 that exhibited the high binding affinity to SGIV-infected cells by truncating the original Q2 into some different specific segments. We first evaluated the specificity and binding affinity of these truncated aptamers to SGIV-infected cells by flow cytometry, fluorescent imaging of cells and aptamer-based enzyme-linked apta-sorbent assay (ELASA). We then performed cytotoxicity analysis, assessment of the inhibitory effects upon SGIV infection and the celluar internalization kinetics of each truncated aptamer. Compared to the initial Q2, one of the truncated aptamer Q2-C5 showed a 3-fold increase in the binding affinity for SGIV-infected cells, and held more effective inhibitory effects, higher internalization kinetics and stability. Hence, the aptamer's truncated methods could be applied in the research of identifying aptamer's key sequences. The shorter, structure optimizing aptamer showed more excellent performance over the originally selected aptamer, which could potentially be applied in developing commercial detection probes for the early and rapid diagnosis of SGIV infection, and highly specific therapeutic drugs against SGIV infection.

Topics: Animals; Antiviral Agents; Aptamers, Nucleotide; Base Pairing; Bass; Biological Transport; DNA Virus Infections; DNA, Single-Stranded; DNA, Viral; Fish Diseases; Nucleic Acid Conformation; Ranavirus; Spleen; Structure-Activity Relationship

Viral nervous necrosis caused by nervous necrosis virus (NNV) is one of the most severe diseases resulting in high fish mortality rates and high economic losses in the giant grouper industry. Various NNV vaccines have been evaluated, such as inactivated viruses, virus-like particles (VLPs), recombinant coat proteins, synthetic peptides of coat proteins, and DNA vaccines. However, a cheaper manufacturing process and effective protection of NNV vaccines for commercial application are yet to be established. Hence, the present study developed a novel subunit vaccine composed of a carrier protein, receptor-binding domain of Pseudomonas exotoxin A, and tandem-repeated NNV coat protein epitopes by using the structural basis of epitope prediction and the linear array epitope (LAE) technique. On the basis of the crystal structure of the NNV coat protein, the epitope was predicted from the putative target cell receptor-binding region to elicit neutralizing immune responses. The safety of the LAE vaccine was evaluated, and all vaccinated fish survived without any physiological changes. The coat protein-specific antibody titers in the vaccinated fish increased after vaccine administration and exerted NNV-neutralizing effects. The efficacy tests revealed that the relative percent survival (RPS) of LAE antigen formulated with adjuvant was above 72% and LAE vaccine was effective for preventing NNV infection in giant grouper. This study is the first to develop an NNV vaccine by using epitope repeats, which provided effective protection to giant grouper against virus infection. The LAE construct can be used as a vaccine design platform against various pathogenic diseases.

Topics: Animals; Bass; Capsid Proteins; Epitopes; Fish Diseases; Nodaviridae; Recombinant Proteins; RNA Virus Infections; Vaccines, Subunit; Viral Vaccines

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Fas-Associated Death Domain Protein; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Poly I-C; Ranavirus; Sequence Alignment

The present study investigated the effects of dietary Spirulina platensis supplementation on growth performance, hematological and serum biochemical parameters, hepatic antioxidant status, immune responses and resistance to the pathogen infection in Coral trout Plectropomus leopardus. The fish were fed for 8-week with diets containing different levels of S. platensis: 0% (C), 2% (SP2), 4% (SP4), 6% (SP6), 8% (SP8) and 10% (SP10) as treatment groups, followed by a Vibrio harveyi infection test for 14 d. The study indicated that dietary supplementation with Spirulina platensis could significantly improve growth performance, and the highest weight gain rate (WGR) and specific growth rate (SGR) were observed in group SP10 (P < .05). Red cell count (RBC), white cell count (WBC), hemoglobin (Hb) and total antioxidant capacity (T-AOC) in the S. platensis supplemented groups were significantly higher than those of group C (P < .05). However, the levels of cholesterol, triglyceride and malondialdehyde (MDA) contents, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities decreased with the increasing of dietary S. platensis levels. Compared with group C, the lysozyme (LYZ) and respiratory burst activities (RBA), and immunoglobulin (Ig) and complement contents in group SP4, SP6, SP8 and SP10 increased significantly than those of group C respectively (P < .05). After challenge with V. harveyi, the survival rate in group SP4, SP6, SP8 and SP10 was significantly higher than that of group C, and the highest survival rate was in group SP10 (P < .05). These results indicated that P. leopardus fed a diet supplemented with S. platensis (especially at 10%) could significantly promote its growth performance, improve its hepatic antioxidant status, and enhance its immune ability and resistance to V. harveyi infection.

Topics: Animal Feed; Animals; Antioxidants; Bass; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Immunologic; Fish Diseases; Liver; Random Allocation; Spirulina; Vibrio; Vibrio Infections

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the central nervous system, is considered one of the most serious viral diseases in marine aquaculture. Although some studies have localized NNV in the retina cells, none has dealt with immunity in the retina. Thus, for the first time, we intravitreally infected healthy specimens of European sea bass (Dicentrarchus labrax) with NNV with the aim of characterizing the immune response in the retina. Ultrastructural analysis detected important retinal injuries and structure degradation, including pycnosis, hydropic degeneration and vacuolization in some cell layers as well as myelin sheaths in the optic nerve fibres. Immunohistochemistry demonstrated that NNV replicated in the eyes. Regarding retinal immunity, NNV infection elicited the transcription of genes encoding proteins involved in the interferon (IFN) and cell-mediated cytotoxicity (CMC) responses as well as B and T cell markers, demonstrating that viral replication influences innate and adaptive responses. Further studies are needed to understand the retina immunity and whether the main retinal function, vision, is affected by nodavirus.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Nodaviridae; Retina; Retinal Diseases; RNA Virus Infections

Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors (T3SEs) and their physiological role. In this study, the T3SE gene hopPmaJ (hop) was cloned from V. alginolyticus wild-type strain HY9901 and the mutant strain HY9901Δhop was constructed by the in-frame deletion method. The results showed that the deduced amino acid sequence of V. alginolyticus HopPmaJ shared 78-98% homology with other Vibrio spp. In addition, the HY9901Δhop mutant showed an attenuated swarming phenotype and a 2600-fold decrease in the virulence to grouper. However, the HY9901Δhop mutant showed no difference in morphology, growth, biofilm formation and ECPase activity. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901Δhop induced a high antibody titer with a relative percent survival (RPS) value of 84% after challenging with the wild-type HY9901. Real-time PCR assays showed that vaccination with HY9901Δhop enhanced the expression of immune-related genes, including MHC-Iα, MHC-IIα, IgM, and IL-1β after vaccination, indicating that it is able to induce humoral and cell-mediated immune response in grouper. These results demonstrate that the HY9901Δhop mutant could be used as an effective live vaccine to combat V. alginolyticus in grouper.

Topics: Amino Acid Sequence; Animals; Bacterial Vaccines; Bass; Fish Diseases; Mutation; Random Allocation; Sequence Homology; Vaccines, Attenuated; Vibrio alginolyticus; Vibrio Infections; Virulence

The eukaryotic initiation factor 2 alpha subunit (eIF2α) is a key translation regulator that plays an important role in different cellular pressures and stimuli, including virus infection. In the present study, an eIF2α homolog (EceIF2α) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EceIF2α encodes a putative protein of 315 amino acid residues, and shares a high degree of similarity with eIF2αs from other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that EceIF2α was distributed in all examined tissues. Both of the expression levels of EceIF2α in the spleen and head kidney of E. coioides were up-regulated when challenged with polyinosine-polycytidylic acid (poly[I:C]). EceIF2α was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) cells. Over-expression of EceIF2α improved the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. In addition, EceIF2α depressed the activation of NK-κB and IFN-β. Furthermore, dephosphorylation inhibitor treatment led to a significant decrease of RGNNV gene transcription. Taken together, these results suggest that EceIF2α might be involved in the fish immune response to virus challenge.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Eukaryotic Initiation Factor-2; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Poly I-C; RNA Virus Infections; Sequence Alignment

Nervous necrosis virus (NNV) infection causes viral nervous necrosis, inflicting serious economic losses in marine fish cultivation. Vaccination is the most effective choice for controlling and preventing viral infection. Virus-like particles (VLPs) are considered a novel vaccine platform because they are not infectious and they induce neutralizing antibodies efficiently. In the present study, we investigated the effect of the recombinant orange-spotted grouper NNV (OSGNNV) capsid proteins produced in Escherichia coli and cell-free self-assembled into VLPs on protective immune responses in orange-spotted grouper following immersion, intramuscular injection and oral immunization. We found the OSGNNV VLPs elicited neutralizing antibody with high efficacy, and provided the fish with full protection against OSGNNV challenge. In addition, the cell-free self-assembled OSGNNV VLPs did not contain residual host cell components and was safer compared with the intracellular assembled VLPs. Thus, oral vaccination is a more convenient and preferred route for fish vaccination. Our results show that the fish fed four times with a diet supplemented with 50-200 μg/g OSGNNV VLPs at 7-day intervals have sufficient protection. These findings demonstrate that cell-free self-assembled OSGNNV VLPs have potential as oral vaccines in grouper.

Topics: Administration, Oral; Animals; Antibodies, Neutralizing; Bass; Capsid Proteins; Escherichia coli; Fish Diseases; Larva; Nodaviridae; RNA Virus Infections; Vaccination; Vaccines, Virus-Like Particle; Viral Vaccines

Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events.. Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions.. The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Iridoviridae; Pennsylvania; Rivers

The innate immune signaling adapter, Mitochondrial antiviral signaling protein (MAVS) coordinates the signals received from two independent RLRs (RIG-1 and MDA5) to induce IFN & interferon stimulatory genes (ISGs). In the present study, we report identification of an orthologue of MAVS from Lates calcarifer (LcMAVS) and its functional role in piscine RLR signaling. The LcMAVS-cDNA was cloned into pcDNA and transfected into SISS cells. LcMAVS was detected to be a 61KDa protein in western blot. Confocal microscopy demonstrated the mitochondrial localization of LcMAVS. In addition, pcDNA-MAVS transfected cells were protected against Nervous Necrosis Virus (NNV) infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral transcript levels. Ectopic expression of LcMAVS resulted in activation of an ISRE-containing promoter (52 folds over control cells) as well as transcriptional expression of IRF-3, IFN-1 and IFN-inducible genes including Mx and ISG15 (p<0.05). These results suggest that LcMAVS is involved in the antiviral immunity as one of the adaptors in fish IFN-activation pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bass; Fish Diseases; Fish Proteins; Host-Pathogen Interactions; Immunity, Innate; Nodaviridae; Perciformes; RNA Virus Infections

Largemouth bass (Micropterus salmoides) rhabdovirus (MSRV) was isolated from infected juveniles of largemouth bass, and the infected fish exhibited corkscrew, irregular swimming, and crooked body. To our knowledge, the potential molecular mechanisms underlying the pathogenesis of MSRV infection remain largely unknown. In the current study, we found that MSRV infection in largemouth bass skin (LBS) cells induced typical apoptosis, evidenced by the presence of apoptotic bodies and caspase-3 activation. To further analyze the host factors involved in MSRV infection in LBS cells, the transcriptomic profiles during MSRV infection were uncovered using deep RNA sequencing technique, and several differentially expressed genes (DEGs) were validated by quantitative PCR. Our results showed that a total of 124483 unigenes were assembled. Among them, 34465 and 27273 had significant hits to those in the NR and SwissProt databases. After MSRV infection, a total of 2432 and 2480 genes which involved in multiples pathways including TNF signaling, NF-κB signaling, Toll-like receptor signaling and RIG-I signaling pathway were differentially expressed in MSRV infected LBS cells compared to mock-infected cells at 12 h, respectively. Furthermore, quantitative PCR showed that the expression levels of 9 differentially expressed genes (DEGs) related to apoptosis and interferon signaling pathway was consistent with that from transcriptomic profiles. Together, our results not only demonstrated that interferon signaling pathway and apoptosis pathway might exerted crucial roles during MSRV infection, but also provided a useful resource for subsequent investigation of other immune-related genes related to virus infection.

Topics: Animals; Apoptosis; Bass; Cell Culture Techniques; Fish Diseases; High-Throughput Nucleotide Sequencing; Immunity, Innate; Interferons; Rhabdoviridae; Rhabdoviridae Infections; Sequence Analysis, RNA; Signal Transduction; Skin; Transcriptome

Nervous necrosis virus (NNV) infection has been considered a serious disease in farmed grouper. Particularly, the persistent infection model conducts the grouper into a carrier state that continues to spread the virus through spawning. This particular model makes disease control more difficult in the aquaculture industry. In the present study, we used RNA-Seq, a high-throughput method based on next-generation sequencing, to profile the expression of genes during the period of NNV persistent infection. We evaluated the transcriptomic changes in the brain tissue of grouper. The inactivated-NNV vaccine was used as a comparison group. Based on the differentially expressed genes, highly immune cell active signaling and surface receptor expression were triggered during persistent infection. The interferon-induced response was also highly expressed in the infected brain tissue. However, critical negative regulatory factors of T-cells, such as PD-L1 and LAG3, were up-regulated. The present transcriptome study revealed a comprehensive view of the state of NNV persistent infection and provided insights into the state of impaired NNV clearance in the grouper.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Immunity, Innate; Nodaviridae; RNA Virus Infections; Transcriptome; Vaccines, Inactivated; Viral Vaccines

Recombinant Epinephelus lanceolatus serum amyloid A (rElSAA) exhibits strong immunostimulant activity and enhances phagocytic clearance of bacteria by macrophages. However, the effects of dietary rElSAA supplementation on growth performance, immunomodulation and disease resistance in giant grouper have not been previously evaluated. To test whether oral administration of rElSAA affects growth, fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 28 days. No statistically significant differences in body weight were observed between groups. Next, we tested whether oral administration of rElSAA may enhance disease resistance. Fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 3, 7, 14, 21 or 28 days, followed by challenge with Vibrio alginolyticus. Survival was then monitored for 4 days. Fish that were fed with rElSAA-containing diet for 28 days showed significantly improved survival after infection. In addition, the expression levels of immune defense-associated genes in hepatic tissue were assessed by quantitative real-time polymerase chain reaction before and after V. alginolyticus infection. Oral administration of rElSAA increased the expression level of toll-like receptor 5, whereas the expression levels of CC chemokine 1, SAA and C reactive protein were decreased. Thus, the data suggest that rElSAA may enhance host immunity by attenuating regulatory T cell-mediated suppression of inflammation. Together, our results demonstrate that rElSAA is a promising candidate as a feed additive for giant grouper, which may effectively enhance disease resistance after being administered for several weeks.

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Escherichia coli; Fish Diseases; Fish Proteins; Gene Expression; Random Allocation; Recombinant Proteins; Serum Amyloid A Protein; Vibrio alginolyticus; Vibrio Infections

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%-92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14-21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

Topics: Agammaglobulinaemia Tyrosine Kinase; Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Protein-Tyrosine Kinases; Sequence Alignment

Onion Allium cepa and ginger Zingiber officinale have health-promoting properties that qualify them as functional foods. The effect of repeated acute stressors was examined in juvenile Brown-marbled Grouper Epinephelus fuscoguttatus that were fed four diets supplemented with onion at 1.8%, ginger at 1.8%, vitamin C at 0.86%, and β-glucan at 0.8% of the diet. The non-supplemented diet served as the control. After 12 weeks of feeding, fish were exposed to stressors and were experimentally infected with a fish pathogen, the bacterium Vibrio harveyi JML1. After repeated exposure to hypoxia, cortisol levels rose significantly in the non-supplemented fish compared to those fed onion, ginger, β-glucan, or vitamin C. Within groups, postexposure cortisol levels in the onion-, ginger-, and vitamin C-fed fish did not change relative to pre-stress levels, whereas significant increases in poststress values were observed in the control and β-glucan groups. The net cortisol increase was also significantly greater in the non-supplemented group compared to the supplemented groups. The net cortisol increase did not vary among the supplemented groups except that the β-glucan-fed group exhibited a higher net increase than the onion-fed group. Similarly, repeated acute exposure to osmotic stress significantly increased the plasma cortisol level in the non-supplemented group compared to groups that received supplements; no differences were found in the supplemented groups except the β-glucan group. Within groups, significant increases in poststress values relative to pre-stress levels were found only in the control and β-glucan groups. Repeated acute exposure to hypoxia significantly increased cumulative mortality in the control group compared to the supplemented groups (except the β-glucan group), whereas repeated exposure to acute osmotic stress significantly increased cumulative mortality only in the control group 10 d after infection with V. harveyi JML1. Based on our collective results, most of the supplemented groups performed better than the control, but the best supplements were onion and ginger in terms of enhancing stress tolerance and increasing survival of Brown-marbled Grouper upon infection with V. harveyi JML1.

Topics: Animals; Ascorbic Acid; Bass; beta-Glucans; Dietary Supplements; Fish Diseases; Hydrocortisone; Hypoxia; Onions; Osmotic Pressure; Vibrio; Vibrio Infections; Zingiber officinale

Evidence of disease and mortalities of young of the year (age-0) Smallmouth Bass Micropterus dolomieu has occurred during the late spring and summer in many parts of the Susquehanna River watershed since 2005. To better understand contributing factors, fish collected from multiple areas throughout the watershed as well as out-of-basin reference populations (Allegheny and Delaware River basins; experimental ponds, Kearneysville, West Virginia) were examined grossly and histologically for abnormalities. Tissue contaminant concentrations were determined from whole-body homogenates, and water contaminant concentrations were estimated using time-integrated passive samplers at selected sites. Observed or isolated pathogens included bacteria, predominantly motile Aeromonas spp. and Flavobacterium columnare; largemouth bass virus, and parasites, including trematode metacercariae, cestodes, and the myxozoan Myxobolus inornatus. Although these pathogens were found in age-0 Smallmouth Bass from multiple sites, no one pathogen was consistently associated with mortality. Chemicals detected in tissue included polychlorinated biphenyl (PCB) congeners, organochlorine, and current-use pesticides. Pyraclostrobin, PCB congeners 170 and 187, cis-chlordane and trans-nonachlor were detected in all Susquehanna watershed samples but rarely in samples from the reference site. The findings support the idea that there is no single cause for disease of age-0 Smallmouth Bass; rather the cumulative effects of co-infections and potential immunomodulation by environmental stressors during a sensitive developmental life stage may lead to mortality. Identifying the most important risk factors will be necessary for more in-depth analyses of individual stressors and better management of the habitat and fish populations.

Topics: Animals; Bacteria; Bass; Coinfection; Fish Diseases; Parasites; Pennsylvania; Risk Factors; Rivers; Seasons; Viruses; Water Pollutants, Chemical

In order to develop live vaccine against Siniperca chuatsi rhabdovirus (SCRV) disease, an avirulent virus strain, designed as Micropterus salmoides rhabdovirus Sanshui (MSRV-SS), was selected from six fish rhabdovirus isolates (SCRV-QY、SCRV-SS、SCRV-GM、CMRV-FS、OMBRV-JM、MSRV-SS) by fish challenge assay. When Chinese perch (Siniperca chuatsi) were intraperitoneally injected live virus strain MSRV-SS, they were completely protected from virulent SCRV-GM challenge with a relative percent survival (RPS) of 100% on 18th day post vaccination. Then, the wild type MSRV-SS was purified by plaque clone assays, and the biological characteristics of the clonal strain designed as MSRV-SS-7 were investigated. The MSRV-SS-7 was avirulent to Chinese perch and its growth characteristic was similar to the MSRV-SS. The immune protection effects of clonal MSRV-SS-7 against virulent SCRV-GM were evaluated by intraperitoneal injection (IP) vaccination and immersion (IM) vaccination, their RPSs were all 100%. Altogether, these results indicate that MSRV-SS-7 is a potential live vaccine candidate against SCRV disease.

Topics: Animals; Bass; Fish Diseases; Perciformes; Rhabdoviridae; Rhabdoviridae Infections; Vaccination; Viral Vaccines

A reverse vaccinology-based survey of potent antigens associated with fish nocardiosis was conducted using the largemouth bass, Micropterus salmoides, with an aim to develop subunit vaccines. The antigens selected from the virulent strain Nocardia seriolae 961113 include the gene products of NGL2579 (GAPDH), NGL5701 (MMP), NGL4377 (OCTase), NGL4486 (ABC transporter), NGL3372 (LLE), NGL3388 (GHf10), NGL6627 (Antigen-85), NGL6696 (Esterase), and NGL6936 (CBP). These antigens were heterologously expressed in E. coli BL21 (DE3) for recombinant protein production. Then fish were vaccinated was these antigens, boosted at 2 weeks, and challenged with N. seriolae at 6 weeks after vaccination. The relative protection survival assay revealed high and significant protection efficacies of 94.45, 50.00, and 44.45 in fish that received the NGL3388 (GHf10), NGL6936 (CBP), and NGL3372 (LLE) vaccines, respectively. There were no apparent relationships or differences in tissue lesions among the administered vaccines. The serum titers against the bacterial preparations were higher for all vaccinated groups than for the control group at 4 weeks after immunization. However, no significant difference in serum titer was found at 6 weeks after immunization. The results of this study demonstrate that subunit vaccines against fish nocardiosis have differential effects, but are highly promising for nocardial prophylaxis.

Topics: Animals; Bacterial Vaccines; Bass; Escherichia coli; Fish Diseases; Nocardia; Nocardia Infections; Recombinant Proteins; Vaccines, Synthetic

Bax inhibitor-1 (BI-1) is a conserved anti-apoptotic protein that suppresses endoplasmic reticulum (ER) stress-induced cell death. However, the function of fish BI-1 is not quite clear. In the present study, a bi-1 homolog (Ecbi-1) from orange spotted grouper, Epinephelus coioides was identified and its roles in viral infection were investigated. EcBI-1 encoded 237 amino acids protein, contained six transmembrane regions and a conservative C-terminus motif. Ecbi-1 predominantly expressed in kidney and spleen of healthy grouper. After SGIV stimulation, Ecbi-1 transcript was significantly increased in vitro. Subcellular localization analysis revealed that EcBI-1 was localized throughout the cytoplasm and co-localized with ER. Furthermore, overexpression of EcBI-1 suppressed SGIV infection induced cell death, caspase-3 activity and viral genes transcription. And C-terminus motif was critical for regulation roles of EcBI-1 during SGIV infection. In addition, EcBI-1 could interact with EcBNIP3 in vitro. Together, our data firstly demonstrated that fish BI-1 play important roles in response to viral infection.

Topics: Amino Acid Sequence; Animals; Apoptosis Regulatory Proteins; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Ranavirus; Sequence Alignment

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Topics: Animals; Antibodies, Viral; Bass; Brain; Fish Diseases; Head Kidney; Immunity, Innate; Nodaviridae; RNA Virus Infections; Transcription, Genetic; Virulence

Developing viral vaccines through the ultraviolet (UV) inactivation of virus is promising technique since it is straightforward and economically affordable, while the resulting viruses are capable of eliciting an adequate antiviral immune response. Nodavirus (NNV) is a devastating virus that mainly affects European sea bass juveniles and larvae, causing serious economic losses in Mediterranean aquaculture. In this work, a potential vaccine consisting on UV-inactivated NNV (iNNV) was generated and administered to healthy juveniles of European sea bass to elucidate whether it triggers the immune response and improves their survival upon challenge. First, iNNV failed to replicate in cell cultures and its intraperitoneal administration to sea bass juveniles also failed to produce fish mortality and induction of the type I interferon (IFN) pathway, indicating that the NNV was efficiently inactivated. By contrast, iNNV administration induced significant serum non-specific antimicrobial activity as well as a specific antiviral activity and immunoglobulin M (IgM) titres against NNV. Interestingly, few changes were observed at transcriptional level in genes related to either innate or adaptive immunity, suggesting that iNNV could be modulating the immune response at protein or functional level. In addition, the iNNV vaccinated group showed improved survival, reaching a relative survival percentage of 57.9%. Moreover, challenged fish that had been vaccinated presented increased serum antibacterial, antiviral and IgM titres, as well as the higher transcription of mhc1a, ifn, isg15 and cd8a genes in brain, while in the head-kidney the transcription of mhc1a, mhc2b and cd8a was down-regulated and mx, isg15 and tcrb was up-regulated. Although the UV-inactivated vaccine against NNV showed promising results, more effort should be addressed to improving this prophylactic method by increasing our understanding of its action mechanisms, thus enabling the mortality rate of NNV to be further reduced.

Topics: Adaptive Immunity; Animals; Aquaculture; Bass; Brain; Fish Diseases; Head Kidney; Immunity, Innate; Immunoglobulin M; Interferon Type I; Nodaviridae; RNA Virus Infections; Vaccination; Vaccines, Inactivated; Viral Vaccines

In the present study, the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a typical carnivorous fish, was chosen as a model to investigate the regulation of glycogen metabolism owning to its characteristic of glucose intolerance. The variation of plasma glucose concentration, glycogen content, and expressions of glycogen metabolism-related genes under acute hyperglycemia stress were measured. Following glucose administration, plasma glucose concentration increased immediately, and the glucose level remained elevated for at least 12 h. The prolonged glucose clearance and hyperglycemia revealed glucose intolerance of this fish species. Meanwhile, the glycogen content in both liver and muscle changed significantly during the clearance of plasma glucose. However, the peak value of hepatic glycogen (1 and 12 h post injection) appeared much earlier than muscle (3 and 24 h post injection). To investigate the regulation of glycogen metabolism from molecular aspect, the complete coding sequence (CDS) of glycogen synthase (GS) and glycogen phosphorylase (GP) in both liver and muscle types were obtained, encoding a polypeptide of 704, 711, 853, and 842 amino acid residues, respectively. The results of gene expression analysis revealed that the expression of liver type and muscle type GS was significantly higher than other time points at 12 and 24 h post glucose injection, respectively. Meanwhile, the highest expressions of GP in both liver and muscle types occurred at 24 h post glucose injection. The response of GS and GP to glucose load may account for the variation of glycogen content at the transcriptional level to some extent.

Topics: Animals; Bass; Blood Glucose; Fish Diseases; Glycogen; Glycogen Synthase; Hyperglycemia; Phosphorylases; Stress, Physiological

Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host-pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress- and innate immune-related genes in European sea bass (

Topics: Animals; Bass; Cytokines; Fish Diseases; Germ-Free Life; Glucocorticoids; Hepcidins; Host-Pathogen Interactions; Immunity, Innate; Larva; Up-Regulation; Vibrio; Vibrio Infections

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK-OmpU fusion protein in addition to the metabolizable Montanide

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Interleukin-1beta; Interleukin-8; Recombinant Proteins; Vibrio; Vibrio Infections

European sea bass (Dicentrarchus labrax) is one of the most important species for European aquaculture. Viral nervous necrosis (VNN), commonly caused by the redspotted grouper nervous necrosis virus (RGNNV), can result in high levels of morbidity and mortality, mainly during the larval and juvenile stages of cultured sea bass. In the absence of efficient therapeutic treatments, selective breeding for host resistance offers a promising strategy to control this disease. Our study aimed at investigating genetic resistance to VNN and genomic-based approaches to improve disease resistance by selective breeding. A population of 1538 sea bass juveniles from a factorial cross between 48 sires and 17 dams was challenged with RGNNV with mortalities and survivors being recorded and sampled for genotyping by the RAD sequencing approach.. We used genome-wide genotype data from 9195 single nucleotide polymorphisms (SNPs) for downstream analysis. Estimates of heritability of survival on the underlying scale for the pedigree and genomic relationship matrices were 0.27 (HPD interval 95%: 0.14-0.40) and 0.43 (0.29-0.57), respectively. Classical genome-wide association analysis detected genome-wide significant quantitative trait loci (QTL) for resistance to VNN on chromosomes (unassigned scaffolds in the case of 'chromosome' 25) 3, 20 and 25 (P < 1e06). Weighted genomic best linear unbiased predictor provided additional support for the QTL on chromosome 3 and suggested that it explained 4% of the additive genetic variation. Genomic prediction approaches were tested to investigate the potential of using genome-wide SNP data to estimate breeding values for resistance to VNN and showed that genomic prediction resulted in a 13% increase in successful classification of resistant and susceptible animals compared to pedigree-based methods, with Bayes A and Bayes B giving the highest predictive ability.. Genome-wide significant QTL were identified but each with relatively small effects on the trait. Tests of genomic prediction suggested that incorporating genome-wide SNP data is likely to result in higher accuracy of estimated breeding values for resistance to VNN. RAD sequencing is an effective method for generating such genome-wide SNPs, and our findings highlight the potential of genomic selection to breed farmed European sea bass with improved resistance to VNN.

Topics: Algorithms; Animals; Bass; Breeding; Chromosome Mapping; Disease Resistance; Fish Diseases; Genome-Wide Association Study; Genotyping Techniques; Nodaviridae; Pedigree; Polymorphism, Single Nucleotide; Quantitative Trait, Heritable; RNA Virus Infections; Sequence Analysis, DNA

The dusky grouper Epinephelus marginatus (Lowe) is an ecologically and commercially important fish species of the Atlantic and Mediterranean coastal rocky habitats. Despite records of didymozoid infections in several grouper species, the identification and pathogenesis of these parasites in E. marginatus are lacking. The aim of this study is to characterize the didymozoids of E. marginatus, particularly their mechanisms of infection and histopathological features. Dusky groupers (n = 205) were caught off Majorca Island (western Mediterranean Sea) and examined for parasites. Of the fish sampled, 45% were infected with white and yellow didymozoid capsules and brown nodules, found on the gills and pseudobranchs. Parasite abundance had a strong positive relationship with the fish length; only fish larger than 20 cm were infected, suggesting infection via consumption of an intermediate host, for which E. marginatus size was a limiting factor. The capsules contained two convoluted viable adult trematodes, identified as Didymodiclinus sp., in close contact with host capillary vessels, with no evidence of the tissue inflammatory response. Conversely, nodules containing degraded parasites were surrounded by an intense inflammatory infiltrate. The findings suggest that Didymodiclinus sp. have the potential to evade the host's immune system by inhibiting the inflammatory response.

Topics: Animals; Bass; Ecological and Environmental Phenomena; Fish Diseases; Gills; Immune Evasion; Inflammation; Mediterranean Sea; Seafood; Trematoda

Member of the dynamin family of large GTPases, dynamin-related protein 1 (Drp1) dependent mitochondrial fission is an intricate process regulating both cellular and organ dynamics. Present study shows that NNV perturbs mitochondrial dynamics by promoting Drp-1 dependent mitochondrial fission, which attenuates MAVS mediated downstream signaling. NNV infected SISS cells revealed induction in Drp1 expression and subsequent translocation into mitochondria. The level of MAVS expression was up-regulated over a period of 24 hpi and declined with the progression of NNV infection at 48 and 72 hpi confirmed by western blot and mRNA transcript analysis. Drp-1 displayed its association with fragmented mitochondria and the transcript abundance was significant post infection along with Mff. Expression levels of IRF-3 IFN-1 and Mx followed a similar pattern with abundant expression at 48 hpi and diminished expression during the further period. Importantly, silencing of Drp1 caused significant elevation in the RLR downstream molecules and reduction in viral RNA expression. These results suggest that NNV-induced mitochondrial fission serve to attenuate host RLR signaling. This provides an illustration of host-pathogen interaction in which the virus evades innate immunity by enhancing mitochondrial fission and perturbs MAVS, and the downstream molecules.

Topics: Animals; Bass; Cell Line; DEAD Box Protein 58; Dynamins; Fish Diseases; Fish Proteins; Mitochondrial Dynamics; Nodaviridae; Reactive Oxygen Species; RNA Virus Infections; Signal Transduction; Spleen

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.

Topics: Animals; Base Sequence; Bass; Carps; Fish Diseases; Genome; Genotype; Molecular Sequence Data; Phylogeny; Republic of Korea; Rhabdoviridae; Rhabdoviridae Infections; Sequence Analysis, DNA; Virulence

Outbreaks of viral nervous necrosis (VNN) in Asian sea bass (Lates calcarifer) at the larval stages via vertical transmission of nervous necrosis virus (NNV) from asymptomatic broodfish remain as a major deterrent during seed production. A five-year study was conducted to produce NNV-specific-free sea bass broodfish reared in land-based tanks through an annual immunization regimen with the formalin-inactivated NNV. We primarily immunized (intraperitoneal injection) sea bass juveniles (5 g) and monitored the neutralizing antibody (Nab) titers in the sera of these fish at scheduled intervals post-immunization. Nab titers in the sera of immunized fish peaked at Month 2 (titer: 1:4480 ± 1185) but thereafter gradually declined and significantly dropped (1:260 ± 83) at Month 12 post-primary immunization. Booster immunization of these fish at Month 12 post-immunization led to abrupt increases in Nab titers in booster immunized (B-Im) fish at Month 1 (1:12800 ± 6704) but thereafter declined and dropped at Month 12 (1:480 ± 165) post-booster immunization. The annual booster injections with the inactivated vaccine or L-15 (Unimmunized [U-Im]) were consecutively conducted for 4 years until the fish became sexually mature. Mature fish from both groups were successively induced to spawn twice (1-month interval) via intramuscular injection with luteinizing hormone-releasing hormone analogue (LHRH-a; 100 µg/kg BW). NNV was not detected by RT-PCR in oocytes and milts, and spawned eggs of B-Im fish. In contrast, oocytes and milts, and spawned eggs of U-Im fish were NNV positive. Spawned eggs of B-Im broodfish exhibited Nab titers ranging from 1:192 ± 34 to 1:240 while such was not detected (<1:40) in eggs of U-Im fish. Taken together, current data clearly demonstrate that annual immunization regimen with inactivated NNV vaccine is a pragmatic approach for sustaining immunocompetent sea bass broodfish reared in land-based tanks and circumvent the risk of vertical transmission of NNV from asymptomatic broodfish to their offspring under stress of repetitive spawning.

Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Bass; Fish Diseases; Infectious Disease Transmission, Vertical; Nodaviridae; Perciformes; RNA Virus Infections; Vaccines, Inactivated

Traditional methodologies to identify fish pathogens require euthanasia before the collection of tissue samples. While these methods are standardized and proven, there are instances where nonlethal alternatives would be preferred. Despite the need to develop nonlethal sampling techniques, few publications have focused on them and even fewer have used these approaches to identify viruses from infections occurring in wild fish populations. In this study, we compared the ability of nonlethal sampling techniques with traditional methods for the detection of Largemouth Bass virus (LMBV) from a wild population of Largemouth Bass Micropterus salmoides from the upper Mississippi River. Largemouth bass virus was isolated from 30% of the Largemouth Bass sampled using traditional methods where tissue samples were inoculated on Bluegill fry (BF-2) cells. Furthermore, when using tissue cell culture to isolate LMBV, there was no significant difference observed in the overall proportion that was positive between the mucus samples and the kidney and spleen samples. Mucus swabs analyzed with molecular methods (conventional PCR and quantitative PCR) were more sensitive than traditional tissue cell culture-based methods as they detected LMBV from >70% of the samples; limitations to these methods (i.e., carryover contamination) were also identified. The results of this study suggest that nonlethal sampling may be a useful option for detecting LMBV from fish populations.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fisheries; New York; Prevalence; Ranavirus; Virology

We examined 151 European sea bass (Dicentrarchus labrax L.) samples from farms and fish markets of Sicily (Southern Italy) for Anisakidae larvae detection. All the samples were examined by visual inspection and modified chloro-peptic digestion. Two nematode larvae were found in the viscera of only one European sea bass sample from a farm located in Greece (FAO 37.3), giving a total prevalence of infestation of 0.7%. No other parasites were found after chloro-peptic digestion of the samples. The larvae were morphologically ascribed, at genus level, to morphotypes I and molecularly identified as Anisakis pegreffii. To the best if our knowledge, this is the first report on the presence of anisakid parasites in farmed European sea bass of Mediterranean Sea. Our findings suggest that the risk of exposure to Anisakidae nematodes in farmed European sea bass remains very low. However, further data on Mediterranean farms are needed to have a detailed risk analysis.

Topics: Animals; Anisakiasis; Anisakis; Aquaculture; Bass; Fish Diseases; Fisheries; Greece; Larva; Mediterranean Sea; Polymerase Chain Reaction; Sequence Analysis, DNA; Sicily

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a g-type lysozyme gene was cloned from Microptenus salmoides. The g-type sequence consisted of 582 bp, which translated into a 193 amino acid (AA) protein (GenBank accession no: MH087462). The predicted molecular weight and theoretical isoelectric point were 21.36 kDa and 6.91 respectively and no signal peptide was observed. The qRT-PCR analysis showed that the g-type lysozyme gene was differentially expressed in various tissues under normal conditions and the highest g-type lysozyme level was observed in liver, gill and spleen while there seemed to be low expression in the muscle, heart and head-kidney. The expression of g-type lysozyme was differentially upregulated in the spleen, gill and intestine after stimulation with heat stress and Aeromonas hydrophila (A. hydrophila). Under heat stress and A. hydrophila injection, the g-type lysozyme mRNA levels all in spleens, gill and intestine tissues increased significantly (P < 0.05), with the maximum levels attained at 12 h, 24 h (or 12 h) and 24 h. Thereafter, they all decreased significantly (P < 0.01) and the expression in gill returned to nearly the basal value within 72 h. Those results suggested that g-type lysozyme was involved in the immune response to heat stress and bacterial challenge. The cloning and expression analysis of the g-type lysozyme provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides. The g-type lysozyme gene perhaps also played an important role in the immune responses against bacterial invasion.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Heat-Shock Response; Immunity, Innate; Muramidase; Phylogeny; Sequence Alignment

A myxozoan parasite, Myxobolus inornatus, is one disease agent identified in young of the year (YOY) smallmouth bass in the Susquehanna River Basin, Pennsylvania. We investigated spatial and temporal variability in M. Inornatus prevalence across the Susquehanna River Basin and at several out-of-basin sites. We examined potential land use drivers of M. Inornatus prevalence including agricultural and developed land use. In 1,267 YOY smallmouth bass collected from 32 sites during 2013-2016, M. Inornatus was documented in 43.6% of samples. Among-site variability in parasite prevalence was greater than among-year variability. The effect of agricultural land use on M. Inornatus prevalence had a high probability of being positively correlated at multiple spatial scales (probability of positive effect > 0.80). The effect of developed land use on M. Inornatus prevalence had a relatively high probability of being negatively correlated at multiple spatial scales (probability of negative effect > 0.70). Our results suggest that land use practices could be related to M. Inornatus infection of smallmouth bass. Further study will be necessary to determine whether disease dynamics are a consequence of effects on the host, alterations of instream habitat mediating invertebrate host dynamics and/or survival and dispersal of the parasite infective stage.

Topics: Animals; Bass; Fish Diseases; Myxobolus; Parasitic Diseases, Animal; Pennsylvania; Prevalence; Seasons; Spatial Analysis

No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of fish mycobacteriosis (also known as "fish tuberculosis"). The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinum pathology in Zebrafish Danio rerio. Two doses of heat-killed, wild-type, virulent M. marinum and two doses of a heat-killed, avirulent M. marinum iipA::kan mutant strain were used in parallel to vaccinate European Seabass Dicentrarchus labrax. The fish were then challenged with live, virulent M. marinum, and the pathogenesis of the infection was monitored. High specific immunoglobulin M (IgM) response and an increase in cytokine tumor necrosis factor alpha (TNF-α) messenger RNA expression levels were observed in all vaccinated fish. At 1 month postchallenge, TNF-α expression levels increased in spleen tissues of fish vaccinated with the virulent type and in those of unvaccinated fish, whereas in the head kidney, expression was up-regulated only in unvaccinated fish. The expression then decreased, and at 2 months postchallenge, expression appeared similar in all vaccination types. The highest survival rate (75%) was recorded in the group of fish that were vaccinated with a high dose of avirulent iipA::kan mutant. The iipA::kan mutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kan mutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis.

Topics: Animals; Bass; Fish Diseases; Hot Temperature; Immunity, Cellular; Immunity, Humoral; Immunoglobulin M; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; RNA, Messenger; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

Interleukins are critical cytokines that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. Here, we identified and analyzed IL-12p40 from the Chinese sea bass Lateolabrax maculatus (LmIL-12p40). The LmIL-12p40 gene is expressed as a 1386-base pair transcript that encodes a polypeptide of 321 amino acids. Transcriptional expression analysis indicated that LmIL-12p40 mRNA was ubiquitously expressed in all tested tissues and had a comparatively high expression level in immune-associated tissues (head-kidney and intestines). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments showed that, after Vibro harveyi and Streptococus agalactiae infection, LmIL-12p40 mRNA expression was significantly up-regulated in the spleen, liver and head-kidney. To further clarify the immune function of LmIL-12p40 after bacterial challenge, the recombinant LmIL-12p40 protein was acquired using a prokaryotic expression method. Furthermore, the LmIL-12p40 dimer (LmIL-12p80) could be produced via protein-protein interactions by incubating p40 monomer expressed from the pET28a vector (pET28a-LmIL-12p40) with p40 monomer expressed from the pGEX4T-1 vector (pGEX4T-1-LmIL-12p40). The antimicrobial activity of the purified LmIL-12p40 and LmIL-12p80 proteins were further studied in vitro using a bacterial growth inhibition test (for both liquid and solid cultures) and in vivo (using a bacterial growth inhibition test with the head-kidney tissues). Furthermore, BL21 (DE3) E. coli cells transformed with the recombinant pET28a-LmIL-12p40 vector were dramatically protected in response to metal toxicity and H

Topics: Animals; Bacteria; Bass; Cell Movement; Escherichia coli; Fish Diseases; Fish Proteins; HEK293 Cells; Humans; Hydrogen Peroxide; Interleukin-12 Subunit p40; Metals, Heavy; Streptococcal Infections; Stress, Physiological; Vibrio Infections

The Patagonian seabass Acanthistius patachonicus (Jenyns, 1840) (Serranidae) is a marine fish valued for commercial and sport fisheries from Argentina. We report a new myxosporean (Cnidaria: Myxozoa) infecting the urinary system of the Patagonian seabass from San Antonio Bay, San Matías Gulf, on the Atlantic Ocean. The mature myxospores were subspherical, 8.2-11.0 μm × 7.9-11.0 μm and 7.7-9.0 μm in thickness; two subspherical polar capsules, 2.4-3.8 μm × 2.3-3.6 μm, with 3 to 4 turns of the polar tubule; openings on different valves in almost opposite directions. Ornamented shell valves exhibited 17-20 concentrically organized surface ridges. SSU rDNA phylogenetics analyses placed the new species in the freshwater urinary tract clade, clustering in a clade formed by Myxobilatus gasterostei (Parisi, 1912), Acauda hoffmani Whipps, 2011, and other Ortholinea spp. Based on spore morphology, site of infection, and molecular data, we described this myxozoan as Ortholinea concentrica n. sp.

Topics: Animals; Argentina; Atlantic Ocean; Bass; DNA, Ribosomal; Female; Fish Diseases; Myxozoa; Parasitic Diseases, Animal; Phylogeny; Spores; Urinary Tract

Members of the genus Neoechinorhynchus Stiles & Hassall, 1905 are endoparasites of freshwater fishes, brackish water fishes, and freshwater turtles distributed worldwide. In North America, 33 species have been described. One of the most widely distributed species in the eastern United States and Canada is Neoechinorhynchus (Neoechinorhynchus) cylindratus, a common acanthocephalan that infects centrarchid fishes. In the current study, adult specimens of N. (N) cylindratus were collected from largemouth bass (Micropterus salmoides) from the Purificación River in northern Mexico. In the same freshwater system, two additional congeneric species (Neoechinorhynchus (Neoechinorhynchus) emyditoides and Neoechinorhynchus (Neoechinorhynchus) panucensis) were collected and analysed. Sequences of the large subunit, internal transcribed spacers ITS1 and ITS2, 5.8S from nuclear DNA, and sequences of the cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA were generated and aligned with other sequences obtained from GenBank. Maximum likelihood and Bayesian inference analyses inferred for each dataset showed that N. (N) panucensis, N. (N) emyditoides and N. (N) cylindratus were nested within several clades, indicating that these species do not share a common ancestor. Our phylogenies also revealed that the genus Neoechinorhynchus is paraphyletic, requiring further taxonomic revision using phylogenetic systematics and re-examination of morphological and ecological data. The presence of several N. (N) cylindratus adults in northern Mexico allowed us to typify this species for the first time using a combination of morphological and molecular characteristics. The current record shows a wide distribution range of N. (N) cylindratus across Canada, the United States and Mexico in the Nearctic region.

Topics: Acanthocephala; Animals; Bass; DNA, Helminth; DNA, Intergenic; Fish Diseases; Fresh Water; Helminthiasis, Animal; Mexico; Phylogeny

Topics: Animal Feed; Animals; Bass; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Methionine; Photobacterium

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, cas1, il1β, tnfα, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae.

Topics: Animals; Apoferritins; Bass; Fish Diseases; Immunity, Innate; Larva; Recombinant Proteins; Vibrio; Vibrio Infections

Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.

Topics: Amino Acid Sequence; Animals; Bass; Cell Differentiation; Cloning, Molecular; Cytokines; DNA, Complementary; Escherichia coli; Escherichia coli Infections; Fish Diseases; Fish Proteins; Macrophages; Phagocytosis; Phylogeny; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Serum Amyloid A Protein

Topics: Animals; Bass; Chromosomes, Bacterial; Collagenases; Fish Diseases; Gram-Negative Bacterial Infections; Hemolysin Proteins; Phospholipases; Photobacterium; Plasmids; Virulence

Four Gram-negative bacterial isolates were recovered from 2 disease outbreaks that occurred in 2013 affecting European sea bass Dicentrarchus labrax fry and sea bream Sparus aurata adults. Main symptoms were erratic swimming, eroded fins and, in the sea bream outbreak, haemorrhages on the body surface; bacteria were always recovered from internal organs, almost in pure culture. On the basis of phenotypic characterization and 16S rRNA gene sequence analysis, the isolates were identified as Lacinutrix venerupis, a bacterium not previously reported as a fish pathogen. The highest 16S rDNA sequence similarities were recorded with the type strain of this species (99.9-100% similarity), while other species showed similarities below 97%, the closest relative being L. mariniflava (96.3% similarity). Phenotypic characterization showed some discrepancies with the L. venerupis type strain (mainly in BIOLOG GN profile); however, DNA-DNA hybridization assays with L. venerupis and L. mariniflava type strains confirmed that these isolates belong to the former species (levels of DNA relatedness were 98-100% and 38-50%, respectively). Finally, a virulence evaluation of the isolates using Senegalese sole Solea senegalensis fry was also performed; significant mortalities (80-100% mortality within 4 d) were recorded after intraperitoneal injection, but only with high doses of bacteria (107colony forming units fish-1). Further studies will be necessary to determine the importance of this species as a fish pathogen.

Topics: Animals; Bass; Disease Outbreaks; DNA, Bacterial; Fish Diseases; Flavobacteriaceae; Flavobacteriaceae Infections; Phylogeny; Sea Bream

BNIP3 is a kind of BH3-only protein that induces both cell death and autophagy. Here, a BNIP3 gene (EcBNIP3) was identified from orange spotted grouper, Epinephelus coioides. EcBNIP3 possessed 236 amino acids residues, contained a conservative BNIP3 domain and a transmembrane region. Besides, EcBNIP3 expressed at a relative high level in heart and spleen. EcBNIP3 transcript was up-regulated after SGIV infection in vitro. Subcellular localization analysis revealed that EcBNIP3 was predominantly localized in the cytoplasm and co-localized with mitochondria. In addition, overexpression EcBNIP3 accelerated SGIV infection induced cell death but inhibited viral genes transcription. Taken together, these results provided new evidence that fish BNIP3 might involved in response to virus infection.

Topics: Amino Acid Sequence; Animals; Apoptosis Regulatory Proteins; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Phylogeny; Ranavirus; RNA, Messenger; Sequence Alignment; Tissue Distribution

Grouper, Epinephelus coioides, fed a diet containing sodium alginate at 0 (control, named C) and 1.0 g kg

Topics: Adjuvants, Immunologic; Alginates; Animal Feed; Animals; Bass; Cold Temperature; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Glucuronic Acid; Gram-Negative Bacterial Infections; Hexuronic Acids; Immunity, Innate; Photobacterium; Random Allocation; Stress, Physiological

Vibrio harveyi is one of the most common threats to farmed grouper, so considerable efforts are in practice to control the pathogen. This study presents a highly effective vaccine against V. harveyi in the orange-spotted grouper with the help of a single intraperitoneal immunization. The vaccine candidate was in form of whole, formalin-inactivated V. harveyi cells combined with a metabolizable ISA763 AVG adjuvant. Our results indicated that the vaccine triggered a remarkably higher expression level of interleukin (IL)-1β, IL-6, IL-8, and IL-10 in the groupers' kidneys and spleens, as recorded after 1 and 3 days of immunization. Antibody titers were significantly elevated in the vaccinated fish. A pivotal observation was that the vaccine highly protected the grouper from a homologous V. harveyi strain challenge with relative percentage survival values of 100% and 91.7% at 6 and 12 weeks post-immunization, respectively. Vaccinated fish also demonstrated strong cross-protection against a heterologous bacterial isolate challenge. Therefore, the inactivated V. harveyi vaccine is a promising candidate that could stimulate good immune responses and confer remarkable protection in farmed groupers.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Bass; Cytokines; Fish Diseases; Fish Proteins; Formaldehyde; Gene Expression; Immunization; Vaccines, Inactivated; Vibrio; Vibrio Infections

The insufficiently known nematode species Philometra margolisi Moravec, Vidal-Martínez et Aguirre-Macedo, 1995 (Philometridae) is redescribed based on light and scanning electron microscopical (SEM) examinations of specimens collected from the gonad of the type host, the red grouper Epinephelus morio (Valenciennes) (Serranidae, Perciformes), in the northern Gulf of Mexico off Florida, USA. Also, new prevalence data for females of P. margolisi were derived from 188 fish, and a subset of these (n = 38) were used to determine prevalence and intensity of male nematodes. The male of this species was studied with SEM for the first time, which revealed some new, taxonomically important morphological features. The male posterior end had a V-shaped caudal mound, four pairs of minute adanal papillae, a pair of large papillae located posterior to the cloacal aperture and a pair of very small phasmids. The distal end of the gubernaculum is unique among all gonad-infecting species of Philometra parasitizing serranids in that its ventral surface is flat, smooth, without the usual two longitudinal grooves; the dorsal lamellate structures on the gubernaculum are also different in this species. In contrast to data in the original species description, the body length of gravid females of P. margolisi was 132-280 mm. Overall prevalence for male nematodes (76.3%) was much higher than for females (15.4%), and female nematode prevalence was higher in samples collected during host spawing season than out of season (27.1% and 3.3%, respectively).

Topics: Animals; Bass; Female; Fish Diseases; Gonads; Gulf of Mexico; Male; Nematoda; Nematode Infections; Prevalence; Species Specificity

The present study reports the identification, and characterization of three new putative piscidin paralogues, ecPis-2, ecPis-3 and ecPis-4, from orange-spotted grouper (Epinephelus coioides). The cDNA of the three piscidins with the 207, 216, and 231 nt open reading frame encoded respectively a 68-, 71-, and 76-amino acid preprotein consisting of the predicted signal peptide, and putative mature peptide and prodomain. The phylogenetic analysis indicated that multiple piscidin paralogues in one fish species are highly diversified, the analysis suggested that the piscidins should be a family belonging to the superfamily of ancient cationic, linear, and amphipathic host defence peptides widespread across invertebrate and vertebrate taxa comprising insect cecropins and ceratotoxins, and the amphibian dermaseptins. The synthetic putative mature peptides, ecPis-2S, ecPis-3S and ecPis-4S, had strong activities against bacterial and fungal species. EcPis-3S exhibited powerful activity against the infective stage of Cryptocaryon irritans, theronts. The full length ecPis-2 and ecPis-4 by removal of signal peptide, ecPis-2L and ecPis-4L respectively, had potency against bacterial, fungal and parasitic species. The peptide ecPis-2S was proved to exist in spleen of orange-spotted grouper by HPLC followed by ESI-LCMS analysis. Basal transcriptions of ecPis-2, ecPis-3 and ecPis-4 were detected not only in the potential sites of pathogen entry such as gills, skin and intestine, but also in tissues such as head kidney, trunk kidney, blood cells, and spleen with highly abundant immune cells, however different paralogues expressed constitutively with different levels in the tissues. In addition, the expression of ecPis-2, ecPis-3 and ecPis-4 was upregulated in orange-spotted grouper challenged by Vibrio Parahaemolyticus, in different tissues at different time point after bacteria injection. These results support ecPis-2, ecPis-3 and ecPis-4 being the important immune-related genes in orange-spotted grouper innate immune system and playing multifunctional and complementary roles following their structural and functional diversification, and expression pattern difference. Finally, this study facilitates the evaluation of ecPis-2S, 2L, ecPis-3S, and ecPis-4S, -4L as potential templates of therapeutic agents against pathogens.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Organ Specificity; Phylogeny; Random Allocation; Sequence Alignment; Vibrio Infections; Vibrio parahaemolyticus

A real-time genotype-specific polymerase chain reaction (PCR) assay combined with high-resolution melting (HRM) analysis was developed to assess the most common genotypes of nervous necrosis viruses or nodaviruses. Nodaviruses are the causal agents of viral nervous necrosis infections, which have been wreaking havoc in the aquaculture industry worldwide, with fish mortality up to 100%. The four different genotypes of nodaviruses correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostics requires analysis of genetic variation among viruses. The aim of the present study was to develop a real-time tetra-primer genotype-specific PCR assay for genotype identification. Four primers were utilized for simultaneous amplification of nodavirus genotype-specific products in a single closed-tube PCR after a reverse-transcription reaction using RNA isolated from fish samples. For high-throughput sample analysis, SYBR Green-based real-time PCR was used in combination with HRM analysis. The assay was evaluated in terms of specificity and sensitivity. The analysis resulted in melting curves that were indicative of each genotype. The detection limit when using reference plasmids was 100 ag/µL for both genotypes, while the sensitivity of the assays when testing a complex mixture was 10 fg/µL for red-spotted grouper nervous necrosis virus (RGNNV) and 100 fg/µL for striped jack nervous necrosis virus (SJNNV). To test the capability of this method under real-world conditions, 58 samples were examined. All samples belonged to the RGNNV genotype, which was fully validated. The results were in full agreement with genotyping by reference methods. The proposed methodology provides a rapid, sensitive, specific, robust and automatable assay for nodavirus genotyping, making it a useful tool for diagnosis and screening for epidemiological studies.

Topics: Animals; Bass; DNA Primers; Fish Diseases; Genotype; Molecular Diagnostic Techniques; Nodaviridae; Real-Time Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral

Topics: Adjuvants, Immunologic; Animal Feed; Animals; Bass; Blood Proteins; Choline; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Proteins; Immunity, Innate; Immunomodulation; Organ Specificity; Vibrio alginolyticus; Vibrio Infections

DNA vaccines had been widely used against microbial infection in animals. The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been increasingly studied in recent years. MyD88 is one of the adapter molecules to activate the signaling cascades and produces inflammatory mediators, and its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, plasmid pcMyD88 was constructed and the capacity of MyD88 as molecular adjuvant was explored by co-injecting with a DNA vaccine encoding AcfA against Vibrio alginolyticus infection in orange spotted grouper. The results suggested that it needed at least 7 days to transported DNA vaccine pcacfA or molecular adjuvant pcMyD88 from the injected muscle to kidney and spleens and stimulate host's immune system for later protection. The co-injection of pcMyD88 with DNA vaccine pcacfA could increase significantly specific antibody levels and the expression levels of the immune-related genes including MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα. Furthermore, pcMyD88 enhanced the immunoprotection of pcacfA against V. alginolyticus infection, with the significantly higher RPS of 83.3% in pcMyD88 + pcacfA group compared with that of pcacfA alone (73.3%) at challenging test of 10 weeks post vaccination. Together, these results clearly demonstrate that MyD88 is an effective adjuvant for the DNA vaccine pcacfA in orange spotted grouper.

Topics: Adjuvants, Immunologic; Animals; Bacterial Vaccines; Bass; Fish Diseases; Immune System; Myeloid Differentiation Factor 88; Random Allocation; Vaccines, DNA; Vibrio alginolyticus; Vibrio Infections

In July 2012, philometrid nematodes were discovered in cultured rockfish (

Topics: Animals; Bass; Fish Diseases; Fisheries; Microscopy, Electron, Scanning; Nematoda; Nematode Infections; Republic of Korea

Fish skin is the largest immunologically active mucosal organ, providing first-line defense against external pathogens. However, the skin-associated immune mechanisms of fish are still unclear. Cryptocaryon irritans is an obligate ectoparasitic ciliated protozoan that infects almost all marine fish, and is believed to be an excellent pathogen model to study fish mucosal immunity. In this study, a de novo transcriptome assembly of Epinephelus coioides skin post C. irritans tail-infection was performed for the first time using the Illumina HiSeq™ 2500 system. Comparative analyses of infected skin (group Isk) and uninfected skin (group Nsk) from the same challenged fish and control skin (group C) from uninfected control fish were conducted. As a result, a total of 91,082 unigenes with an average length of 2880 base pairs were obtained and among them, 38,704 and 48,617 unigenes were annotated based on homology with matches in the non-redundant and zebrafish database, respectively. Pairwise comparison resulted in 10,115 differentially-expressed genes (DEGs) in the Isk/C group comparison (4,983 up-regulated and 5,132 down-regulated), 2,275 DEGs in the Isk/Nsk group comparison (1,319 up-regulated and 956 down-regulated) and 4,566 DEGs in the Nsk/C group comparison (1,534 up-regulated and 3,032 down-regulated). Seven immune-related categories including 91 differentially-expressed immune genes (86 up-regulated and 5 down-regulated) were scrutinized. Both DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and immune-related gene expression analysis were used, and both analyses showed that the genes were more significantly altered in the locally-infected skin than in the uninfected skin of the same challenged fish. This suggests the skin's local immune response is important for host defense against this ectoparasite infection. Innate immune molecules, including hepcidin, C-type lectin, transferrin, transferrin receptor protein, serum amyloid A, cathepsin and complement components were significantly up-regulated (fold-change ranged from 3.3 to 12,944) in infected skin compared with control skin. The up-regulation of chemokines and chemokine receptors and activation of the leukocyte transendothelial migration pathway suggested that leucocytes intensively migrated to the local infected sites to mount a local immune defense. Toll-like receptors (TLRs) 1, 2, 5 and 5S were most significantly up-regulated in the infected skin, suggesting that these

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Gene Expression; Immunity, Mucosal; Random Allocation; Signal Transduction; Skin Diseases; Tail; Transcriptome

A computational model equipped with the main immunological features of the sea bass (Dicentrarchus labrax L.) immune system was used to predict more effective vaccination in fish. The performance of the model was evaluated by using the results of two in vivo vaccinations trials against L. anguillarum and P. damselae.. Tests were performed to select the appropriate doses of vaccine and infectious bacteria to set up the model. Simulation outputs were compared with the specific antibody production and the expression of BcR and TcR gene transcripts in spleen. The model has shown a good ability to be used in sea bass and could be implemented for different routes of vaccine administration even with more than two pathogens. The model confirms the suitability of in silico methods to optimize vaccine doses and the immune response to them. This model could be applied to other species to optimize the design of new vaccination treatments of fish in aquaculture.. The method is available at http://www.iac.cnr.it/∼filippo/c-immsim/.. nromano@unitus.it.. Supplementary data are available at Bioinformatics online.

Topics: Animals; Aquaculture; Bass; Computer Simulation; Fish Diseases; Immune System; Vaccination

Co-stimulatory molecules (CD83, CD80 and CD86), belong to immunoglobulin superfamily, are type I membrane glycoprotein, which express on antigen presenting cells and provide the second signal for the activation of T lymphocytes. In the present study, we cloned the grouper's CD83 (675 bp) and CD80/86 (876 bp). Homology analysis showed that both EcCD83 and EcCD80/86 shares the highest amino acid similarity (51% and 47%) for the overall sequence with puffer fish (Takifugu rubripes). Some conserved features and important functional residues in mammalian CD83, CD80 and CD86 were also identified from these molecules of teleosts including grouper, suggesting the function of both molecules may be conserved among vertebrates. In transfected HEK293T cells, both molecules localized on the membrane surface. Tissue distribution analysis showed both EcCD83 and EcCD80/86 mRNAs were mainly expressed in immune organs, and EcCD80/86 was extremely higher expressed in mucosal immune tissues including skin and gill than systematic immune organs, which indicates these co-stimulatory molecules may prime T cell activation in local mucosal tissues. In Cryptocaryon irritans infected groupers, the expression level of EcCD83 and EcCD80/86 were both seen significant up-regulation in the skin at most tested time points.

Topics: Amino Acid Sequence; Animals; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Bass; CD83 Antigen; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Immunoglobulins; Membrane Glycoproteins; Phylogeny; Sequence Alignment

The unique receptor XCR1 of the XC subfamily of chemokines is specially expressed in CD8α-like dendritic cells. This receptor has one ligand in mice (XCL1) and two ligands in humans (XCL1 and XCL2). In mammals, the XCR1-XCL1 complex performs a vital role in regulating the localization and function of T cells, dendritic cells, and other cell types. In this study, three XCR1-like receptors (EcXCR1, EcXCR1L, and EcCCR12) were identified from a transcriptome database of orange-spotted grouper. The open reading frames (ORFs) of EcXCR1, EcXCR1L, and EcCCR12 predictably encode 337, 348, and 358 amino acids, respectively. All receptors are seven trans-membrane proteins, and contain conserved functional regions, and conserved sites, that are crucial for the role of chemokine receptors in mammals. Conserved features include four cysteine residues in the extracellular regions, a "DRY" motif in the second intracellular loop, and common characteristics at the N-terminus that are important for ligand interaction. In healthy grouper, EcXCR1, EcXCR1L, and EcCCR12 were broadly expressed in all the tissues tested. EcXCR1 was expressed at high levels in the liver, and EcXCR1L, and EcCCR12 in the thymus. After grouper infection with Cryptocaryon irritans, EcXCR1 and EcCCR12 were up-regulated in the skin and the spleen, and EcCCR12 in the skin, gill, and spleen. EcXCR1L expression changed only slightly. These results imply that EcXCR1 and EcCCR12 may be involved in host defense against parasite infection. A polyclonal antibody was produced against EcCCR12, and used to detect EcCCR12-positive cells in peripheral blood. These results will contribute considerably to elucidate the biological role of piscine XCR1-like receptors and their ligands system in the future.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Membrane Proteins; Phylogeny; Sequence Alignment

P38 mitogen-activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec-P38γ and Ec-P38δ, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec-p38γ and Ec-p38δ sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr-Gly-Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (α, β, γ, and δ) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec-p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up-regulated in the tissues examined, with the highest expressions of Ec-p38α (5.2 times) and Ec-p38δ (4.2 times) occurring in the skin, while Ec-p38β (24.8 times) and γ (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec-p38γ/Ec-p38δ and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-p38γ and Ec-p38δ were conserved, the p38 subtypes showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly up-regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen-caused inflammation.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Immunity, Innate; p38 Mitogen-Activated Protein Kinases; Phylogeny; Sequence Alignment

The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub-lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up-regulation of interleukin-1β (IL-1β) and TNFα, major histocompatibility complex-II (MHCII) and cyclooxygenase-2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24 h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti-iNOS and anti-TNFα presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti-TNFα isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti-iNOS but not to anti-TNFα. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up-regulates the expression of several posterior gut inflammation-related genes conditioning fish growth and GALT capacity of response after bacterial infection.

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Oils; Intestinal Diseases; Vibrio; Vibrio Infections

First parasitological surveys of Myxozoa are performed on the sparid saddled seabream Oblada melanura (Linnaeus, 1758) and the serranid painted comber Serranus scriba (Linnaeus, 1758) caught from the Bay of Bizerte and the Gulf of Tunis respectively in Northeast Tunisia, Western Mediterranean. In this study, 6 bivalvulid myxosporean species belonging to the 3 genera Ceratomyxa Thélohan, 1892, Myxodavisia Zhao, Zhou, Kent & Whipps, 2008 and Zschokkella Auerbach, 1910, are isolated infecting their hosts. Two species Ceratomyxa sp. 1 ex O. melanura (Prevalence (P) = 36%) and Ceratomyxa sp. 2 ex O. melanura (P = 13%) infected the saddled seabream and four species Ceratomyxa sp. 1 ex S. scriba (P = 11.7%), Ceratomyxa sp. 2 ex S. scriba (P = 6.7%), Myxodavisia sp. (P = 8.3%) and Zschokkella sp. (P = 5.6%) infected the painted comber. These myxosporeans differ, in vegetative stages and/or in mature spores, from all the previously known congeneric species, and are described here on the basis of their morphological and morphometric features, their host and tissue specificities and their biogeographical distribution. This is the first report of myxosporean infections in O. melanura and S. scriba. The occurrence of two ceratomyxid species in each host species supports that the genus Ceratomyxa is host-specific not only in sparids but also in serranids, which agrees with data previously obtained from Sparidae in Mediterranean Sea and from Serranidae in GBR, Australia. A member of the myxosporean genus Myxodavisia is recorded from the Mediterranean Sea for the first time, and Zschokkella spp. infections have not previously been recorded from a host in Serranidae. During the examination, a several cases of Co-infection among myxosporeans, both with two and three species, are provided and statistically studied. Indeed, 5% of the breams and 9.4% of the combers are infected with more than one myxosporean parasite. The relationship between myxosporean infections and some biological parameters are pointed out. A higher prevalence of myxosporean infection is coincided with the peak period of spawning activity in May-June for S. scriba. For both hosts, analysis using Fulton's condition factor (K) has revealed no significant difference between infected and non-infected fishes. Clinically, no external signs of disease have been occurred in infected hosts, but some changes in the bile fluid, colour, and viscosity and in the gall bladder 's tissue are examined in S. scriba.

Topics: Animals; Australia; Bass; Cnidaria; Fish Diseases; Mediterranean Sea; Myxozoa; Parasitic Diseases, Animal; Phylogeny; Sea Bream; Tunisia

Among other functions, CCL25/CCR9 has an important role in regulating the trafficking of developing T cells in the thymus, and in homing memory T cells to the small intestine. The function of this chemokine-receptor complex is not well studied in fish. We identified a CCL25-like (EcCCL25, 108 aa) and two CCR9-like sequences (EcCCR9aa 373 aa; and EcCCR9b, 375 aa) from a transcriptome database of orange-spotted grouper (Epinephelus coioides). EcCCL25, EcCCR9a, and EcCCR9b shared conserved structural features with homologs from mammals and from other fish, and a consistent relationship with phylogenetic trees and sequence identities. In healthy grouper, EcCCL25, EcCCR9a, and EcCCR9b were highly expressed in the thymus, and the gills, were expressed at lower levels in the stomach, and had different expression levels in other tissues. After infection with Cryptocaryon irritans, EcCCL25 expression was up-regulated at early time points in the spleen and head kidney, and in the skin, and gills at later time points; EcCCR9a expression was increased in the gill, spleen, and head kidney. After infection with C. irritans, EcCCR9b expression was reduced in all tissues tested. These results suggested that grouper CCL25/CCR9a complex may be involved in host defense against C. irritans infection.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Chemokines, CC; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Immunity, Innate; Phylogeny; Receptors, CCR; Sequence Alignment

Haptoglobin (Hp) molecule has been cloned and characterized in two marine teleosts (gilthead seabream and European sea bass), obtaining putative proteins of 319 residues encoded by an ORF of 960 bp in both species. However, the matrix of similarity revealed low identities among bony fish species 78.9% (seabream-sea bass), 43% (seabream/seabass-zebrafish) and lower than 20% with sharks and human. The protein sequences showed a signal peptide from the position 1 to 23, a trypsin domain from 47 to 297, and several predicted disulfide bridges and glycosylation sites. The expression of hp transcript levels during ontogeny showed a progressive increase of expression in seabream whilst remained almost unaltered in sea bass. By tissues, this gene was found constitutively expressed with the highest levels on liver in both species. The main results on hp transcript levels showed the up-regulation in gilthead seabream suffering from naturally occurring lymphocystis disease; and the down-regulation and up-regulation after nodavirus infection in the resistant gilthead seabream and the susceptible European sea bass, respectively. These findings demonstrate for the first time an important role of haptoglobin against viral infections, operating differently in two of the most important marine farmed fish species.

Topics: Animals; Bass; Down-Regulation; Fish Diseases; Fish Proteins; Haptoglobins; Nodaviridae; Sea Bream; Up-Regulation; Virus Diseases

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl

Topics: Administration, Oral; Animal Feed; Animals; Aquaculture; Bass; Calcium Chloride; Drug Delivery Systems; Fish Diseases; Fluorescein-5-isothiocyanate; Germ-Free Life; Larva; Particle Size; Seawater; Serum Albumin, Bovine

Recent increases in emergent infectious diseases have raised concerns about the sustainability of some marine species. The complexity and expense of studying diseases in marine systems often dictate that conservation and management decisions are made without quantitative data on population-level impacts of disease. Mark-recapture is a powerful, underutilized, tool for calculating impacts of disease on population size and structure, even in the absence of etiological information. We applied logistic regression models to mark-recapture data to obtain estimates of disease-associated mortality rates in three commercially important marine species: snow crab (Chionoecetes opilio) in Newfoundland, Canada, that experience sporadic epizootics of bitter crab disease; striped bass (Morone saxatilis) in the Chesapeake Bay, USA, that experience chronic dermal and visceral mycobacteriosis; and American lobster (Homarus americanus) in the Southern New England stock, that experience chronic epizootic shell disease. All three diseases decreased survival of diseased hosts. Survival of diseased adult male crabs was 1% (0.003-0.022, 95% CI) that of uninfected crabs indicating nearly complete mortality of infected crabs in this life stage. Survival of moderately and severely diseased striped bass (which comprised 15% and 11% of the population, respectively) was 84% (70-100%, 95% CI), and 54% (42-68%, 95% CI) that of healthy striped bass. The disease-adjusted yearly natural mortality rate for striped bass was 0.29, nearly double the previously accepted value, which did not include disease. Survival of moderately and severely diseased lobsters was 30% (15-60%, 95% CI) that of healthy lobsters and survival of mildly diseased lobsters was 45% (27-75%, 95% CI) that of healthy lobsters. High disease mortality in ovigerous females may explain the poor recruitment and rapid declines observed in this population. Stock assessments should account for disease-related mortality when resource management options are evaluated.

Topics: Animals; Bacterial Physiological Phenomena; Bass; Brachyura; Connecticut; Dinoflagellida; Fish Diseases; Fisheries; Host-Parasite Interactions; Logistic Models; Longevity; Maryland; Mycobacterium; Mycobacterium Infections; Nephropidae; Newfoundland and Labrador; Virginia

Vibrio harveyi is a major bacterial pathogen that causes serious vibriosis in cultured groupers, leading to massive deaths. In this study, we evaluated the immune responses and protective efficacy of vaccines containing V. harveyi formalin-killed cells (FKC) formulated with CpG ODN 1668-enriched plasmids (p30CpG and p60CpG) in the orange-spotted grouper. Results indicated that antibody titres were remarkably increased in vaccinated fish 2 weeks post-immunisation. Expression level of major histocompatibility complex (MHC) class II, CD 8, and toll-like receptor 9 was significantly upregulated in the spleen of fish immunised with CpG ODN 1668-adjuvanted vaccines, as recorded at 6 weeks after immunisation. Additionally, the FKC + p60CpG-vaccinated fish displayed greater mRNA levels of MHC I and tumor necrosis factor-alpha. Of note, the relative percent survival after V. harveyi challenge was significantly higher in FKC + p60CpG-vaccinated fish (96.2%) than in FKC + p30CpG-vaccinated (79.8%) and FKC-vaccinated fish (59.9%). These results demonstrate that the FKC + CpG ODN 1668 vaccines are promising candidates that could enhance both innate and adaptive immune responses, conferred remarkable protection, and CpG ODN 1668 is a potential adjuvant for vaccines against V. harveyi.

Topics: Adjuvants, Immunologic; Animals; Bacterial Vaccines; Bass; Fish Diseases; Oligodeoxyribonucleotides; Random Allocation; Vaccines, Inactivated; Vibrio; Vibrio Infections

Changes in innate immunity parameters and epinecidin mRNA transcript levels were examined to characterize the non-specific immune response of E. coioides to pathogenic V. harveyi JML1 isolated from affected cage-cultured fish. After fish had been injected with bacteria at a dose causing 30% mortality, blood and tissue samples were collected at 0, 6, 12, 24, 48, 72, 96, 120, and 240 h post-infection (hpi) for assessment of indices such as the oxidative burst (OB) and phagocytic index (PI) of head kidney cells, and lysozyme activity (LYS) and total immunoglobulin (Total Ig) levels of the plasma. The epinecidin mRNA transcript levels (EGE) from skin, gills, liver, kidney, and spleen tissues were also determined by gel-based RT-PCR. Lastly, daily mortality (DM), liver total bacterial load (TBC), and presumptive Vibrio count (TVC) were monitored up to 240 hpi. The results revealed that bacteria proliferated rapidly in fish tissue, reaching peak densities at 24 hpi for both TBC and TVC but was on a downward trend thereafter. The pattern in fish mortality closely correlated with TBC and TVC. Total Ig, OB, and PI in E. coioides were suppressed in the early part of infection when V. harveyi load was high but recovered and later increased as bacterial density declined. LYS and EGE were consistently high and their activities were not hampered by bacterial infection. The study demonstrated that V. harveyi JML1 interacts with E. coioides by transiently inhibiting some immune parameters resulting in mortalities. However, consistently high LYS, upregulated EGE, and resurgent PI, OB and Total Ig conferred resistance and subsequent recovery in the fish. The study provides new insights on the interaction between E. coioides and V. harveyi JML1 that can aid in formulating health management strategies for groupers. Further studies on prophylactic interventions to enhance the innate immune response in grouper during infection with V. harveyi JML1 are suggested.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Fish Proteins; Immunity, Innate; Vibrio; Vibrio Infections

Interleukin-17 receptors (IL17Rs) mediate the activation of several downstream signal pathways to induce inflammatory response and contribute to the pathology of many autoimmune diseases. In this study, six IL17Rs (IL17RA1, RA2, RB, RC, RD and RE) were cloned and characterized from Epinephelus coioides, an orange-spotted grouper. Multiple sequence alignment and structural analysis revealed that all members of IL17Rs were low in sequence identity with each other. But their structures were conservative in grouper, which contain signal peptide, extracellular FNIII domain (IL17RA1/RA2/RB) or IL-17_R_N domain (IL17RC/RD/RE), transmembrane domain and SEFIR domain in their intracellular region. The analysis of tissue distribution showed these six genes were ubiquitously and differentially expressed in all major types of tissues. What's more, it is interesting to find their high expression in immune tissues (liver, gill, skin and thymus). IL17RA1 and IL17RA2 were significantly down-regulated at all time-points in gill and spleen after Cryptocaryon irritans infection, however, there was no significant change in other grouper IL17Rs. It suggests that the C. irritans may escape from the host immunity or the host prevents serious inflammation by inhibiting the expression of ILl7Rs.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Random Allocation; Receptors, Interleukin-17; Sequence Analysis, DNA

Tripartite motif-containing protein 35 (TRIM35) has been demonstrated to exert critical roles in cancer, cell death and other multiple cell processes. However, the precisely roles of TRIM35 during virus infection still remained largely unknown. In the current study, we cloned a TRIM35 gene from orange spotted grouper (EcTRIM35) and uncovered its roles in response to nodavirus infection. EcTRIM35 encoded a 456-aa protein which showed 65% and 32% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Structure prediction and amino acid alignment analysis indicated that EcTRIM35 contained three conserved domains, including RING domain, B-BOX and SPRY domain. In healthy grouper, the high expression level of EcTRIM35 could be detected in liver, spleen and intestine. After infection with red-spotted grouper nervous necrosis (RGNNV) and Singapore grouper iridovirus (SGIV) in GS cells, the transcript of EcTRIM35 was significantly up-regulated with the infection time increased. Under fluorescence microscopy, the bright fluorescence aggregates were observed in EcTRIM35 transfected cells, but the fluorescence distribution was obviously altered in the EcTRIM35-ΔRING transfected cells. After incubation with RGNNV, the overexpression of EcTRIM35 in vitro significantly enhanced the viral replication, evidenced by the enhancement of cytopathic effect (CPE) severity and the up-regulation of the viral gene transcription. Moreover, the ectopic expression of EcTRIM35 significantly decreased the expression of interferon signaling molecules or effectors. Further studies elucidated that EcTRIM35 overexpression significantly weakened the MAVS-, MITA- or TBK1-induced interferon immune response, but showed no effects on MDA5-induced immune response. Thus, our results will shed new lights on the roles of fish TRIM35 in innate immune response against grouper virus infection.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; Sequence Alignment; Tripartite Motif Proteins

Signal Transducer and Activator of Transcription 1 (STAT1) has been demonstrated to function as a critical mediator in multiple cell processes, such as cell proliferation, cell death, and innate immune response. Interestingly, two orthologues of human STAT1, including STAT1a and STAT1b genes have been identified in different fish. However, the detailed roles of fish STAT1a in virus replication still remained largely uncertain. Here, we cloned a STAT1a from orange-spotted grouper Epinephelus coioides (EcSTAT1a) and characterized its roles during fish virus infection. EcSTAT1a encoded a 751-aa peptide which shared 97% and 93% identity to STAT1 from mandarin fish (Siniperca chuatsi) and Malabar grouper (Epinephelus malabaricus), respectively. Amino acid alignment analysis showed that EcSTAT1a contained a STAT-int domain, a STAT-alpha domain, a STAT-bind domain (DNA binding domain), a SH2 domain and a STAT1-TAZ2 bind domain. In examined tissues from healthy grouper, the expression of EcSTAT1a was predominant in intestine, gill and liver. In grouper cells, the relative expression levels of EcSTAT1a was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) or Singapore grouper iridovirus (SGIV) infection. Under fluorescence microscopy, we found that EcSTAT1a mainly localized in the cytoplasm. The ectopic expression of EcSTAT1a in vitro significantly delayed the cytopathic effect (CPE) progression evoked by RGNNV and SGIV. Further studies showed that the expression levels of viral genes, including SGIV major capsid protein (MCP), VP19, ICP-18, LITAF and RGNNV coat protein (CP), RNA-dependent RNA polymerase (RdRp) were all significantly reduced in EcSTAT1a overexpressing cells compared to the control vector transfected cells, suggested that EcSTAT1a exerted antiviral activity against iridovirus and nodavirus. Furthermore, overexpression of EcSTAT1a significantly increased the expression of interferon related cytokines or effectors and pro-inflammatory factors. Together, our results elucidated that EcSTAT1a might function as a critical antiviral factor by regulating the host interferon immune and inflammation response.

Topics: Amino Acid Sequence; Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; Sequence Alignment; STAT1 Transcription Factor

Vibrio harveyi is a gram-negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange-spotted grouper (Epinephelus coioides) at 1 and 2 days post-infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P < 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune-related pathway functions, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, NF-κB signaling pathways, and Jak-STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Transcriptome; Vibrio; Vibrio Infections

Due to high-density aquafarming in Taiwan, groupers are commonly infected with two different iridoviruses: Megalocytivirus (grouper iridovirus of Taiwan, TGIV) and Ranavirus (grouper iridovirus, GIV). Iridoviral diseases cause mass mortality, and surviving fish retain these pathogens, which can then be horizontally transferred. These viruses have therefore become a major challenge for grouper aquaculture. In this study, comparisons of the biological responses of groupers to infection with these two different iridoviruses were performed. A novel approach for transcriptomic analysis was proposed to enhance the discovery of differentially expressed genes and associated biological pathways. In this method, suitable and available reference species are selected from the NCBI taxonomy tree and the Ensembl and KEGG databases instead of either choosing only one model species or adopting the NCBI non-redundant dataset as references. Our results show that selection of multiple appropriate model species as references increases the efficiency and performance of analyses compared to those of traditional approaches. Using this method, 17 shared pathways and 5 specific pathways were found to be significantly differentially expressed following infection with the two iridoviruses, among which 11 pathways were additionally identified based on the proposed method of multiple reference species selection. Among the pathways responsive to infection with a specific iridovirus, the spliceosomal pathway (ko03040; p-value = 0.0011) was exclusively associated with TGIV infection, while the glycolysis/gluconeogenesis pathway (ko00010; p-value = 0.0032) was associated with GIV infection. These findings and designed corresponding biological experiments may facilitate a deeper understanding of the mechanisms by which both TGIV and GIV cause fatal infections, as well as the ways in which they induce different pathologies and symptoms. We believe that the proposed novel mechanism for de novo transcriptomic analysis provides superior and comprehensive functional annotations and that the resulting shared and specific pathways identified may help immunologists develop specific vaccines against various types of iridovirus in the near future.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Gene Expression Profiling; Iridoviridae; Ranavirus; Random Allocation; Transcriptome

Antiviral immune responses are triggered by the innate immune recognition of viral infection. Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (TRIF) is an adapter in responding to activation of Toll-like receptors, which provides early clearance of viral pathogens. Our study focuses on the functional characterization of grouper TRIF (EcTRIF) based on the comparison of its sequence and functional evolution from grouper fish to mammals. The results show that the open reading frame of EcTRIF encoded a protein of 580 amino acids. Real-time PCR analysis indicates that EcTRIF was constitutively expressed in all the analyzed tissues in healthy grouper. EcTRIF was significantly induced in spleen post-LPS and poly (I:C) stimulation. Fluorescence microscopy shows that EcTRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in the Golgi apparatus. Luciferase reporter assays confirmed that EcTRIF was able to activate the IFN and NF-κB promoter. Overexpression of EcTRIF in grouper brain cells inhibited the replication of red-spotted grouper nervous necrosis virus (RGNNV). These results indicate that EcTRIF plays an important role in modulating antiviral innate immune responses. Our results have applications in functional studies on TRIF in teleost fish and immune evolution.

Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Nodaviridae; Phylogeny; Poly I-C; RNA Virus Infections; Sequence Alignment

Human DEAD box ATP-dependent RNA helicase DDX3X has been demonstrated to exert crucial functions in carcinogenesis and antiviral immune response. However, to our knowledge, few information focused on the functions of fish DDX3X. In this study, we cloned and characterized a DDX3X homolog from orange spotted grouper (Epinephelus coioides) (EcDDX3X). EcDDX3X encoded a 733-amino acid protein which shared 97% and 76% identity to spiny damselfish (Acanthochromis polyacanthus) and human (Homo sapiens), respectively. Amino acid alignment analysis showed that EcDDX3X contained conserved DExDc and Helic C domains. The transcription levels of EcDDX3X were significantly increased in poly I:C transfected cells and red-spotted grouper nervous necrosis virus (RGNNV) infected cells. Under fluorescence microscopy, the green fluorescence was observed evenly in the cytoplasm in EcDDX3X transfected cells. The ectopic expression of EcDDX3X significantly inhibited the replication of RGNNV, evidenced by the decreased numbers of the vacuoles evoked by RGNNV infection, and the reduced transcription levels of RGNNV coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes. In contrast, the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells was not significantly affected by EcDDX3X overexpression. Further studies showed that overexpression of EcDDX3X in vitro significantly increased the expression levels of several interferon associated cytokines or effectors. Moreover, the regulatory effect of EcDDX3X on interferon immune response was dependent on its N terminal region, but not the DExDc and Helic C domain. In addition, we also found that overexpression of EcDDX3X significantly increased the interferon promoter activity, and the activation of interferon immune response was regulated by both IRF3 and IRF7. Together, our results firstly showed that fish DDX3X exerted crucial roles in antiviral immunity against RNA virus infection via upregulating interferon antiviral responses.

Topics: Amino Acid Sequence; Animals; Bass; DEAD-box RNA Helicases; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Nodaviridae; Phylogeny; Poly I-C; Ranavirus; RNA Virus Infections; Sequence Alignment

ADARs are RNA editing catalysts that bind double-stranded RNA and convert adenosine to inosine, a process that can lead to destabilization of dsRNA structures and suppression of mRNA translation. In mammals, ADAR1 genes are involved in various cellular pathways, including interferon (IFN)-mediated response. However, the function of fish ADAR1 remains unclear. We report here the cloning of ADAR1 in Malabar grouper (Epinephelus malabaricus) (MgADAR1) and its response to various immune stimulants. The MgADAR1 cDNA is 5371-bp long, consisting of an open reading frame encoding a putative protein of 1381 amino acids, a 235-nt 5'-terminal untranslated region (UTR), and a 990-nt 3'-UTR. The deduced amino acid sequence exhibits signature features of a chitin synthesis regulation domain, two Z-DNA-binding domains (Z alpha), three dsRNA binding motifs (DSRM) and one tRNA-specific and dsRNA adenosine deaminase domain (ADEAMc). MgADAR1 mRNA expressed ubiquitously in tissues of healthy Malabar grouper, with elevated levels in the brain, gills and eyes. In response to poly (I: C), the MgADAR1 mRNA level was significantly up-regulated in the brain and spleen, but not head kidney. Upon nervous necrosis virus (NNV) infection the level of MgADAR1 increased in the brain, whereas Mx increased in the brain, spleen and head kidney. Induction of MgADAR1 by poly (I: C) and NNV was also observed in vitro. Additionally, the expression of MgADAR1 was upregulated by recombinant grouper IFN in grouper cells. These data indicate an intricate interplay between ADAR1 and NNV infection in grouper as MgADAR1 might be regulated in a tissue-specific manner.

Topics: Adenosine Deaminase; Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Nodaviridae; Phylogeny; Poly I-C; Sequence Alignment

Hepcidin is a small cysteine rich peptide that regulates the sole known cellular iron exporter, ferroportin, effectively controlling iron metabolism. Contrary to humans, where a single hepcidin exists, many fish have two functionally distinct hepcidin types, despite having a single ferroportin gene. This raises the question of whether ferroportin is similarly regulated by the iron regulator Hamp1 and the antimicrobial Hamp2. In sea bass (Dicentrarchus labrax), iron overload prompted a downregulation of ferroportin, associated with an upregulation of hamp1, whereas an opposite response was observed during anemia, with no changes in hamp2 in either situation. During infection, ferroportin expression decreased, indicating iron withholding to avoid microbial proliferation. In vivo administration of Hamp1 but not Hamp2 synthetic peptides caused significant reduction in ferroportin expression, indicating that in teleost fish with two hepcidin types, ferroportin activity is mediated through the iron-regulator Hamp1, and not through the dedicated antimicrobial Hamp2. Additionally, in vitro treatment of mouse macrophages with fish Hamp1 but not Hamp2 caused a decrease in ferroportin levels. These results raise questions on the evolution of hepcidin and ferroportin functional partnership and open new possibilities for the pharmaceutical use of selected fish Hamp2 hepcidins during infections, with no impact on iron homeostasis.

Topics: Animals; Bass; Cation Transport Proteins; Fish Diseases; Fish Proteins; Hepcidins; Infections; Iron Overload

The reliable production of marine fish larvae is one of the major bottlenecks in aquaculture due to high mortalities mainly caused by infectious diseases. To evaluate if the compound poly-β-hydroxybutyrate (PHB) might be a suitable immunoprophylactic measure in fish larviculture, its capacity to improve immunity and performance in European sea bass (Dicentrarchus labrax) yolk-sac larvae was explored. PHB was applied from mouth opening onwards to stimulate the developing larval immune system at the earliest possible point in time. Larval survival, growth, microbiota composition, gene expression profiles and disease resistance were assessed. PHB administration improved larval survival and, furthermore, altered the larva-associated microbiota composition. The bacterial challenge test using pathogenic Vibrio anguillarum revealed that the larval disease resistance was not influenced by PHB. The expression profiles of 26 genes involved e.g. in the immune response showed that PHB affected the expression of the antimicrobial peptides ferritin (fer) and dicentracin (dic), however, the response to PHB was inconsistent and weaker than previously demonstrated for sea bass post-larvae. Hence, the present study highlights the need for more research focusing on the immunostimulation of different early developmental stages for gaining a more comprehensive picture and advancing a sustainable production of high quality fry.

Topics: Animals; Aquaculture; Bass; Disease Resistance; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation, Developmental; Host-Pathogen Interactions; Hydroxybutyrates; Larva; Multivariate Analysis; Polyesters; Time Factors; Vibrio; Vibrio Infections; Yolk Sac

Nervous necrosis virus (NNV) causes high mortalities in several marine species. We aimed to evaluate the innate cell-mediated cytotoxic (CMC) activity of head-kidney leucocytes (HKLs) isolated from naïve European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), a very susceptible and resistant fish species to NNV, respectively, against fish cell lines infected with NNV. Seabream HKLs showed significantly increased innate CMC activity against NNV-infected cells, compared to those uninfected, while sea bass HKLs failed to do so. Thus, we performed a RNA-seq study to identify genes related to the CMC activity of sea bass leucocytes. Thus, we found that sea bass HKLs incubated with DLB-1 cells alone (CMC_DLB1) or with NNV-infected DLB-1 cells (CMC_DLB1-NNV) showed very similar transcriptomic profiles and the GO analysis revealed that most of the up-regulated genes were related to immunity. Strikingly, when the CMC samples with and without NNV were compared, GO analysis revealed that most of the up-regulated genes in CMC_DLB1-NNV samples were related to metabolism and very few to immunity. This is also in agreement with the functional data. These data point to the escape of CMC activity by NNV infection as an important factor involved in the high susceptibility to nodavirus infections of European sea bass.

Topics: Animals; Bass; Cell Line; Fish Diseases; Immunity, Innate; Leukocytes; Nodaviridae; RNA Virus Infections; Sea Bream

Ortholinea labracis n. sp. is described and its life cycle is inferred from a Southern Portuguese fish farm, with basis on microscopic and molecular procedures. This myxosporean parasite infects the urinary bladder of the European seabass Dicentrarchus labrax and the intestinal epithelium of a marine oligochaete of the genus Tectidrilus. Myxospores subspherical in valvular view and ellipsoidal in sutural view measuring 7.6 ± 0.3 (6.8-8.7) μm in length, 7.2 ± 0.2 (6.7-7.7) μm in width and 6.5 ± 0.4 (5.8-7.7) μm in thickness. Two polar capsules, 3.0 ± 0.2 (2.6-3.4) μm long and 2.4 ± 0.1 (2.0-2.9) μm wide, located at the same level, but with divergent orientation and opening to opposite sides of the suture line. Sequencing of the SSU rRNA gene revealed a similarity of 100% between the analysed myxospores and triactinomyxon actinospores. The phylogenetic setting of O. labracis n. sp. shows subgrouping in correlation with tissue tropism, but identifies this parasite as another exception to the main division of Myxosporea into the main freshwater and marine lineages.

Topics: Animals; Bass; DNA, Ribosomal; Fish Diseases; Intestinal Mucosa; Microscopy, Electron, Transmission; Myxozoa; Oligochaeta; Parasitic Diseases, Animal; Portugal; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Urinary Bladder

Topics: Animals; Bass; Dermatitis; Environmental Monitoring; Fish Diseases; Libya; Population Surveillance; Retrospective Studies; Social Media

A new enteric microsporidian was found to be associated with the mass mortality of hatchery-bred juvenile groupers, Epinephelus spp., in China. The outbreak usually occurred during the rainy season between May and November when water temperature ranged from 26 to 30 °C and salinity from 28 to 34 ppt, although this microsporidian can be detected year round. External clinical signs included severe emaciation, white faeces syndrome, anorexia, sinking to the bottom of culture ponds and mass mortality (up to 90%). Upon necropsy, severe intestinal oedema and thin and transparent intestinal wall could be observed. The mature spores are tiny, measuring 1.3-1.5 (1.35 ± 0.13) × 1.6-2.4 (2.16 ± 0.31) μm and can be found in the cytoplasm and the nucleoplasm of most enteric epithelial cells of host. Epidemiological investigation showed that this species was distributed throughout most of the culture area of grouper fingerlings in Fujian, Guangdong, Hainan and Guangxi provinces in China, with maximum prevalence of 95%. Molecular analysis based on the partial small subunit rRNA sequence (1045 bp) placed this species within the Enterocytozoonidae, but sequence identities to other species were below 90%. The exact taxonomic position warrants study of the ultrastructural characteristics of the developmental stages.

Topics: Animals; Bass; China; Disease Outbreaks; Fish Diseases; Microsporidia; Microsporidiosis; Phylogeny; RNA, Protozoan; Sequence Analysis, DNA

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adaptor molecule in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In our previous study, the molecular characteristics of EtTRAF6 (TRAF6 from Epinephelus tauvina), the tissue distributions, expression patterns after challenging with bacterial and viral pathogens were investigated. Here we identified EtTRAF6 as an important regulator of virus-triggered signaling pathway. Overexpression of EtTRAF6-ORF and truncated forms of EtTRAF6, including EtTRAF6-C (delete the MATH domain), EtTRAF6-N (delete the RING domain) and EtTRAF6-MATH, inhibited IFN-β activity strongly in grouper spleen (GS) cells. Overexpression of EtTRAF6 repressed virus-induced production of type I IFNs. When EtTRAF6 cotransfected with EcIRF3 or EcIRF7, EtTRAF6 inhibited IRF-induced activation of IFN-β. Over-expressed EtTRAF6 inhibited the transcription of SGIV genes significantly in GS cells. Although TRAF6 has a role in apoptosis regulation, it is not known if EtTRAF6 has any role in apoptosis regulation. Strikingly, when over-expressed in fathead minnow (FHM) cells, EtTRAF6 protected them from cell death induced by SGIV. Therefore, these results suggest that TRAF6 may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Ranavirus; Sequence Analysis, DNA; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

The main aims of this study were to construct glutathione peroxidase (GPx) DNA vaccine of Vibrio harveyi ZJ0603 and to investigate its immune protective efficiency as a vaccine candidate on the orange-spotted grouper (Epinephelus coioides) treated with V. harveyi. Base on the cloning of ZJ0603 GPx gene, a DNA vaccine, named as pcDNA-GPx, was constructed by inserting GPx gene into pcDNA3.1 (+) plasmid. Orange-spotted groupers were immunized with the pcDNA-GPx plasmid by injection intramuscularly. The relative percent of survival (RPS) of fish vaccinated with the DNA vaccine against pathogenic V. harveyi infection was 77.5%. The expression of DNA vaccine was analyzed in the tissues of orange-spotted grouper by PCR and RT-PCR. The results indicated that pcDNA-GPx distributed and expressed in the head kidney, liver, spleen, gill and injected muscle at 7 and 28 days after vaccination. Significant specific antibody responses were also detected in the vaccinated orange-spotted groupers by indirect ELISA method. In a conclusion, DNA vaccine pcDNA-GPx showed an effective immune protection to the orange-spotted grouper treated with V. harveyi. The GPx can be used as a candidate DNA vaccine for the control of vibriosis.

Topics: Animals; Bacterial Vaccines; Bass; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Glutathione Peroxidase; Polymerase Chain Reaction; Vaccines, DNA; Vibrio; Vibrio Infections

Tripartite motif-containing 32 (TRIM32) has been demonstrated to pay vital roles in cancer, genetic disorders and antiviral immunity. However, the molecular functions of fish TRIM32 still remained largely unknown. Here, a novel TRIM32 gene from orange spotted grouper (EcTRIM32) was cloned and characterized. EcTRIM32 encoded a 685-aa protein which showed 93%, and 60% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Amino acid alignment showed that EcTRIM32 contained a conserved RING-finger domain, a BBOX domain and NHL domain. In healthy grouper, the transcript of EcTRIM32 was predominantly detected in brain, liver, intestine, spleen and skin. After injection with Singapore grouper iridovirus (SGIV) and polyI:C, the relative expression of EcTRIM32 in grouper spleen was differently regulated, suggested that EcTRIM32 was involved in antiviral immune response. In transfected grouper spleen (GS) cells, EcTRIM32 displayed bright fluorescence aggregates or spots in the cytoplasm. Notably, the deletion RING domain altered its precise localization and distributed throughout the cytoplasm in GS cells. In EcTRIM32 overexpressing cells, the replication of SGIV or red-spotted grouper nervous necrosis virus (RGNNV) was significantly inhibited compared to the vector control cells. Moreover, the overexpression of EcTRIM32 positively regulated the interferon immune response, evidenced by the significant increase of the expression level of interferon related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), interferon-induced 35-kDa protein (IFP35), MXI, TIR-domain-containing adaptor-inducing interferon-β (TRIF) and melanoma differentiation-associated protein 5 (MDA5). Further studies showed that overexpression of EcTRIM32 significantly enhanced the MDA5-mediated interferon immune response, but decreased stimulator of interferon genes (STING)-mediated interferon immune response. Meanwhile, the expression levels of pro-inflammation cytokines, including TNFα, IL-6 and IL-8 were up-regulated by the ectopic expression of EcTRIM32. We speculated that the regulation of IRF7, and pro-inflammation cytokines by EcTRIM32 overexpression might contribute critical roles in SGIV infection. In addition, the deletion of RING domain not only significantly weakened the antiviral roles of EcTRIM32, but also obviously affected the regulatory effects of EcTRIM32 on interferon immune a

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Innate; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; RNA, Messenger; Sequence Homology, Amino Acid; Tripartite Motif Proteins

Interferon regulatory factors (IRFs) are transcription mediators which play vital roles in multiple biological processes, such as antiviral defense, immune response, cell growth regulation and apoptosis. A fish specific IRF, termed IRF11, has been identified in previous study through searching fish genome databases. Herein, a transcript of IRF11, EcIRF11 was cloned from orange-spotted grouper, Epinephelus coioides. The EcIRF11 cDNA sequence has 1573 bp in length, encoding a putative protein of 261 amino acids, with a high degree of similarity found between EcIRF11 and its teleost counterparts. Comparative analyses in teleost genomes revealed that IRF11 may have an ancient origin at least 450 million years ago, and the locus harbouring IRF11 might have experienced chromosomal rearrangement and/or inversion during evolution. Expression analysis revealed that the other two members, IRF1 and IRF2 also in the IRF1 subgroup (SG) as IRF11, exhibited high expression levels in early experimental infection phase in response to viral stimulation of poly I:C and to bacterial stimulation of Vibrio parahaemolyticus infections in the fish, while EcIRF11 is not transcriptionally modulated at the examined time points except in kidney at 6 h following poly I:C stimulation. Taken together, the results obtained in this study indicate that IRF11 might have been originated from the same ancestor as IRF1 and IRF2, but exhibits distinct basal and induced expression, implying its different function which needs further characterization.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Interferon Regulatory Factors; Phylogeny; RNA, Messenger; Sequence Alignment; Vibrio Infections; Vibrio parahaemolyticus

Intersex in wild fish populations has received considerable attention in the scientific literature and public media. Conventional detection of testicular oocytes, the presence of immature oocytes within testis of male fish, commonly employs transverse sectioning of excised testis and is lethal. The present study used a nonlethal laparoscopic technique to collect biopsies of testis from black bass, entering the body cavity via the genital pore. Detection of testicular oocytes was compared between biopsy and conventional methods using 79 smallmouth bass (Micropterus dolomieu) from 8 sites and 68 largemouth bass (Micropterus salmoides) from 4 sites. The 2 methods performed similarly at sites where testicular oocyte severity was moderate or high (6 of 8 smallmouth bass sites), whereas transverse sectioning resulted in superior testicular oocyte detection at sites where severity was low (2 of 8 smallmouth bass sites and all 4 largemouth bass sites). In smallmouth bass, testicular oocyte prevalence by transverse and biopsy methods was strongly correlated across sites (r

Topics: Animals; Bass; Disorders of Sex Development; Fish Diseases; Male; Oocytes; Prevalence; Severity of Illness Index; Testis

Diplectanid monogeneans are gill parasites that can infect fish in huge numbers and thus become harmful, especially in maricultured fish. It is therefore useful to have taxonomic tools, such as keys, to identify species. The following diplectanid species from groupers of the Mediterranean Sea were studied: five species of Pseudorhabdosynochus Yamaguti, 1958, including P. riouxi (Oliver, 1986) Kritsky & Beverley-Burton, 1986 from the dusky grouper Mycteroperca marginata, P. enitsuji Neifar & Euzet, 2007, P. bouaini Neifar & Euzet, 2007, P. dolicocolpos Neifar & Euzet, 2007 and P. sinediscus Neifar & Euzet, 2007 from the goldblotch grouper M. costae, and Echinoplectanum echinophallus (Euzet & Oliver, 1965) Justine & Euzet, 2006 from the dusky grouper. New material was obtained from fish collected from off Tunisia and Libya and compared to the type-material and voucher specimens in museum collections. Identifications of fish were confirmed by barcoding of cytochrome c oxidase subunit I (COI) sequences. The sclerotized vagina was considered the most important structure for systematics. The three species P. riouxi, P. bouaini, and P. enitsuji share a common general structure of the sclerotized vagina with a conspicuous spherical secondary chamber. We thus propose the 'Pseudorhabdosynochus riouxi group' to accommodate them. Pseudorhabdosynochus dolicocolpos has an elongate vaginal structure that is completely different from all its congeneric species reported from the Mediterranean Sea, and Pseudorhabdosynochus sinediscus has a sclerotized vagina in which the secondary chamber is not visible, and a haptor without squamodiscs. A taxonomic key to diplectanid species on Mycteroperca spp. in the Mediterranean Sea is proposed; it includes ten species of Pseudorhabdosynochus and one species of Echinoplectanum.

Topics: Animals; Bass; Cestode Infections; Female; Fish Diseases; Male; Mediterranean Sea; Platyhelminths

Toll-like receptor 5 (TLR5) is an important receptor that interacts with bacterial flagellin and regulates host immune response in mammal. Recent studies demonstrate that piscine contains two types of TLR5, namely membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), and both of which perform crucial role in flagellin response. In the present study, a TLR5M and a TLR5S sequence was cloned from orange-spotted grouper (Epinepheluscoioides), and their ORFs are respectively 2466 bp (821 aas) and 1935 bp (644 aas). EcTLR5M has the typical TLR structure of a LRR domain, a transmembrane region and a TIR domain, while EcTLR5S only contains a LRR domain like other species' TLR5S. Both molecules have 23 LRR motifs, a LRR-NT and a LRR-CT in the LRR domain, similar to those of other species. Phylogenetic and sequence alignment indicated that both EcTLR5s respectively displayed closer relationship and higher sequence identity with those in other fish species. In healthy grouper, EcTLR5M was highly expressed in the skin, head kidney and spleen, while EcTLR5S was mainly detected in the liver. Ciliate Cryptocaryon irritans infection could significantly up-regulate the expression level of EcTLR5s in the gill and spleen from day 1 to day 3, and higher expression fold change was observed in the spleen. Taken together, the present studies contributed to understanding the function of piscine TLR5M/S and clarify their possible role in fish immune response against ciliate infection.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; DNA, Complementary; Expressed Sequence Tags; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; RNA, Messenger; Sequence Analysis, DNA; Toll-Like Receptor 5

Betanodavirus, also referred to nervous necrosis virus (NNV), is the causative agent of the fatal disease, viral nervous necrosis and has brought significant economic losses in marine and freshwater cultured fish, especially larvae and juveniles. Here, we used an established invasion model with virus-like particle (VLP)-cells, mimicking orange-spotted grouper nervous necrosis virus (OGNNV), to investigate the crucial events of virus entry. VLP were observed in the perinuclear regions of Asian sea bass (SB) cells within 1.5 h after attachment. VLP uptake was strongly inhibited when cells were pretreated with biochemical inhibitors (chlorpromazine and dynasore) blocking clathrin-mediated endocytosis (CME) or transfected with siRNA against clathrin heavy and light chains. Inhibitors against key regulators of caveolae/raft-dependent endocytosis and macropinocytosis had no effect on VLP uptake. In contrast, disruption of cellular cholesterol by methyl-β-cyclodextrin or reduction of cholesterol fluidity by Cholera toxin B subunit significantly decreased VLP entry. Furthermore, VLP entry is dependent on low pH and cytoskeleton, demonstrated by inhibitor (chloroquine, ammonia chloride, cytochalasin D, wiskostatin, and nocodazole) perturbation. Therefore, OGNNV VLP enter SB cells via CME depending on dynamin-2, cholesterol and its fluidity, low pH, and cytoskeleton. In addition, ten more cell lines were screened for VLP entry and VLP can only enter NNV-sensitive cells, GB and SSN-1, via CME, indicating that CME is the common endocytosis pathway for VLP. These results may provide the data for NNV entry without the influence of the viral genome, an ideal model for exploring the behaviour of betanodavirus in cells, and valuable references to vaccine development.

Topics: Animals; Bass; Cholesterol; Clathrin; Cytoskeleton; Endocytosis; Fish Diseases; Hydrogen-Ion Concentration; Nodaviridae; RNA Virus Infections

High percentage of dietary vegetable oil (VO) induced negative effects on immunity in numerous fish species. The present study was conducted to investigate whether VO could exert anti-immunological effects by regulating non-specific immunity, liver antioxidant capacity and nuclear factor kappa beta (NF-κB) signaling in Japanese sea bass (Lateolabrax japonicus). Three iso-nitrogenous and iso-lipid diets were formulated by replacing 0% (FO, the control), 50% (FV) and 100% (VO) of fish oil with vegetable oil. Each diet was randomly fed to triplicate groups of fish for 10 weeks. Results showed that the alternative complement pathway (ACP) activity and the disease resistance were significantly lower in fish fed VO diets compared with the control group (P < 0.05). Liver superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GPx) enzyme activities, as well as total antioxidant capacity (T-AOC) significantly decreased in fish fed VO diets (P < 0.05). Meanwhile, significantly low level of liver SOD1 and CAT mRNA, nuclear factor erythroid 2-related factor 2 (Nrf2) of both mRNA and protein were observed in fish fed VO diets when compared with fish fed FO diets (P < 0.05). However, the transcription level of TNFα and IL1β was significantly higher in the liver of fish fed VO diets, which might be attributed to the activation of NF-κB signaling pathway since the protein expression of p65, one of the key members of NF-κB family, was significantly increased (P < 0.05). These results suggested that dietary VO could lower the ACP activity, disease resistance and liver antioxidant capacity, but it could also exacerbate inflammatory response by activating NF-κB signaling pathway in Japanese sea bass.

Topics: Animals; Antioxidants; Bass; Dietary Supplements; Fish Diseases; Fish Proteins; Immunity, Innate; Inflammation; Liver; NF-kappa B; Plant Oils; Random Allocation

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

Topics: Animals; Australia; Base Composition; Bass; Chlamydia; Chlamydia Infections; DNA, Bacterial; Fish Diseases; Genome, Bacterial; Genomics; Gills; RNA, Ribosomal, 16S; Type III Secretion Systems; Virulence Factors

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Topics: Animal Fins; Animals; Bass; Cell Line; Fish Diseases; High-Throughput Nucleotide Sequencing; MicroRNAs; Nodaviridae; RNA Virus Infections; Sequence Analysis, RNA; Time Factors

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post-infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10

Topics: Animals; Bass; Fish Diseases; Hybridization, Genetic; Phylogeny; Sequence Analysis, DNA; Skin Ulcer; Vibrio; Vibrio Infections; Virulence

T cell activation is a complicated process accompanying with the activation of T cell receptor (TCR) signaling pathway, which is not well described in teleost fish. The initiation of this pathway depends on the interaction of membrane TCR co-receptors (e.g. CD4/8, CD3 and CD45) and a series of cytoplasmic protein tyrosine kinases (e.g. Lck, Fyn and ZAP70). Cyptocaryon irritans is a ciliate pathogen of marine fish white spot disease causing huge economic lost in marine aquaculture. This parasite can infect fish gill and skin and is considered to be a good pathogen model for fish gill and skin mucosal immunity. Our previous studies showed the locally mucosal antibody response was important for fish defense against this parasite. While how TCR signaling pathway involved in T cell activation to help B cell activation in C. irritans infected fish is still not known. In the present study, we cloned a grouper TCR co-receptor gene EcCD3ε (537 bp) and its three kinase genes, including EcLck (1512 bp), EcFyn (1605 bp) and EcZAP70 (1893 bp). Homology analysis showed that they all shared the highest identity with corresponding genes from Takifugu rubripes (EcCD3ε 41%, EcLck 88%, EcFyn 98% and EcZAP70 93%), and their conserved motifs involved in the signaling transduction were analyzed. The tissue distribution analysis showed these four genes were high expressed in thymus, and it is interesting to find their comparative high expression in skin, gill and midgut mucosal immune tissues. In C. irritans infected grouper, the expression of three TCR co-receptors (EcCD4-1, EcCD3ε and EcCD45) and three kinases (EcLck, EcFyn and EcZAP70) was tested in skin, gill, head kidney and spleen at 0, 12 h, 24 h, 2 d, 3 d, 5 d and 7 d. All six genes were significantly up-regulated in skin at most tested time points, which indicate the possibility of skin local T cell activation to support the local antibody response. Compared to three TCR co-receptors, significantly up-regulation of three kinases were seen in the spleen, and the spleen fold changes of these three kinases were much higher than head kidney, which indicates spleen maybe the major systematic immune organs for T cell activation in C. irritans infected fish.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Mucosal; Phylogeny; Receptors, Antigen, T-Cell; RNA, Messenger; Sequence Analysis, DNA; Signal Transduction

Two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) were recorded for the first time from marine perciform fishes off Tunisia and Libya: Philometra rara n. sp. from the rare, deep-water Haifa grouper Hyporthodus haifensis (Serranidae) off Libya and Philometra saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Pomatomidae) off Tunisia. Identification of both fish species was confirmed by molecular barcoding. Light and scanning electron microscope studies of Ph. rara n. sp. showed that it is characterized by the length of spicules (216-219 μm) and the gubernaculum (90-93 μm), the gubernaculum/spicules length ratio (1:2.32-2.43), and mainly by the shape and structure of the distal end of the gubernaculum (shovel-shaped with a wide median smooth field in dorsal view), appearing as having a dorsal protuberance in lateral view, and by the structure of the male caudal mound (dorsally interrupted); large subgravid females (70-137 mm long) are characterized by the presence of four oval submedian cephalic elevations, each of them bearing a pair of cephalic papillae of the outer circle. The finding of Ph. saltatrix off Tunisia confirms that this species is widespread throughout the Mediterranean region. A molecular analysis of our Ph. saltatrix specimens and other available philometrid cytochrome c oxidase 1 (COI) sequences showed that most species have robust clades. Sequences of Ph. saltatrix from Tunisia diverge from Ph. saltatrix from Brazil and the USA, suggesting that speciation is currently occurring between populations from both sides of the Atlantic Ocean.

Topics: Animals; Bass; DNA Barcoding, Taxonomic; Dracunculoidea; Female; Fish Diseases; Fishes; Libya; Male; Mediterranean Sea; Microscopy, Electron, Scanning; Prevalence; Spirurida Infections; Tunisia

Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization.. Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four C. In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity, Mucosal; Immunoglobulins; Models, Molecular; Nodaviridae; Phylogeny; RNA Virus Infections; Sequence Alignment

Topics: Animals; Bass; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Histocompatibility Antigens Class II; Signal Transduction; Spleen; Vibrio Infections; Vibrio parahaemolyticus

Topics: Amino Acid Sequence; Animals; Bass; Cell Line; Fish Diseases; Models, Molecular; Nodaviridae; Protein Structure, Tertiary; Ribavirin; RNA Virus Infections; RNA-Dependent RNA Polymerase; Sequence Alignment; Virus Replication

The parasites of 536 European sea bass, Dicentrarchus labrax, were studied between January 2012 and December 2013 in six Corsican fish farms. The indicator value (IndVal) method, which combines measures of fidelity and specificity, has been used in this study. Because of its resilience to changes in abundance, IndVal is a particularly effective tool for ecological bioindicator. The IndVal method showed how season can influence the occurrence of parasite species in cultured sea bass and also identified parasites as bioindicators relative to fish farm location. The combination of specificity and fidelity highlighted several parasite species as significant indicators. A randomization test identified five parasite species as having a significant indicator value for season (the monogenean Diplectanum aequans; the copepods Lernanthropus kroyeri and Caligus minimus; the isopod Ceratothoa oestroides, and the myxosporidian Ceratomyxa labracis). If gills parasites are compared, they can be seen to be indicator species for two different seasons. The only Monogenea species D. aequans had fidelity and specificity more pronounced in winter, whereas both copepod species and the Isopoda revealed highest rates of infestation corresponding with an increase of water temperature. Four species have a significant indicator value for site location (D. aequans, L. kroyeri, C. minimus, and C. oestroides). The fact that the farm 6 was isolated on the east coast of Corsica may not have allowed the parasite to infect other farms. The presence of copepods on a single farm can also be explained according to salinity variations. Data for species composition and infection levels should help to improve the monitoring and management of parasitism in cultured sea bass populations.

Topics: Animals; Bass; Copepoda; Fish Diseases; Fisheries; France; Gills; Isopoda; Microscopy, Electron, Scanning; Myxozoa; Seasons; Temperature; Trematoda

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.

Topics: Animals; Bass; Cell Line; Disorders of Sex Development; Endocrine Disruptors; Estrogens; Fish Diseases; Lakes; Male; New England; Receptors, Androgen; Receptors, Glucocorticoid; Rivers; Seasons; Testis; Vitellogenins; Yeasts

Largemouth bass, Micropterus salmoides, is a native fish species with special importance for sport fishing competitions in Nuevo León, Mexico. However, no study has investigated the parasitic fauna of M. salmoides, and no reports are available on monogenean parasites in this fish species. Therefore, we described the monogenean parasites of M. salmoides and the effects of season and fish condition factor in five reservoirs: La Boca (LB), El Cuchillo-Solidaridad (CS), Sombreretillo (S), Laguna Salinillas (LS) and Cerro Prieto (CP). The monogeneans infecting M. salmoides were Clavunculus unguis and Acolpenteron ureteroecetes (collected in all localities), as well as Syncleithrium fusiformis, Haplocleidus furcatus, Clavunculus bifurcatus and Urocleidus principalis (CS). Clavunculus unguis had the highest prevalence in fish from all reservoirs. The abundance of monogeneans was generally greater in late spring to autumn than in winter. Although season was not correlated with abundance (r s = 0.0934, P <  0.0154), the months of highest temperature (from May to September) were positively correlated with parasite abundance. A significant association was observed between fish condition factor and the presence of monogeneans (P <  0.05), except for A. ureteroecetes. Our findings include five new geographic records for C. unguis, S. fusiformis, H. furcatus and C. bifurcatus.

Topics: Animals; Bass; Cestode Infections; Fish Diseases; Mexico; Platyhelminths; Seasons; Temperature

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Vaccines; Bass; Cross Protection; Dihydrolipoamide Dehydrogenase; Fish Diseases; Proteome; Recombinant Proteins; Vibrio; Vibrio Infections

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 μg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 μg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Viral; Bass; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunity, Innate; Isoenzymes; Nodaviridae; Reproducibility of Results; RNA Virus Infections; Sensitivity and Specificity

The nervous necrosis virus (NNV) is an aquatic virus that can infect more than 30 species including the grouper, which is a valuable fish species in Taiwan. NNV causes up to 90-100% mortality in the aquaculture industry. Interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins to protect the host against viruses and possess very unique specific characteristics in fish. The cross-reactivity of heterologous IFNs on grouper cells and larvae has not been well-studied to date. To evaluate and compare the anti-NNV effect of different fish IFNs in grouper, we successfully synthesized, subcloned, expressed and purified several fish type I IFNs in the present study: grouper (gIFN), salmon (sIFN), seabass (sbIFN) and tilapia (tpIFN). The gIFN and sIFN proteins up-regulated myxovirus resistance protein (Mx) gene expression in grouper kidney (GK) cells, but similar effects were not observed for sbIFN and tpIFN. Following co- and pre-treatment with the 4 types of IFNs with NNV infection in GK cells, sIFN exhibited the strongest antiviral ability to suppress NNV gene replication (especially at 24 h) and significantly reduced the cytopathic effect (CPE) at 72 h, followed by gIFN. Unsurprisingly, sbIFN and tpIFN had no significant effect on CPE but slightly suppressed NNV gene replication. The cytotoxicity of these four fish IFNs on GK cells was also examined for the first time. In the in vivo test, we confirmed that gIFN and sIFN had a significant protective effect against NNV when administered by intraperitoneal (IP) injection and the oral route in Malabar grouper (Epinephelus malabaricus) larvae. This study compared the protective effects of IFNs from various fish species against NNV and demonstrated crosstalk between sIFN and grouper cells for the first time. These results provide information concerning the efficacy of fish IFNs for possible therapeutic applications.

Topics: Administration, Oral; Animals; Bass; Escherichia coli; Fish Diseases; Fish Proteins; Injections, Intraperitoneal; Interferon Type I; Nodaviridae; Organisms, Genetically Modified; Recombinant Proteins; RNA Virus Infections; Sequence Analysis, DNA

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Topics: Animals; Bass; Cells, Cultured; Fish Diseases; Fish Proteins; Gene Expression Regulation; Genome, Viral; Interferon Type I; Nodaviridae; Poly I-C; Virus Replication

Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.

Topics: Animals; Bass; Capsid Proteins; Fish Diseases; Gene Expression Regulation, Viral; Models, Molecular; Mutagenesis, Insertional; Nodaviridae; Peptides; Protein Conformation; RNA Virus Infections; Viral Vaccines; Virus Internalization

A thorough understanding of host-microbe interactions is crucial for more efficient disease management in the marine larviculture industry. As demonstrated in terrestrial animal research, gnotobiotic systems (involving animals cultured in germ-free conditions or inoculated with known microorganisms) are excellent tools to extend our understanding of the mechanisms involved in host-microbe interactions and allow the evaluation of new treatments for diseases. In this study, we introduce a germ-free European sea bass Dicentrarchus labrax larval model, independent of the continuous addition of antimicrobial agents. This model has an experimental set-up that allows addition of live feed to the larvae without compromising the germ-free status. This model will facilitate and render aquaculture research more effective in terms of mitigation fish larval diseases.

Topics: Animals; Bacterial Infections; Bass; Disinfectants; Fish Diseases; Germ-Free Life; Larva; Ovum

Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT-PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock-infected group. VERv presence was confirmed by rRT-PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.

Topics: Adaptation, Physiological; Animals; Bass; Brain Diseases; Fish Diseases; Nodaviridae; Retinal Diseases; RNA Virus Infections; Salinity

The tripartite motif (TRIM)-containing proteins exert important immune regulatory roles through regulating different signaling pathways in response to different stimuli. TRIM39, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which could regulate cell cycle progression and apoptosis. However, the antiviral activity of TRIM39 is not explored. Here, a TRIM39 homolog from grouper, Epinephelus coioides (EcTRIM39) was cloned, and its effects on cell cycle progression and fish virus replication were investigated. The full-length EcTRIM39 cDNA was composed of 2535 bp and encoded a polypeptide of 543 amino acids with 70% identity with TRIM39 homologs from bicolor damselfish. Amino acid alignment analysis indicated that EcTRIM39 contained a RING finger, B-box and SPRY domain. Expression profile analysis revealed that EcTRIM39 was abundant in intestine, spleen and skin. Upon different stimuli in vivo, the EcTRIM39 transcript was obviously up-regulated after challenging with Singapore grouper iridovirus (SGIV), and polyinosinic-polycytidylic acid (poly I:C). Using fluorescence microscopy, we found that EcTRIM39 localized in the cytoplasm and formed aggregates in grouper spleen (GS) cells. The ectopic expression of EcTRIM39 in vitro affected the cell cycle progression via mediating G1/S transition. Moreover, the RING domain was essential for its accurate localization and effect on cell cycle. In addition, overexpression of EcTRIM39 significantly inhibited viral gene transcription of SGIV and red-spotted grouper nervous necrosis virus (RGNNV) in vitro, and the mutant of RING exerted the opposite effect. Together, our results demonstrated that fish TRIM39 not only regulated the cell cycle progression, but also acted as an important regulator of fish innate immune response against viruses.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Iridovirus; Nodaviridae; Phylogeny; Poly I-C; RNA Virus Infections; RNA, Messenger; Sequence Alignment; Tripartite Motif Proteins

This study investigates, for the first time, the intestinal responses of European sea bass Dicentrarchus labrax chronically exposed to microplastics through ingestion. Fish (n = 162) were fed with 3 different treatment diets for 90 days: control, native polyvinyl chloride (PVC) and polluted polyvinyl chloride (PVC) pellets. Intestines were fixed and processed for histological analysis using standard techniques. Histopathological alterations were examined using a score value (from 0 to 4). The distal part of intestine in all samples proved to be the most affected by pathological alterations, showing a gradual change varying from moderate to severe related to exposure times. The histological picture that characterizes both groups especially after 90 days of exposure, suggests that the intestinal functions can be in some cases totally compromised. The worst condition is increasingly evident in the distal intestine of fish fed with polluted PVC pellets respect to control groups (p < 0.05) to different exposure times. These first results underline the need to assess the impact of increasing microplastics pollution on the marine trophic web.

Topics: Animals; Bass; Fish Diseases; Intestinal Diseases; Plastics; Water Pollutants, Chemical

Piscine nodavirus (betanodavirus) is the causative agent of viral nervous necrosis (VNN) in a variety of cultured fish species, particularly marine fish. In the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment. The virus in seawater was concentrated by an iron-based flocculation method and subjected to isolation with E-11 cells. A real-time reverse transcriptase PCR (RT-PCR) assay was used to quantify the virus in water. After spiking into seawater was performed, a betanodavirus strain (red spotted grouper nervous necrosis virus [RGNNV] genotype) was effectively recovered in the E-11 cells at a detection limit of approximately 10(5)copies (equivalent to 10(2)50% tissue culture infective doses [TCID50])/liter seawater. In an experimental infection of juvenile sevenband grouper (Epinephelus septemfasciatus) with the virus, the virus was isolated from the drainage of a fish-rearing tank when the virus level in water was at least approximately 10(5)copies/liter. The application of this method to seven band grouper-rearing floating net pens, where VNN prevailed, resulted in the successful isolation of the virus from seawater. No differences were found in the partial sequences of the coat protein gene (RNA2) between the clinical virus isolates of dead fish and the cell-cultured virus isolates from seawater, and the viruses were identified as RGNNV. The infection experiment showed that the virus isolates from seawater were virulent to seven band grouper. These results showed direct evidence of the horizontal transmission of betanodavirus via rearing water in marine aquaculture.

Topics: Animals; Bass; Fish Diseases; Nodaviridae; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Seawater; Sequence Analysis, DNA; Virus Cultivation

Fish-pathogenic Vibrio can cause large-scale crashes in marine larval rearing units and, since the use of antibiotics can result in bacterial antibiotic resistance, new strategies for disease prevention are needed. Roseobacter-clade bacteria from turbot larval rearing facilities can antagonize Vibrio anguillarum and reduce mortality in V. anguillarum-infected cod and turbot larvae. In this study, it was demonstrated that antagonistic Roseobacter-clade bacteria could be isolated from sea bass larval rearing units. In addition, it was shown that they not only antagonized V. anguillarum but also V. harveyi, which is the major bacterial pathogen in crustaceans and Mediterranean sea bass larvae cultures. Concomitantly, they significantly improved survival of V. harveyi-infected brine shrimp. 16S rRNA gene sequence homology identified the antagonists as Phaeobacter sp., and in silico DNA-DNA hybridization indicated that they could belong to a new species. The genomes contained genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA), and its production was confirmed by UHPLC-TOFMS. The new Phaeobacter colonized live feed (Artemia) cultures and reduced Vibrio counts significantly, since they reached only 10(4)CFUmL(-1), as opposed to 10(8)CFUmL(-1) in non-Phaeobacter treated controls. Survival of V. anguillarum-challenged Artemia nauplii was enhanced by the presence of wild type Phaeobacter compared to challenged control cultures (89±1.0% vs 8±3.2%). In conclusion, TDA-producing Phaeobacter isolated from Mediterranean marine larviculture are promising probiotic bacteria against pathogenic Vibrio in crustacean live-feed cultures for marine fish larvae.

Topics: Animals; Antibiosis; Artemia; Base Sequence; Bass; DNA, Bacterial; Fish Diseases; Genome, Bacterial; Larva; Mediterranean Sea; Probiotics; RNA, Ribosomal, 16S; Roseobacter; Sequence Analysis, DNA; Tropolone; Vibrio

Due to the mounting awareness of the risks associated with the use of antibiotics in aquaculture, treatment with probiotics has recently emerged as the preferred environmental-friendly prophylactic approach in marine larviculture. However, the presence of unknown and variable microbiota in fish larvae makes it impossible to disentangle the efficacy of treatment with probiotics. In this respect, the recent development of a germ-free culture model for European sea bass (Dicentrarchus labrax L.) larvae opened the door for more controlled studies on the use of probiotics. In the present study, 206 bacterial isolates, retrieved from sea bass larvae and adults, were screened in vitro for haemolytic activity, bile tolerance and antagonistic activity against six sea bass pathogens. Subsequently, the harmlessness and the protective effect of the putative probiotic candidates against the sea bass pathogen Vibrio harveyi were evaluated in vivo adopting the previously developed germ-free sea bass larval model. An equivalence trial clearly showed that no harmful effect on larval survival was elicited by all three selected probiotic candidates: Bacillus sp. LT3, Vibrio lentus and Vibrio proteolyticus. Survival of Vibrio harveyi challenged larvae treated with V. lentus was superior in comparison with the untreated challenged group, whereas this was not the case for the larvae supplemented with Bacillus sp. LT3 and V. proteolyticus. In this respect, our results unmistakably revealed the protective effect of V. lentus against vibriosis caused by V. harveyi in gnotobiotic sea bass larvae, rendering this study the first in its kind.

Topics: Animals; Antibiosis; Aquaculture; Bacillus; Bass; Fish Diseases; Germ-Free Life; Probiotics; Vibrio; Vibrio Infections

The genus Edwardsiella is composed of a diverse group of facultative anaerobic, gram-negative bacteria that can produce disease in a wide variety of hosts, including birds, reptiles, mammals, and fish. Our report describes the isolation and identification of Edwardsiella piscicida associated with chronic mortality events in 2 separate captive largemouth bass (Micropterus salmoides) populations in New York and Florida. Wet-mount biopsies of skin mucus, gill, kidney, and spleen from several affected largemouth bass contained significant numbers of motile bacteria. Histologic examination revealed multifocal areas of necrosis scattered throughout the heart, liver, anterior kidney, posterior kidney, and spleen. Many of the necrotic foci were encapsulated or replaced by discrete granulomas and associated with colonies of gram-negative bacteria. Initial phenotypic and matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis against existing spectral databases of recovered isolates identified these bacteria as Edwardsiella tarda Subsequent molecular analysis using repetitive sequence mediated and species-specific PCR, as well as 16S rRNA, rpoB, and gyrB sequences, classified these isolates as E. piscicida As a newly designated taxon, E. piscicida should be considered as a differential for multiorgan necrosis and granulomas in largemouth bass.

Topics: Animals; Bass; Edwardsiella; Enterobacteriaceae Infections; Fish Diseases; Florida; New York; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Species Specificity

Based on light and scanning electron microscopical studies of nematode specimens (males and mature females) collected from the ovary of groupers (Serranidae, Perciformes) in the Mediterranean Sea off Tunisia (near Tunis and Sfax), two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) are reported: Philometra inexpectata n. sp. from the mottled grouper Mycteroperca rubra and P. jordanoi (López-Neyra, 1951) from the dusky grouper Epinephelus marginatus. Identification of both fish species was confirmed by molecular barcoding. The new species is mainly characterized by the length of equally long spicules (147-165 μm), the gubernaculum (63-93 μm long) bearing at the tip two dorsolateral lamellar parts separated from each other by a smooth median field, a V-shaped mound on the male caudal extremity, the presence of a pair of large caudal papillae located posterior to the cloaca and by the body length of the males (1.97-2.43 mm). Philometra inexpectata n. sp. is the fifth known gonad-infecting philometrid species parasitizing serranid fishes in the Mediterranean region. The males of P. jordanoi were examined by scanning electron microscopy for the first time; this detailed study revealed some new taxonomically important morphological features, such as the number and arrangement of cephalic and caudal papillae, presence of amphids and phasmids and mainly the lamellate structures at the posterior end of the gubernaculum. A key to gonad-infecting species of Philometra parasitic in serranid fishes is provided.

Topics: Animals; Bass; Dracunculoidea; Female; Fish Diseases; Food Parasitology; Male; Mediterranean Sea; Ovary; Species Specificity; Spirurida Infections

Epinephelus lanceolatus, considered to be an aquaculture fish species of high economic value in East Asia, is one of the largest groupers in the Epinephelus genus. Vibrio alginolyticus is a bacterial species that causes high morbidity in marine fish; infection can cause exophthalmia, ulcers, septicemia, and corneal opaqueness in fish. Epinephelus lanceolatus larvae infected with Vibrio alginolyticus were subjected to transcriptome analysis to study the immune regulation pathway. Grouper larvae were injected with 2.6 × 10(4) CFU/fish in 20 μl of V. alginolyticus and control larvae were injected with TSB; RNA samples were then collected at 4, 6, 8, 10, 12, 16, 24, and 48 h after infection. Extracted RNA was subjected to reverse transcription, and used to examine the immune gene response of E. lanceolatus by Real-time PCR. Samples taken at 6 h were subjected to next-generation sequencing, resulting in a total read value of 28,705,411 and total base number of 2,152,905,850. The unigene number was 100,848, and 5913 unigenes were filtered using FPKM>0.3, 2FC, p < 0.05. Gene Ontology (GO) analysis of the filtered genes revealed a total of 30 GO numbers in the cellular component, and 58 GO numbers for both biological processes and molecular functions. Of the GO group related to immune pathways, 27 unigenes related to biological processes involving the immune response, 31 related to the immune system, 9 related to the inflammatory response, and 43 related to the response to stress were identified. KEGG pathway analysis only detected 1 to 4 genes, and as such, we selected the GO analysis results for further analysis using GeneSpring. This demonstrated that V. alginolyticus probably stimulates TLR5 activity via the bacterial flagellum, through an MyD88-dependent pathway; the resulting production of IL-1β and IL-8 through the NFκB pathway induces pro-inflammatory and/or chemotactic effects. Alternatively, serum amyloid A may stimulate neutrophils that induce the secretion of MMP9 from infected tissues, resulting in the cleavage and activation of IL-8. IL-8, in turn, would enhance neutrophil chemotaxis. Infection also induced expression of genes encoding C3, C6, C7, C8, and C9, which induce the complement system and form the membrane attack complex to lyse the bacteria membrane. The qPCR results indicated that TLR5 is significantly increased between 10 and 16 h, IL-1β between 8 and 16 h, IL-8 between 8 and 12 h, and C6 between 4 and 16 h, as compared to levels in the

Topics: Animals; Bass; Cytokines; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Immunity, Innate; Real-Time Polymerase Chain Reaction; Toll-Like Receptor 5; Vibrio alginolyticus; Vibrio Infections

Apoptosis plays vital roles in many physiological process and immune response. BOK is one of the central regulators in apoptosis. In this study, a new BOK homolog (Ec-BOK) was cloned and characterized from Orange-spotted grouper, Epinephelus coioides. Ec-BOK encoded a 210 amino acid peptides which shared 97% identity to Stegastes partitus BOK protein, contained four BH domains and one transmembrane region. Ec-BOK widely expressed in all analyzed tissues with the higher expressions in kidney and spleen. Its expression level was up-regulated after SGIV infection in vitro. Further analysis revealed that overexpression of Ec-BOK inhibited viral genes transcriptions and virus replication in fish cell. Our findings suggested that Ec-BOK might play a role in the immune response against virus.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Organ Specificity; Phylogeny; Ranavirus; RNA, Messenger; Sequence Alignment

Known life cycles of myxosporean parasites have two hosts, but very few life cycles have been disclosed, especially in the marine environment. Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992 is a systemic parasite from the European seabass, Dicentrarchus labrax (Linnaeus, 1758), a highly valuable commercial fish. It affects its health, leading to aquaculture production losses. During 2013 and 2014, an actinospore survey was conducted in a total of 5942 annelids collected from a fish farm in Algarve and from the Aveiro Estuary, in Portugal. A new tetractinomyxon actinospore was found in a capitellid polychaete, belonging to the genera Capitella collected at the fish farm. The tetractinomyxons were pyriform measuring 11·1 ± 0·7 µm in length and 7·2 ± 0·4 µm in width, and presented three rounded polar capsules measuring 2·4 ± 0·3 µm in diameter. The molecular analysis of the 18S rRNA gene sequences from the tetractinomyxons revealed a similarity of 100% with the DNA sequences deposited in the GenBank from S. dicentrarchi myxospores collected from the European seabass and the spotted seabass in the same fish farm and 99·9% similarity with the DNA sequence obtained from the myxospores found infecting the European seabass in the Aveiro Estuary. Therefore, the new tetractinomyxons are inferred to represent the actinospore phase of the S. dicentrarchi life cycle.

Topics: Animals; Aquaculture; Bass; Estuaries; Fish Diseases; Life Cycle Stages; Myxozoa; Parasitic Diseases, Animal; Polychaeta; Portugal

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DEAD-box RNA Helicases; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Nodaviridae; Phylogeny; Ranavirus; RNA Virus Infections; RNA, Messenger; Sequence Alignment

Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyAch gene, a chromosome I-borne gene that encodes HlyAch hemolysin, whereas the three nonhemolytic isolates contained hlyAch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyAch-positive and hlyAch-negative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones.. The geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges.

Topics: Animals; Aquaculture; Bacterial Typing Techniques; Bass; Black Sea; Fish Diseases; Genes, Bacterial; Genetic Variation; Genotype; Gram-Negative Bacterial Infections; Photobacterium; Polymerase Chain Reaction; Sequence Analysis, DNA; Virulence Factors

A continuous cell line designated BMGB (brown-marbled grouper brain) was established from the brain tissues of the brown-marbled grouper Epinephelus fuscoguttatus and characterized. BMGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic protein and keratin and were persistently infected by betanodavirus, as confirmed through immunocytochemistry, polymerase chain reaction and immunoblot analyses. Because few intact virions were present in the BMGB cell culture fluid, the cytopathic effect (CPE) was not observed when the culture fluid was inoculated with GBC1 cells. However, BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocytivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. Because the cells contained several unusual or degraded viral proteins, the persistent infection of betanodavirus in the BMGB cells may have resulted from a mechanism that destroys the viral proteins rather than the result of Mx protein expression. Despite the persistent betanodavirus infection, BMGB cells proliferated in a manner similar to other normal tropic fish cells and supported the propagation of several piscine viruses; however, the yield was lower than that of normal cells. The BMGB cells will be useful for investigating virus and host cell interaction.

Topics: Animals; Bass; Brain; Cell Line; Disease Susceptibility; Fish Diseases; Fish Proteins; Myxovirus Resistance Proteins; Nodaviridae; Polymerase Chain Reaction; RNA Virus Infections; Sequence Analysis, DNA

Anemia is a common disorder, characterized by abnormally low levels of red blood cells or hemoglobin. The mechanisms of anemia development and response have been thoroughly studied in mammals, but little is known in other vertebrates, particularly teleost fish. In this study, different degrees of anemia were induced in healthy European sea bass specimens (Dicentrarchus labrax) and at pre-determined time points hematological parameters, liver iron content and the expression of genes involved in iron homeostasis and hematopoiesis, with particular attention on hepcidins, were evaluated. The experimental anemia prompted a decrease in hamp1 expression in all tested organs, in accordance to an increased need for iron absorption and mobilization, with slight increases in hamp2 in the kidney and intestine. The liver was clearly the major organ involved in iron homeostasis, decreasing its iron content and showing a gene expression profile consistent with an increased iron release and mobilization. Although both the spleen and head kidney are involved in erythropoiesis, the spleen was found to assume a more preponderant role in the recovery of erythrocyte levels. The intestine was also involved in the response to anemia, through the increase of iron transporting genes. Administration of Hamp1 or Hamp2 mature peptides showed that only Hamp1 affects hematological parameters and liver iron content. In conclusion, the molecular mechanisms of response to anemia present in sea bass are similar to the ones described for mammals, with these results indicating that the two hepcidin types from teleosts assume different roles during anemia.

Topics: Anemia; Animals; Bass; Erythropoiesis; Fish Diseases; Gene Expression Regulation; Hepcidins; Iron; Protein Isoforms

As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Galectin 1; Immunity, Innate; Lipopolysaccharides; Phylogeny; Poly I-C; Ranavirus; RNA, Messenger; Sequence Alignment

Asian seabass is an important food fish in Southeast Asia. Viral nervous necrosis (VNN) disease, triggered by nervous necrosis virus (NNV) infection, has caused mass mortality of Asian seabass larvae, resulting in enormous economic losses in the Asian seabass industry. In order to better understand the complex molecular interaction between Asian seabass and NNV, we investigated the transcriptome profiles of Asian seabass epithelial cells, which play an essential role in immune regulation, after NNV infection. Using the next generation sequencing (NGS) technology, we sequenced mRNA from eight samples (6, 12, 24, 48 h post-inoculation) of mock and NNV-infected Asian seabass epithelial cell line, respectively. Clean reads were de novo assembled into a transcriptome consisting of 89026 transcripts with a N50 of 2617 bp. Furthermore, 251 differentially expressed genes (DEGs) in response to NNV infection were identified. Top DEGs include protein asteroid homolog 1-like (ASTE1), receptor-transporting protein 3 (RTP3), heat shock proteins 30 (HSP30) and 70 (HSP70), Viperin, interferon regulatory factor 3 (IRF3) and other genes related to innate immunity. Our data suggest that abundant and diverse genes corresponding to NNV infection. The results of this study could also offer vital information not only for identification of novel genes involved in Asian seabass-NNV interaction, but also for our understanding of the molecular mechanism of Asian seabass' response to viral infection. In addition, 24807 simple sequence repeats (SSRs) were detected in the assembled transcriptome, providing valuable resources for studying genetic variations and accelerating quantitative trait loci (QTL) mapping for disease resistance in Asian seabass in the future.

Topics: Animals; Bass; Cell Line; Epithelial Cells; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Microsatellite Repeats; Nodaviridae; RNA Virus Infections; RNA, Messenger; Transcriptome

Singapore grouper iridovirus (SGIV) is a devastating aquaculture virus responsible for heavy economic losses to grouper, Epinephelus sp. aquaculture. The aim of this study was to develop a rapid and sensitive detection method for SGIV infections in infected groupers.. We previously generated DNA aptamers against SGIV-infected cells. In this study, we established and characterized a novel aptamer (Q3)-based enzyme-linked apta-sorbent assay (ELASA) for the detection of SGIV infection in Epinephelus coioides. The Q3-based ELASA could detect SGIV infection rapidly in vitro and in vivo, with high specificity and stability. Q3-based ELASA specifically recognized SGIV-infected cells, but not other-virus-infected cells or uninfected cells. Q3-based ELASA detected SGIV infection in a dose-dependent manner at Q3 concentrations as low as 125 nmol l(-1) . The results in relation to SGIV-infected cells (5 × 10(4) ), incubation time (1 min) and incubation temperature (37°C) demonstrated that Q3-based ELASA could detect SGIV infection quickly and stably, superior to antibody-based enzyme-linked immunosorbent assay. Q3-based ELASA could detect the presence of SGIV infection in kidney, liver and spleen samples in vivo, at dilutions of 1/50, 1/100 and 1/50 respectively. The complete detection process took 1-2 h.. Q3-based ELASA could be a useful tool for diagnosing SGIV infection.. This is the first developed aptamer-based ELASA for detecting SGIV infection, and is widely applicable in grouper aquaculture industry in light of its rapidity, and high specificity and stability.

Topics: Animals; Aptamers, Nucleotide; Bass; DNA Virus Infections; Fish Diseases; Immunoassay; Iridovirus

In the period 2013-2015, wild dusky grouper, Epinephelus marginatus (Lowe), caught in Libyan coastal waters and ranging in size from 42 to 92 cm in total length, were observed to have distinctive skin lesions of unknown aetiology. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digenean trematode parasites carrying similar eggs were also seen within the blood vessels of the deep and superficial dermis. The cause of this distinctive condition, termed dusky grouper dermatitis (DGD), and its potential impact upon already threatened Mediterranean wild dusky grouper populations and upon cultured grouper more widely have yet to be established.

Topics: Animals; Bass; Dermatitis; Endangered Species; Fish Diseases; Libya

Betanodaviruses are the causative agents of a highly infectious disease of fish known as viral nervous necrosis (VNN). To date, 4 different nervous necrosis virus (NNV) genotypes have been described, but natural reassortant viruses have also been detected, which further increase viral variability. Water temperature plays an important role in determining the appearance and the severity of VNN disease. We assessed the effect of temperature (20°, 25° and 30°C) on mortality and virus load in the brain of European sea bass Dicentrarchus labrax experimentally infected with 4 genetically different betanodaviruses, namely red-spotted grouper NNV (RGNNV), striped jack NNV (SJNNV) and the reassortant strains RGNNV/SJNNV and SJNNV/RGNNV. The RGNNV/SJNNV virus possesses the polymerase gene of RGNNV and the coat protein gene of SJNNV, and vice versa for the SJNNV/RGNNV virus. The obtained results showed that the RGNNV strain is the most pathogenic for juvenile sea bass, but clinical disease and mortality appeared only at higher temperatures. The SJNNV strain is weakly pathogenic for D. labrax regardless of the temperature used, while virus replication was detected in the brain of survivors only at 20°C. Finally, reassortant strains caused low mortality, independent of the temperature used, but the viral load in the brain was strongly influenced by water temperature and the genetic type of the polymerase gene. Taken together, these data show that nodavirus replication in vivo is a composite process regulated by both the genetic features of the viral strain and water temperatures.

Topics: Animals; Bass; Fish Diseases; Genotype; Nodaviridae; RNA Virus Infections; Temperature

The tripartite motif (TRIM)-containing proteins have attracted particular attention to their multiple functions in different biological processes. TRIM13, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which plays critical roles in diverse cellular processes including cell death, cancer and antiviral immunity. In this study, a TRIM13 homolog from orange spotted grouper, Epinephelus coioides (EcTRIM13) was cloned and characterized. The full-length of EcTRIM13 cDNA encoded a polypeptide of 399 amino acids which shared 81% identity with TRIM13 homolog from large yellow croaker (Larimichthys crocea). Amino acid alignment analysis showed that EcTRIM13 contained conserved RING finger and B-box domain. Expression patterns analysis indicated that EcTRIM13 was abundant in liver, spleen, kidney, intestine and gill. Moreover, the transcript of EcTRIM13 in grouper spleen was differently regulated after injection with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C). Under fluorescence microscopy, we observed the tubular structure in wild type EcTRIM13 transfected cells, but the RING domain mutant resulted in the fluorescence distribution was changed and the bright punctate fluorescence was evenly situated throughout the cytoplasm, suggesting that the RING domain was essential for its accurate localization. Overexpression of EcTRIM13 in vitro obviously increased the replication of red spotted grouper nervous necrosis virus (RGNNV), and the enhancing effect of EcTRIM13 on virus replication was affected by the RING domain. Furthermore, the ectopic expression of EcTRIM13 not only negatively regulated the interferon promoter activity induced by interferon regulator factor (IRF) 3, IRF7, and melanoma differentiation-associated protein 5 (MDA5), but also decreased the expression of several interferon related factors. In addition, the overexpression of EcTRIM13 also differently regulated the transcription of pro-inflammatory factors. Together, our results firstly demonstrated that fish TRIM13 exerted negative regulation of antiviral response against nodavirus infection.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Phylogeny; Poly I-C; Ranavirus; RNA, Messenger; Sequence Alignment; Tripartite Motif Proteins

Tripartite motif-containing 25 (TRIM25) has been demonstrated to exert crucial roles in the regulation of innate immune signaling. However, the roles of fish TRIM25 in antiviral immune response still remained uncertain. Here, a novel fish TRIM25 gene from orange spotted grouper (EcTRIM25) was cloned and its roles in grouper virus infection were elucidated. EcTRIM25 encoded a 734-aa protein which shared 68% identity to large yellow croaker (Larimichthys crocea). Amino acid alignment showed that EcTRIM25 contained three conserved domains, including a RING-finger domain, a B box/coiled-coil domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM25 was predominantly detected in skin, spleen and intestine. After stimulation with Singapore grouper iridovirus (SGIV) or poly I:C, the relative expression of EcTRIM25 in grouper spleen was significantly increased at the early stage of injection. Subcellular localization analysis showed that EcTRIM25 distributed throughout the cytoplasm in grouper cells. Notably, the deletion RING domain affected its accurate localization and displayed microtubule like structures or bright aggregates in GS cells. After incubation with SGIV or red spotted grouper nervous necrosis virus (RGNNV), overexpression of full length of EcTRIM25 in vitro significantly decreased the viral gene transcription of SGIV and RGNNV. Consistently, the deletion of RING domain obviously affected the inhibitory effect of EcTRIM25. Furthermore, overexpression of EcTRIM25 significantly increased the expression level of interferon related signaling molecules, including interferon regulatory factor (IRF) 3, interferon-induced 35-kDa protein (IFP35), MXI, IRF7 and myeloid differentiation factor 88 (MyD88), suggesting that the positive regulation of interferon immune response by EcTRIM25 might affected RGNNV replication directly. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcTRIM25. We proposed that the regulation of IRF7, MyD88 and pro-inflammation cytokines might contribute more important roles in SGIV infection. In addition, the RING domain of EcTRIM25 also played critical roles in the regulation of interferon immune and inflammation response. Together, our results will provide new evidences that the RING domain was essential for the antiviral action of fish TRIM25 against iridovirus and nodavirus infection.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Phylogeny; Poly I-C; Ranavirus; RNA, Messenger; Sequence Alignment; Tripartite Motif Proteins

A new microsporidian infecting the connective tissue of the coelomic cavity of the blacktail comber Serranus atricauda, in the Madeira Archipelago (Portugal), is described on the basis of morphological, ultrastructural, and molecular features. The microsporidian formed large whitish xenomas adhering to the peritoneal visceral organs of the host. Each xenoma consisted of a single hypertrophic cell, in the cytoplasm of which mature spores proliferated within parasitophorous vacuoles surrounded by numerous collagen fibers. Mature spores were ellipsoidal and uninucleated, measuring an average of 6.5 ± 0.5 μm in length and 3.4 ± 0.6 μm in width. The anchoring disk of the polar filament was subterminal, laterally shifted from the anterior pole of the spore. The isofilar polar filament coiled in 18-19 turns, forming two rows that surrounded the posterior vacuole. The latter occupied about one third of the spore length. The polaroplast surrounding the apical and uncoiled portion of the polar filament displayed two distinct regions: a lamellar region and an electron-dense globule. Molecular analysis of the rRNA genes, including the internal transcribed spacer region, and phylogenetic analysis using maximum likelihood and neighbor joining demonstrated that this microsporidian parasite clustered with some Glugea species. Based on the differences found both at the morphological and molecular levels, to other members of the genus Glugea, the microsporidian infecting the blacktail comber is considered a new species, thus named Glugea serranus n. sp.

Topics: Animals; Bass; Fish Diseases; Glugea; Microscopy, Electron, Transmission; Microsporidiosis; Phylogeny; Portugal; Sequence Analysis, DNA

The increasing frequency of jellyfish outbreaks in coastal areas has led to multiple ecological and socio-economic issues, including mass mortalities of farmed fish. We investigated the sensitivity of the European sea bass (Dicentrarchus labrax), a widely cultured fish in the Mediterranean Sea, to the combined stressors of temperature, hypoxia and stings from the jellyfish Pelagia noctiluca, through measurement of oxygen consumption rates (MO2), critical oxygen levels (PO2crit), and histological analysis of tissue damage. Higher levels of MO2, PO2crit and gill damage in treated fish demonstrated that the synergy of environmental and biotic stressors dramatically impair farmed fish metabolic performances and increase their health vulnerability. As a corollary, in the current scenario of ocean warming, these findings suggest that the combined effects of recurrent hypoxic events and jellyfish blooms in coastal areas might also threaten wild fish populations.

Topics: Animals; Aquaculture; Bass; Bites and Stings; Fish Diseases; Gills; Oxygen Consumption; Scyphozoa; Stress, Physiological

As crucial components of the toll-like receptor (TLR) and interleukin-1 (IL-1) receptor (IL-1R) signaling pathways, interleukin-1 receptor associated kinase (IRAK) family members play essential roles in an animal's immune response. In this study, an IRAK family member, designated EcIRAK-1, was identified in the orange-spotted grouper Epinephelus coioides, and its role in signal transduction investigated. The full-length EcIRAK-1 gene is 2822 bp, encoding a 760-amino-acid protein that has the typical characteristics of mammalian IRAK-1, including an N-terminal death domain, a ProST domain, a central kinase domain, and C-terminal C1 and C2 domains. EcIRAK-1 shares 42%-79% sequence identity with other fish IRAK-1 proteins, and the death and kinase domains are more conserved than the other domains. Several important amino acids and motifs of mammalian IRAK-1 are also conserved in the grouper and other piscine IRAK-1s. In healthy grouper, EcIRAK-1 was broadly expressed in all the tissues tested, with the highest expression in the gill and skin. After infection with Cryptocaryon irritans, EcIRAK-1 expression increased in the gill and spleen. After its exogenous expression in HEK293T cells, EcIRAK-1 significantly activated nuclear factor kappaB (NF-κB). The death domain, ProST domain, and some conserved amino acids, such as T58, T207, K237, and T387, in EcIRAK-1 are required for its signaling function. These data demonstrate that piscine IRAK-1 has the same structural characteristics as its mammalian counterpart and that its function is conserved among vertebrates.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; HEK293 Cells; Humans; Interleukin-1 Receptor-Associated Kinases; NF-kappa B; Phylogeny; RNA, Messenger; Sequence Alignment; Signal Transduction

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

Topics: Animals; Bass; Cloning, Molecular; Cytokines; DNA, Complementary; Fish Diseases; Fish Proteins; Nodaviridae; Poly I-C; RNA Virus Infections; RNA, Messenger; Sequence Analysis, DNA; Sequence Analysis, Protein; Transcription, Genetic; Ubiquitins

The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Fish Proteins; Hepcidins; Vibrio; Vibrio Infections

Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus, we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti-NNV vaccine and to understand its mechanisms.

Topics: Administration, Oral; Animals; Bass; Cells, Cultured; Chitosan; Cytotoxicity, Immunologic; Fish Diseases; Immunity, Cellular; Interferons; Intestines; Nodaviridae; RNA Virus Infections; Transcription, Genetic; Vaccination; Vaccines, DNA

Nervous necrosis virus (NNV) is a kind of the betanodaviruses, which can cause viral nervous necrosis (VNN) and massive mortality in larval and juvenile stages of orange-spotted grouper (Epinephelus coioides). Due to the lack of viral genomes, virus-like particles (VLPs) are considered as one of the most promising candidates in vaccine study to control this disease. In this study, a type of VLPs, which was engineered on the basis of orange-spotted grouper nervous necrosis virus (OGNNV), was produced from prokaryotes. They possessed the similar structure and size to the native NNV. In addition, synthetic oligodeoxynucleotide (ODN) containing CpG motif was added in vaccines, and the expression patterns of several genes were analyzed after injecting with VLP and VLP with adjuvant (VA) to assess the regulation effect of vaccine for inducing immune responses. RT-PCR assays showed that six related genes in healthy tissues were ubiquitously expressed in all nine tested tissues. The vaccine alone was able to enhance the expression of genes, including MHCIa, MyD88, TLR3, TLR9 and TLR22 after vaccination, indicating that the vaccine was able to induce immune response in grouper. In liver, spleen and kidney, the gene expressions of VA group were all significantly higher than that of VLP group at 72 h post-stimulation, showing that the fish of VA challenge group obtained the longer-lasting protective immunity and resistance to pathogen challenge than that of VLP group. The data indicated that the efficacy of vaccine could be further enhanced by CpG ODN after vaccination and provided the reference for the development of future viral vaccine in grouper.

Topics: Animals; Bass; Fish Diseases; Gene Expression; Genes, Viral; Nodaviridae; Oligodeoxyribonucleotides; RNA Virus Infections; Vaccination; Vaccines, Virus-Like Particle; Viral Vaccines

Laboratory of genetics and physiology 2 (LGP2), a member of RIG-I like receptor (RLR) family, plays crucial roles in modulating cellular antiviral response during viral infection. However, the detailed roles of LGP2 in different virus infection were controversial up to now. Here, we cloned a LGP2 gene from orange-spotted grouper (EcLGP2) and investigated its roles in response to grouper virus infection. EcLGP2 encoded a 678-aa protein which shared 83% identity to sea perch (Lateolabrax japonicas). Amino acid alignment showed that EcLGP2 contained three conserved domains, including a DEAD/DEAH box helicase domain, a helicase superfamily C-terminal domain and a C-terminal domain of RIG-I. In healthy grouper, the transcript of EcLGP2 could be predominantly detected in kidney, gill, fin, spleen and skin. Subcellular localization analysis showed that EcLGP2 distributed throughout the cytoplasm in grouper cells. Notably, the intracellular distribution of EcLGP2 was altered at the late stage of Singapore grouper iridovirus (SGIV) infection, but remained unchanged during red-spotted grouper nervous necrosis virus (RGNNV) infection. Moreover, overexpression of EcLGP2 in vitro significantly enhanced the viral replication of SGIV and RGNNV, evidenced by the acceleration of CPE occurrence and the up-regulation of the viral gene transcription or protein synthesis. Further studies indicated that overexpression of EcLGP2 decreased the expression level of interferon related molecules or effectors, including IRF3, IRF7, ISG15, IFP35, MXI, MXII, and MDA5, suggesting that the negative feedback of interferon immune response by EcLGP2 might contribute to the enhancement of RGNNV infection. Moreover, the expression levels of pro-inflammation cytokines, including IL-8 and TNFα were significantly decreased, but that of IL-6 was increased by the ectopic expression of EcLGP2. Thus, our results will contribute greatly to understanding the roles of fish LGP2 in innate immune response during iridovirus and nodavirus infection.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DEAD Box Protein 58; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Innate; Nodaviridae; Ranavirus; RNA Virus Infections; RNA, Messenger; Sequence Alignment; Up-Regulation

Reoviruses (family Reoviridae) infect vertebrate and invertebrate hosts with clinical effects ranging from inapparent to lethal. Here, we describe the discovery and characterization of Largemouth bass reovirus (LMBRV), found during investigation of a mortality event in wild largemouth bass (Micropterus salmoides) in 2015 in WI, USA. LMBRV has spherical virions of approximately 80 nm diameter containing 10 segments of linear dsRNA, aligning it with members of the genus Orthoreovirus, which infect mammals and birds, rather than members of the genus Aquareovirus, which contain 11 segments and infect teleost fishes. LMBRV is only between 24 % and 68 % similar at the amino acid level to its closest relative, Piscine reovirus (PRV), the putative cause of heart and skeletal muscle inflammation of farmed salmon. LMBRV expands the known diversity and host range of its lineage, which suggests that an undiscovered diversity of related pathogenic reoviruses may exist in wild fishes.

Topics: Animals; Bass; Fish Diseases; Genome, Viral; Phylogeny; Reoviridae; Reoviridae Infections

Photobacterium damselae is a Gram negative bacterium causes photobacteriosis, a worldwide septicemic disease in aquaculture including sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogenicity of bacterial subspecies and the disease pathological changes in natural and experimental infections have thus far yielded inconsistency of effective preventive measures. This study aimed to represent a comprehensive analysis of the potential pathogenic capacities of the two subspecies of P. damselae in cultured sea bass and sea bream in the Northwestern region of Egypt. Diseased 321 sea bass and 257 sea bream, in addition to 99 healthy sea bass fingerlings were sampled from three farms located along the Mediterranean Sea. P. damselae subspecies were isolated from diseased fish and characterized using bacteriological, molecular, and antimicrobial susceptibility methods. Healthy fish were challenged by a virulent P. damselae subsp. piscicida, monitored for disease signs and mortality, and the histopathological abnormalities and hematological disorders were carried out. Clinical signs and gross lesions in naturally infected sea bass and sea bream showed great similarities with absence of a subspecies-specific characteristic sign or lesion. The two subspecies were recovered through the entire year from individual fish sample, suggests a coexistence of two subspecies endemic infection. In diseased sea bass, 38.32% and 16.20% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. However in diseased sea bream, 44.47% and 26.46% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. High mortalities and devastating clinicopathologic abnormalities represented by sever clinical signs, hematological disorders and histological abnormalities strengthen the pathogenicity of P. damselae subspecies in the two fish species and therefore, a vaccination strategy against both subspecies should be taken into account.

Topics: Animals; Aquaculture; Bass; Egypt; Fish Diseases; Gram-Negative Bacterial Infections; Histocytochemistry; Mediterranean Sea; Photobacterium; Sea Bream; Survival Analysis

B cell antigen receptor (BCR) plays a crucial role in B cell development and antibody production. It comprises membrane immunoglobulin non-covalently associated with CD79a/CD79b heterodimer. After B cell activation, initial extracellular signals are transduced by BCR complex and amplified by two protein tyrosine kinases, LYN and SYK, which then trigger various pathways. In the present study, we cloned grouper genes for BCR accessory molecules, EcCD79a (669 bp) and EcCD79b (639 bp), as well as two protein tyrosine kinases, EcLYN (1482 bp) and EcSYK (1854 bp). Homology analysis showed that all four molecules had a relatively high amino acid identity compared with those in other animals. Among them, they all shared the highest identity with Takifugu rubripes (EcCD79a 49%, EcCD79b 52%, EcLYN 82% and EcSYK 77%). The conserved features and important functional residues were analyzed. Together with IgM and IgT, tissue distribution analysis showed that all six molecules were mainly expressed in immune organs, particularly systematic immune organs. In groupers infected with Cryptocaryon irritans, up-regulation of EcCD79a and b, EcIgM and EcIgT were not seen in the early stage skin and gill until 14-21 days. Up-regulation of EcCD79a was seen in head kidney at most time points, while EcCD79a and b were only significantly up-regulated in day 14 spleen. Significant up-regulation of EcIgT were seen in day 21 head kidney and day 1, day14 spleen. Significant up-regulation of EcIgM were seen in day 1 head kidney and 12 h spleen. In addition, two protein kinase genes, EcLYN and EcSYK, were up-regulated in the skin at most time points, which suggested that B cells may be activated at the skin local infection site.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Innate; Phylogeny; RNA, Messenger; Sequence Analysis, Protein; Signal Transduction

A high prevalence of skin pigmented lesions of 15% was recently reported in coral trout Plectropomus leopardus, a commercially important marine fish, inhabiting the Great Barrier Reef. Herein, fish were sampled at two offshore sites, characterised by high and low lesion prevalence. A transcriptomic approach using the suppressive subtractive hybridisation (SSH) method was used to analyse the differentially expressed genes between lesion and normal skin samples. Transcriptional changes of 14 genes were observed in lesion samples relative to normal skin samples. These targeted genes encoded for specific proteins which are involved in general cell function but also in different stages disrupted during the tumourigenesis process of other organisms, such as cell cycling, cell proliferation, skeletal organisation and cell migration. The results highlight transcripts that are associated with the lesion occurrence, contributing to a better understanding of the molecular aetiology of this coral trout skin disease.

Topics: Animals; Bass; Coral Reefs; Environmental Monitoring; Fish Diseases; Fisheries; Pigments, Biological

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Histones; Immunity, Innate; Mitogens; Nodaviridae; Pathogen-Associated Molecular Pattern Molecules; Photobacterium; Phylogeny; Random Allocation; RNA Virus Infections; Sea Bream; Sequence Analysis, DNA; Tissue Distribution; Transcription, Genetic; Vibrio; Vibrio Infections

Increased reports uncovered that mammalian tripartite motif-containing 62 (TRIM62) exerts crucial roles in cancer and innate immune response. However, the roles of fish TRIM62 in antiviral immune response remained uncertain. In this study, a TRIM62 gene was cloned from orange spotted grouper (EcTRIM62) and its roles in grouper RNA virus infection was elucidated in vitro. EcTRIM62 shared 99% and 83% identity to bicolor damselfish (Stegastes partitus) and human (Homo sapiens), respectively. Sequence alignment indicated that EcTRIM62 contained three domains, including a RING-finger domain, a B-box domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM62 was predominantly detected in brain and liver, followed by heart, skin, spleen, fin, gill, intestine, and stomach. Subcellular localization analysis indicated that bright fluorescence spots were observed in the cytoplasm of EcTRIM62-transfected grouper spleen (GS) cells. During red-spotted grouper nervous necrosis (RGNNV) infection, overexpression of EcTRIM62 significantly enhanced the severity of CPE and increased viral gene transcriptions. Furthermore, the ectopic expression of EcTRIM62 significantly decreased the transcription level of interferon signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), melanoma differentiation-associated protein 5 (MDA5), myxovirus resistance gene MXI, and MXII, suggesting that the negative regulation of interferon immune response by EcTRIM62 might directly contributed to its enhancing effect on RGNNV replication. Furthermore, our results also demonstrated that overexpression of EcTRIM62 was able to differently regulate the expression levels of pro-inflammation cytokines. In addition, we found the ectopic expression of EcTIRM62 negatively regulated MDA5-, but not mediator of IRF3 activation (MITA)-induced interferon immune response. Further studies showed that the deletion of RING domain and SPRY domain significantly affected the action of EcTRIM62, including the enhancing effect on virus replication and regulation of interferon immune response. Thus, our studies firstly demonstrated that EcTRIM62 negatively regulated the innate antiviral immune response against fish RNA viruses.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Innate; RNA Virus Infections; RNA Viruses; RNA, Messenger; Sequence Alignment; Tripartite Motif Proteins

This investigation is aimed to improve the knowledge on the physiological alterations occurring at morphological and molecular level in European sea bass naturally infected by A. ocellatum and reared at different salinities. European sea bass juveniles (Dicentrarchus labrax) weighing 20 ± 0.5 g were divided in three aquaponics systems: CTRL, reared at 20 ppt salinity; AFI, reared in freshwater (0 ppt) and infected with the dinoflagellate Amyloodinium ocellatum; ASI, reared at 20 ppt salinity and infected with A. ocellatum. Beta vulgaris plants were introduced in each of the aquaponic systems. Temperature was increased 1 °C every second day from 18 to 25 °C during the experiment. At the end of the trial, liver, brain, intestine and gills were sampled for molecular and histological analyses. A. ocellatum affected D. labrax growth (insulin-like growth factor I, IGF-I) and appetite (Neuropeptide Y, NPY) signals in ASI. Immune system was activated in ASI by the presence of parasites by producing higher levels of Interleukin-1 (IL-1) and Tumor Necrosis Factor α (TNFα). Peroxisome proliferator-activated receptor α (PPAR α), codifying for a protein involved in lipid metabolism, was upregulated in ASI because of the necessity to produce energy to maintain homeostasis. On the contrary, A. ocellatum did not cause signs of infection in AFI as confirmed by gene expression and histological analysis, that were similar to CTRL. However, in freshwater reared fish, a modification of lipid metabolism was observed through a reduction in PPARα gene expression and hepatic lipid content.

Topics: Animals; Bass; Dinoflagellida; Fish Diseases; Fresh Water; Gene Expression; Protozoan Infections, Animal; Salinity; Seawater

Two isoforms of piscidin from Malabar grouper (Epinephelus malabaricus), EmPis-1 and EmPis-2, were cloned and studied. EmPis-1 and EmPis-2 showed the different in the 3'UTR features of mRNA and gene expression patterns. AUUUA-motif-containing ARE was found in mRNA of EmPis-1, but not in that of EmPis-2. EmPis-1 and EmPis-2 expressed not only in the potential sites of pathogen entry, but also in grouper's immune-related tissues such as head kidney (HD), peripheral blood leukocytes (PBL) and spleen. The expression level of EmPis-1 was higher than that of EmPis-2 in most fish tissues. Expression of both EmPis-1 and EmPis-2 were upregulated by V. parahaemolyticus significantly in the PBL, HD and spleen. Besides, expression of EmPis-1 was upregulated in gills. The putative mature peptides of EmPis-1 and EmPis-2, which were predicted to adopt an amphipathic α-helical conformation, posessed excellent microbicidal activities against both gram-negative and -positive bacteria. The hemolytic activity of the putative mature peptides of EmPis-1 and EmPis-2 increased in a dose-dependent manner to both grouper erythrocytes and rabbit erythrocytes. Interestingly, grouper erythrocytes were less vulnerable than rabbit erythrocytes to the peptides. Grouper piscidins excluded the signal peptide were not the inactive precursors but possessed high microbicidal activity evidenced by minimum bactericidal concentration (MBC) assay and by the scanning electron microscope (SEM) observation. The present phylogenetic analysis did not support the suggestion that piscidins are ancient AMPs widespread across invertebrate and vertebrate taxa, and that piscidins are included in the cecropin superfamily. Collectively, the present data improve our understanding of the piscidin family, and give greater insights into EmPis-1 and EmPis-2 of the grouper immune system.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; DNA, Complementary; Escherichia coli; Escherichia coli Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Phylogeny; Protein Conformation; Protein Isoforms; Sequence Alignment; Staphylococcal Infections; Staphylococcus aureus; Tissue Distribution; Vibrio Infections; Vibrio parahaemolyticus

Giant groupers (Epinephelus lanceolatus), an important aquaculture fish in Asia, are attacked by nervous necrosis virus (NNV), belonging to betanodavirus. Environmental salinity can affect fish immunity and physiology. We examined whether decreasing salinity from 30 to 15 ppt during acclimation of groupers could affect survival with NNV infection and the associated factors. Although NNV infection decreased muscle moisture, up-regulated the gene expression of Na(+)-K(+)-2Cl(-) cotransporter isoform 2, and elevated plasma cortisol level in groupers, these factors were not related to the higher mortality of groupers reared at 30-ppt salinity (S30-groupers), compared to 15-ppt reared groupers (S15-groupers). Infected S30-groupers exhibited high leukocyte count and innate immune gene expression level. Moreover, NNV-infected dead S30-groupers showed high IL-1β gene expression level but low NNV load in the brain. The high or excess IL-1β gene expression levels in the brain of NNV-infected S30-groupers may be the factor in high mortality.

Topics: Amino Acid Sequence; Animals; Bass; Blood Chemical Analysis; Fish Diseases; Fish Proteins; Gene Expression; Immunity, Innate; Longevity; Muscles; Nodaviridae; Organ Specificity; Phylogeny; RNA Virus Infections; Salinity; Seawater; Sequence Alignment

The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, β-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.

Topics: Animals; Bass; Brain Diseases; Fish Diseases; Nodaviridae; Retinal Diseases; RNA Virus Infections; Vaccines, Inactivated; Vaccines, Synthetic; Viral Vaccines

Mitogen-activated protein kinase 7 (MKK7) is one of the major stress-activated protein kinase (SAPK)-activating kinases in response to environmental or physiological stimuli. Here a MKK7 named as Ec-MKK7 was identified from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-MKK7 was 1853 bp, with an open reading frame (ORF) of 1272 bp encoding a putative protein of 423 amino acids. A characteristic S-K-A-K-T motif was contained in the domain of dual-specificity protein kinase, mitogen-activated protein kinase kinase 7 (PKc_MKK7). Intracellular localization showed that Ec-MKK7 was localized in both the cytoplasm and the nucleus of grouper spleen (GS) and/or grouper brain (EAGB) cells. Moreover, Ec-MKK7 was universally expressed in all examined tissues and showed expression modulation to challenges of lipopolysacchride (LPS), Singapore grouper iridovirus (SGIV) and polyriboinosinic polyribocytidylic acid (poly I:C) in vivo. A gene targeting strategy over-expressing Ec-MKK7 was performed to examine the activities of MKK7 to viral infection in vitro. Our data showed that Ec-MKK7 was involved in the evasion and replication of SGIV but played an antiviral role to the infection of nervous necrosis virus (NNV). All results demonstrated that Ec-MKK7 could play important roles in grouper innate immunity and show distinct functions on virus infection.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; MAP Kinase Kinase 7; Nodaviridae; Organ Specificity; Poly I-C; Ranavirus; RNA Virus Infections; RNA, Messenger; Sequence Alignment

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Interferon Type I; Iridoviridae; Lipopolysaccharides; Poly I-C; Ranavirus; Sequence Analysis, DNA; Specific Pathogen-Free Organisms

The myxosporean parasite Ceratomyxa diplodae Lubat et al. 1989 sensu Sitjà-Bobadilla & Álvarez-Pellitero, 1993, originally described from the annular seabream Diplodus annularis in the Adriatic Sea, has subsequently been reported from several other sparid hosts, and also a moronid fish, the European seabass Dicentrarchus labrax from the Mediterranean Sea. Here, molecular identity and additional morphological data are given for this parasite infecting the gall bladder of D. labrax in a southern Portuguese fish farm. In the bile, disporic plasmodia were spherical to subspherical with a smooth surface membrane. Most myxospores were crescent-shaped, 5.1 ± 0.5 (4.8-6.7) µm long (mean ± SD) and 21.9 ± 1.0 (20.4-23.9) µm thick; a few were more arcuate, 5.7 ± 0.4 (5.3-6.3) µm long and 17.3 ± 1.0 (16.3-19.1) µm thick. The wall consisted of 2 symmetrical valves united along a slightly curved suture line, with moderately tapering to rounded ends. Two spherical polar capsules, measuring 2.9 ± 0.3 (2.5-3.4) µm in diameter, contained a polar filament forming 8 to 9 coils organized in 2 rows. Host species, tissue tropism, and myxospore morphology supported species identification. Phylogenetic analyses of the small subunit ribosomal RNA sequence positioned the parasite among most sparid-infecting Ceratomyxa spp., suggesting the existence of a common ancestor for these species. The acquisition of molecular data from infections of C. diplodae in its original host and in other sparids is essential in order to ascertain if the morphological and biological variations found among reports of this parasite are intra- or inter-specific.

Topics: Animals; Bass; DNA; Fish Diseases; Myxozoa; Parasitic Diseases, Animal; Phylogeny

A total of 169 specimens of the orange-spotted grouper Epinephelus coioides (Hamilton) were collected from fishermen and marine fish farms in the Gulf of Tonkin, Vietnam. Five different species of Prosorhynchus Odhner, 1905 were recorded, including P. tonkinensis n. sp. The new species differs from all other Prosorhynchus species in the presence of an indented posterior extremity. It can be distinguished from the most closely related P. atlanticus Manter, 1940 and P. crucibulum Rudolphi, 1819 by the extension of the uterus always to the level of the ovary, the width and premouth distance in the former, and the arched vitellarium and smaller egg size in the latter, and a different host and geographical region. Prosorhynchus sp. A (not fully identified in this study) has been earlier reported from E. coioides from New Caledonia (see Prosorhynchus sp. B of Bray and Justine, 2013), P. luzonicus Velasquez, 1959 is reported throughout South-East Asia, and Prosorhynchus sp. B (no further identification possible based on a single specimen in this study) and P. maternus Bray & Justine, 2006 are reported for the first time from Vietnam. The present study demonstrates a close relationship of the Prosorhynchus species composition in Vietnam with the Indo-Australian region, warranting further comparative studies among the different epinephelids.

Topics: Animals; Bass; Female; Fish Diseases; Male; Species Specificity; Trematoda; Trematode Infections; Vietnam

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA, Complementary; Fish Diseases; Gene Expression Regulation; Humans; Interleukin-1beta; Kidney; Mice; Nocardia; Nocardia Infections; Phylogeny; Protein Structure, Secondary; Rats; Sequence Alignment; Spleen; Transcriptome; Tumor Necrosis Factor-alpha

Nervous necrosis virus (NNV) belongs to genus Betanodavirus (family Nodaviridae). It is highly pathogenic to various marine fishes. In the present study, cultured NNV suspension was placed in dialysis tube at molecular weight cut off (MWCO) of 10

Topics: Animals; Anions; Bass; Cell Line; Chromatography; Dialysis; Fish Diseases; Nodaviridae; RNA Virus Infections

PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Lipopolysaccharides; Phylogeny; PTEN Phosphohydrolase; RNA, Messenger; Sequence Alignment; Tissue Distribution; Vibrio alginolyticus; Vibrio Infections

Heat shock transcription factor 1 (HSF1) regulates heat shock proteins (HSPs), which assist in protein folding and inhibit protein denaturation following stress. HSF1 was firstly cloned from orange-spotted grouper and exists as two isoforms, one with (osgHSF1a) and one without (osgHSF1b) exon 11. Heat exposure increased the expression of osgHSF1b while cold exposure increased that of osgHSF1a. Both isoforms were mainly expressed in the brains, eyes, and fins. Expression of osgHSF1b was higher than osgHSF1a during development. Poly I:C and LPS could also induce osgHSF1 isoforms expression differentially. Exposure to nervous necrosis virus (NNV) increased the level of both osgHSF1 isoforms at 12 h. GF-1 cells with overexpression of osgHSF1 isoforms enhanced viral loads within 24 h, whereas both pharmacological inhibition and RNA interference of HSF1 reduced virus infection. This study shows that osgHSF1 can support the early stage of virus infection and provides a new insight into the molecular regulation of osgHSF1 between the influence of temperatures and immunity.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA-Binding Proteins; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Heat Shock Transcription Factors; Hot Temperature; Immunity; Lipopolysaccharides; Nodaviridae; Phylogeny; Poly I-C; Protein Isoforms; RNA Virus Infections; Sequence Alignment; Transcription Factors

Tripartite motif 16 (TRIM16), has been demonstrated to act as a tumor suppressor through affecting cell proliferation and migration or tumorigenicity in carcigenesis. However, the roles of TRIM16 in immune response were unknown up to now. Here, we cloned a TRIM16-like gene (TRIM16L) from orange spotted grouper (EcTRIM16L) and investigated its roles in response to virus infection. EcTRIM16L encoded a 478 amino acid peptide which showed 72% and 29% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Sequence alignments indicated that EcTRIM16L shared the different gene structures with human TRIM16, evidenced by the presence of RING domain, but absence of the B-box domain. In transfected grouper cells, the green fluorescence mainly distributed in cytoplasm, and the deletion of SPRY domain affected the accurate localization of EcTRIM16L. In response to different stimuli, including infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis (RGNNV), and transfection with b-DNA or poly I:C, the transcript of EcTRIM16L were differently regulated in grouper spleen cells. After incubation with SGIV, the ectopic expression of EcTRIM16L significantly enhanced the viral replication, demonstrated by the increase of cytopathic effect (CPE) severity and viral gene transcriptions. Simultaneously, we also found that overexpression of EcTIRM16L in vitro significantly weakened the expression of interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, and melanoma differentiation-associated protein 5 (MDA5). Moreover, the ectopic expression of EcTRIM16L significantly decreased both MDA5-and mediator of IRF3 activation (MITA)-induced interferon immune responses. Further studies showed that the RING domain played more important roles in the molecular action of EcTIRM16L during grouper virus infection. Our data, for the first time, demonstrated that fish TRIM16L exerted negative regulation on the interferon immune response against DNA virus infection.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Immunity, Innate; Phylogeny; Poly I-C; Ranavirus; RNA, Messenger; Sequence Alignment; Tripartite Motif Proteins

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the "nervous system development" as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity.

Topics: Animals; Bass; Chromosome Mapping; Fish Diseases; Gene Expression Profiling; Genetic Linkage; Genome-Wide Association Study; Genomics; Microsatellite Repeats; Polymorphism, Single Nucleotide; Prognathism; Quantitative Trait Loci; Transcriptome

The mechanisms through which brown-marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown-marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two-dimensional gel electrophoresis (2-DE). The results showed that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.

Topics: alpha-Macroglobulins; Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunoglobulin Light Chains; Lectins, C-Type; Parvalbumins; Vibrio Infections; Vibrio parahaemolyticus

Topics: Air Sacs; Animals; Bass; Exophiala; Fish Diseases; Phaeohyphomycosis; Queensland

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.

Topics: Animals; Bass; China; DNA Virus Infections; Fish Diseases; Genes, Viral; Iridovirus; Microscopy, Electron, Transmission; Phylogeny

Topics: Animals; Bass; Fish Diseases; Signal Transduction; Spleen; Transcriptome; Vibrio Infections; Vibrio parahaemolyticus

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Topics: Animals; Bass; Fish Diseases; Polymerase Chain Reaction; Streptococcal Infections; Streptococcus agalactiae

The F-lectin, a fucose-binding protein found from invertebrates to ectothermic vertebrates, is the last lectin family to be discovered. Here, we describe effects of two different types of stressors, bacterial infection and confinement stress, on the modulation of European sea bass Dicentrarchus labrax (L.) F-lectin (DlFBL), a well-characterized serum opsonin, using a specific antibody. The infection of the Vibrio alginolyticus bacterial strain increased the total haemagglutinating activity during the 16-day testing period. The DlFBL value showed an upward regulation on the first, second and last days and underwent a slight downward regulation 4 days post-challenge. In contrast, the effect of confinement and density stress showed a decrease in the plasma concentration of lectin, ranging from 50% to 60% compared with the control. The modulation of DlFBL is in line with the hypothesis that humoral lectins could be involved and recruited in the initial recognition step of the inflammation, which leads to agglutination, and the activation of mechanisms responsible for killing of the pathogens.

Topics: Animals; Bass; Fish Diseases; Lectins; Stress, Physiological

Six new species of Leptorhynchoides from the southeastern United States are described. These new species were once part of the Leptorhynchoides thecatus complex of species that was previously recognized on the basis of DNA sequence data. Multivariate morphometric analysis including discriminant function analysis and decision tree analysis indicated that each of the species is morphologically distinct. Both analyses classified more than 90% of specimens correctly and most misclassifications occurred between members of 2 pairs of species that are morphologically similar. The most discriminating continuous characters were: trunk length, number of longitudinal rows of hooks, length of the longest hook, and testes width. Hook asymmetry and missing hooks on the proboscis were also important taxonomic characters. The discriminant function and the decision tree generated from the data were used to classify new specimens, yielding a 96% and 84% correct classification rate, respectively. The new taxonomic designations account for much of the previously recognized variability in host use, habitat use, and development as determined by survey data. With the addition of these 6 new taxa, 10 species currently are recognized within the genus.

Topics: Acanthocephala; Animals; Bass; Decision Trees; Discriminant Analysis; Female; Fish Diseases; Fresh Water; Helminthiasis, Animal; Male; North America

In this study, we investigated the impact of the host factors mucin, bile salts and cholesterol on the virulence of the economically important aquatic pathogen Vibrio anguillarum towards sea bass larvae. Pretreatment of V. anguillarum with either one of the host factors (at 10 mg l(-1)) prior to inoculation into the sea bass rearing water increased virulence of the bacterium, although the effect of cholesterol was not significant. Each of the three host factors significantly increased several virulence-related phenotypes in V. anguillarum, i.e. protease activity, flagellar motility, biofilm formation and exopolysaccharide production, whereas there was no effect on growth of the bacterium under these conditions. Furthermore, the host factors increased the expression of genes involved in these phenotypes, i.e. the metalloprotease empA, the flagellar transcriptional regulator fleQ, the flagellin gene flaA, the chemotaxis methyltransferase gene cheR, the exopolysaccharide biosynthesis gene wbfD and the exopolysaccharide export gene wza. Our results indicate that V. anguillarum uses host mucin, bile salts, and cholesterol as cues to promote the expression of several important virulence traits that enhance the success of transmission from one host to another.

Topics: Animals; Bass; Bile Acids and Salts; Cholesterol; Fish Diseases; Germ-Free Life; Larva; Mucins; Phenotype; Vibrio; Vibrio Infections; Virulence

Since European sea bass (Dicentrarchus labrax) larvae occurred in coastal and estuarine waters at early life stages, they are likely to be exposed to reduced dissolved oxygen waters at a sensitive developmental stage. However, the effects of hypoxia at larval stage, which depend in part on fish species, remain very poorly documented in European sea bass. In the present study, the impacts of an experimental exposure to a chronic moderate hypoxia (40 % air saturation) between 30 and 38 days post-hatching on the physiological and developmental traits of European sea bass larvae were assessed. This study was based on the investigation of survival and growth rates, parameters related to energy metabolism [Citrate Synthase (CS) and Cytochrome-c Oxidase (COX) activities], and biological indicators of the maturation of digestive function [pancreatic (trypsin, amylase) and intestinal (Alkaline Phosphatase "AP" and Aminopeptidase-N "N-LAP") enzymes activities]. While condition of hypoxia exposure did not induce any significant mortality event, lower growth rate as well as CS/COX activity ratio was observed in the Hypoxia Treatment group. In parallel, intestinal enzyme activities were also lower under hypoxia. Altogether, the present data suggest that sea bass larvae cope with moderate hypoxia by (1) reducing processes that are costly in energy and (2) regulating mitochondria functions in order to respond to energy-demand conditions. Both these effects are associated with a delay in the maturation of the digestive function.

Topics: Alkaline Phosphatase; Aminopeptidases; Amylases; Analysis of Variance; Animals; Bass; Electron Transport Complex IV; Energy Metabolism; Fish Diseases; Hypoxia; Larva; Mitochondria; Survival Analysis; Trypsin

Antimicrobial polypeptides (AMPPs) are humoral components of the vertebrates and invertebrates innate immune system. Their potent broad spectrum antimicrobial activities have drawn the attention of the scientific community to their potential use not only as an alternative to antibiotics but also as functional targets for immunostimulants in order to enhance the host immunity. Fish synthesize a great number of these peptides but in European sea bass, an important fish species in the Mediterranean aquaculture, only a few AMPPs have been studied and these surveys have highlighted their functional role as predictive markers of stressful conditions. Many aspects concerning AMPP mode of action in the host during bacterial infections are still unknown. In this work a 72 h time course experiment, performed on juvenile sea bass intraperitoneally (i.p.) injected with a sub-lethal dose of Vibrio anguillarum, was aimed to investigate the mRNA expression of four specific AMPP genes and interleukin-1β (IL-1β) in skin, gills, spleen, and head kidney. AMPP genes were: dicentracin (DIC), histone-like protein 1 (HLP-1), histone-like protein 2 (HLP-2) and hemoglobin-like protein (Hb-LP). The delta-delta C(T) method in real-time RT-PCR allowed to gain more knowledge about temporal dynamics, preferential sites of expression as well as immunological and physiological role of these molecular markers. DIC was significantly up-regulated mainly in head kidney at 1.5-3 h post-infection (p.i.). HLP-1 showed an extended-time overexpression in gills and a significant up-regulation in spleen. HLP-2 was interestingly overexpressed in gills at 24 h p.i., while Hb-LP showed a significant up-regulation in skin for all the 72 h trial as well as lower but always significant values either in gills or in spleen. Different was the response of IL-1β that showed a dramatic up-regulation in spleen and head kidney at 8 h p.i. whilst in gills it displayed a severe inhibition. During this survey the i.p. stimulus surely conditioned the AMPP expression in skin and gills, especially as regards the DIC that as piscidin-related gene has an important defensive role in the mucosal tissues. However, two unconventional AMPP genes such as HLP-2 and Hb-LP, strictly related to the physiological mechanisms of fish, were less affected in terms of expression by the route of infection, being more evident in peripheral loci. These findings might suggest them as potential markers to be analyzed within plans of health

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Benzothiazoles; Diamines; Fish Diseases; Fish Proteins; Gene Expression Regulation; Organ Specificity; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Vibrio; Vibrio Infections

MCSF and its well-known receptor MCSFR had been well studied in humans, regulating the differentiation, proliferation, and survival of the mononuclear phagocyte system. IL-34, which is an alternative ligand of MCSF receptor, was recently identified as a novel cytokine and functionally overlaps with MCSF. However, the functional study of these receptors and their ligands in fish are largely unknown. In the present study, the cDNA of two potential grouper MCSFR ligands have been cloned, EcIL-34 (657 bp) and EcMCSF2 (804 bp), as well as an additional copy of grouper MCSFR, EcMCSFR2 (3141 bp). Sequence analysis showed that these three molecules had higher identities with other fish counterparts compared to mammals and their conserved structures and important functional residues were also analyzed. Tissue distribution analysis showed that EcIL-34 is dominant in brain, gill and spleen compared to EcMCSF2, which is dominant in head kidney, trunk kidney, skin, heart and muscle. EcMCSFR1 was dominant in the most tissues except head kidney and liver compared to EcMCSFR2. The different tissue distribution patterns of these two grouper MCSF receptors and their two ligands indicate the different mononuclear phagocyte differentiation and activation modes in different tissues. In Cryptocaryon irritans infected grouper, EcIL-34 and EcMCSFR2 were the most strongly up-regulated ligand and receptor in the infected sites, gill and skin. Their up-regulation confirmed the proliferation and activation of phagocytes in C. irritans infected sites, which would improve the antigen presentation and elicit the host local specific immune response. In C. irritans infected grouper head kidney, both ligands EcIL-34 and EcMCSF2 (especially EcMCSF2) were up-regulated, but both receptors EcMCSFR1 and EcMCSFR2 were down-regulated, which indicated that the phagocytes differentiation and proliferation may have occurred in this hemopoietic organ, and after that they migrated to the infected cites. The down-regulation of EcIL-34 and EcMCSF2 and no significant change of EcMCSFR1 and EcMCSFR2 in most time point of grouper spleen showed it was less involved in phagocytes response to C. irritans infection.

Topics: Amino Acid Sequence; Animals; Bass; Ciliophora; Ciliophora Infections; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Interleukins; Macrophage Colony-Stimulating Factor; Organ Specificity; Phagocytes; Phylogeny; Receptor, Macrophage Colony-Stimulating Factor; RNA, Messenger; Sequence Alignment

The nuclear phosphoprotein c-Jun is a member of the AP1 family of transcription activating complex, can be induced by various extracellular stimuli such as virus infection. In this study, the c-Jun gene (Ec-c-Jun) was cloned from orange-spotted grouper, Epinephelus coioides. The full-length Ec-c-Jun cDNA is composed of 2046 bp and encodes a polypeptide of 328 amino acids with 81% identity of zebrafish. Amino acid alignment analysis indicated that Ec-c-Jun contained three conserved domains including a transactivation domain (TAD), a DNA-binding domain (DBD) and leucine zipper domain (LZD). RT-PCR results showed that Ec-c-Jun transcript was most abundant in spleen, kidney, heart and gill. The expression of Ec-c-Jun was up-regulated after challenged with Singapore grouper iridovirus (SGIV). To investigate the roles of Ec-c-Jun during SGIV infection, we constructed its dominant-negative mutant (DN-Ec-c-Jun) by deleting the major TAD that lacks amino acids 3-122. Fluorescence microscopy observation revealed that Ec-c-Jun and DN-Ec-c-Jun were expressed predominantly in the nucleus in transfected cells. Interestingly, the green fluorescence of Ec-c-Jun was congregated and co-localized with virus assembly sites at the late stage of SGIV infection. However, in DN-Ec-c-Jun transfected cells, no virus assembly sites were observed, and the distribution of fluorescence remained unchanged. Moreover, overexpression of DN-Ec-c-Jun in vitro delayed the occurrence of CPE induced by SGIV infection and inhibited the virus gene transcription. In addition, ectopic expression of DN-Ec-c-Jun was able to inhibit SGIV induced c-Jun/AP1 promoter activity in GS cells. Thus, we proposed that c-Jun transcription factor was essential for SGIV replication in vitro. Our results will contribute to understanding the crucial roles of JNK signaling pathway in fish virus infection.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Molecular Sequence Data; Phylogeny; Proto-Oncogene Proteins c-jun

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is an important signaling pathway activated by interferons in response to virus infection. Fish STAT3 has been demonstrated to be involved in Singapore grouper iridovirus (SGIV) infection and virus induced paraptosis, but its effects on the replication of other fish viruses still remained uncertain. Here, the roles of grouper STAT3 (Ec-STAT3) in red spotted grouper nervous necrosis virus (RGNNV) infection were investigated. The present data showed that the distribution of phosphorylated Ec-STAT3 was altered in RGNNV infected fish cells, and the promoter activity of STAT3 was significantly increased during virus infection, suggesting that STAT3 activation was involved in RGNNV infection. Using STAT3 specific inhibitor, we found that inhibition of Ec-STAT3 in vitro did not affect the transcription and protein synthesis of RGNNV coat protein (CP), however, the severity of RGNNV induced vacuolation and autophagy was significantly increased. Meanwhile, at the late stage of virus infection, RGNNV induced necrotic cell death was significantly decreased after inhibition of Ec-STAT3. Further studies indicated that Ec-STAT3 inhibition significantly increased the transcript level of autophagy related genes, including UNC-51-like kinase 2 (ULK2) and microtubule-associated protein 1 light chain 3-II (LC3-II) induced by RGNNV infection. Moreover, the expression of several pro-inflammatory factors, including TNFα, IL-1β and IL-8 were mediated by Ec-STAT3 during RGNNV infection. Together, our results not only firstly revealed that STAT3 exerted novel roles in response to fish virus infection, but also provided new insights into understanding the roles of STAT3 in different forms of programmed cell death.

Topics: Animals; Bass; Cell Death; Cells, Cultured; Fish Diseases; Fish Proteins; Nodaviridae; RNA Virus Infections; STAT3 Transcription Factor

Complement component C8 beta was a key molecule in the complement system, mediating the MAC formation and the bacterial lysis. In this study, the full-length C8 beta (EcC8 beta) was obtained, containing a 5'UTR of 25 bp, an ORF of 1764 bp and a 3'UTR of 198 bp. The EcC8 beta gene encoded a protein of 587 amino acids with an estimated molecular mass of 65.87 kDa and a predicted isoelectric point (pI) of 6.35. The deduced amino acid sequence showed that EcC8 beta consisted of the conserved residues and the domains known to be critical for C8 beta function. The quantitative real-time PCR analysis revealed that EcC8 beta transcript was expressed in all the examined tissues, while the strong expression was observed in the liver. In addition, complement C3 was the central molecule in the complement system, converging the upstream complement signals and mediating the MAC assembly pathway, while C8 beta was indispensable for active MAC formation. Following the Vibrio challenge, the increased expression of EcC3 transcript and EcC8 beta transcript was observed in the liver and kidney. These results indicated that EcC8 beta may be an important immune-related gene, playing an important role in the immune defense against the bacterial infection via the complement pathway.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; Complement C8; Fish Diseases; Gene Expression Profiling; Molecular Sequence Data; Phylogeny; Sequence Homology; Vibrio alginolyticus; Vibrio Infections

PPAR gamma was a key nuclear receptor, playing an important role in the immune defense and the anti-inflammatory mechanism. In this study, the full-length PPAR gamma (EcPPAR gamma) was obtained, containing a 5'UTR of 133 bp, an ORF of 1602 bp and a 3'UTR of 26 bp besides the poly (A) tail. The EcPPAR gamma gene encoded a protein of 533 amino acids with an estimated molecular mass of 60.02 KDa and a predicted isoelectric point (pI) of 6.26. The deduced amino acid sequence showed that EcPPAR gamma consisted of the conserved residues and the domains known to be critical for the PPAR gamma function. The quantitative real-time PCR analysis revealed that EcPPAR gamma transcript was expressed in all the examined tissue, while the strong expression was observed in intestine, followed by the expression in liver, gill, spleen heart, kidney and muscle. Vibrio challenge could stimulate the inflammatory response in grouper and induce a sharp increase of pro-inflammatory cytokines expression, lipid peroxidation and DNA damage, while the up-regulation of vibrio-induced inflammation could also increase the non-specific immune defense. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPPAR gamma transcript in immune tissues. Subcellular localization analysis revealed that EcPPAR gamma was distributed in the nucleus. Furthermore, overexpression of EcPPAR gamma could down-regulated the expression of IL1b, IL6, TNF1 and TNF2. In addition, the administration of PPAR gamma antagonist, GW9662, could up-regulate the expression of pro-inflammatory genes, including IL1b, IL6, TNF1 and TNF2. Together, these results indicated that EcPPAR gamma serving as a negative regulator of pro-inflammatory cytokines may play an important role in the immune defense against vibrio-induced inflammation in grouper.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cell Line; Cloning, Molecular; Cytokines; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Hydroxyl Radical; Molecular Sequence Data; Organ Specificity; Phylogeny; PPAR gamma; Sequence Alignment; Superoxide Dismutase; Vibrio alginolyticus; Vibrio Infections

Apolipoproteins, synthesized mainly in liver and intestine and bounded to lipids, play important roles in lipid transport and uptake through the circulation system. In this study, an apolipoprotein A-I gene homologue was cloned from orange-spotted grouper Epinephelus coioides (designed as Ec-ApoA-I) by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-ApoA-I was comprised of 1278 bp with a 792 bp open reading frame (ORF) that encodes a putative protein of 264 amino acids. Quantitative real-time PCR (qPCR) analysis revealed that Ec-ApoA-I was abundant in liver and intestine, and the expression in liver was significantly (P < 0.01) up-regulated after the stimulation of LPS, Poly(I:C), Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). Recombinant Ec-ApoA-I (rEc-ApoA-I) was produced in Escherichia coli BL21 (DE3) expression system exhibited bacteriolyticactivity against Microcococcus lysodeikticus and Aeromonas hydrophila. Intracellular localization revealed that Ec-ApoA-I distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. Overexpression of Ec-ApoA-I in grouper Brain (GB) cells could inhibit the replication of SGIV. These results together indicated that Ec-ApoA-I perhaps is involved in the responses to bacterial and viral challenge.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antiviral Agents; Apolipoprotein A-I; Bacteria; Base Sequence; Bass; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Iridovirus; Lipopolysaccharides; Mice; Molecular Sequence Data; Organ Specificity; Phylogeny; Poly I-C; Recombinant Proteins

Pseudorhabdosynochus regius n. sp. is described from the gills of the mottled grouper Mycteroperca rubra caught off Senegal, Tunisia and Libya (type-locality: off Dakar, Senegal). The species is distinguished from its congeners by the structure of its sclerotised vagina (length 26-35 μm), which exhibits a trumpet in continuity with the primary canal, a straight primary canal, and primary and secondary chambers included in a common sclerotised mass along the primary canal. The species is also characterised by small squamodiscs (length 20-40 μm) with 10-11 rows of rodlets. Its closest relatives (based on the structure of the sclerotised vagina) are species mostly found in the Mediterranean Sea and parasites on species of Mycteroperca. A second species of Pseudorhabdosynochus Yamaguti, 1958 is reported from the same host and localities but not described. A list of diplectanids from groupers in the Mediterranean Sea is provided. We point out that a recent article was not compliant with the new Article 8.5.3 of the International Code of Zoological Nomenclature; for this reason, three species, P. nhatrangensis Dang, Bristow, Schander & Berland, 2013, P. vietnamensis Dang et al., 2013 and P. brunei Dang et al., 2013, are invalid.

Topics: Animals; Atlantic Ocean; Bass; Cestode Infections; Diagnosis, Differential; Female; Fish Diseases; Helminthiasis, Animal; Male; Mediterranean Sea; Platyhelminths; Species Specificity; Terminology as Topic; Vagina

Topics: Animals; Anti-Bacterial Agents; Bass; Cichlids; Drug Resistance, Bacterial; Fish Diseases; Fisheries; Malaysia; Streptococcal Infections; Streptococcus agalactiae; Tilapia

Antimicrobial peptides (AMPs) have a crucial role in the fish innate immune response, being considered a fundamental component of the first line of defence against pathogens. Moreover, AMPs have not been studied in the fish gonad since this is used by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with nodavirus (VNNV), which shows vertical transmission through the gonad and/or gonadal fluids, but no study has looked into the gonad of infected fish. In this framework, we have characterized the antimicrobial response triggered by VNNV in the testis of European sea bass, a very susceptible species of the virus, and in the gilthead seabream, which acts as a reservoir, both in vivo and in vitro, and compared with that present in the serum and brain (target tissue of VNNV). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and bactericidal) and the expression of AMP genes such as complement factor 3 (c3), lysozyme (lyz), hepcidin (hamp), dicentracin (dic), piscidin (pis) or β-defensin (bdef) in the gonad of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon VNNV infection, and even differs to the brain response. Furthermore, the in vitro data suggest that some AMPs are locally regulated playing a local immune response in the gonad, while others are more dependent of the systemic immune system. Data are discussed in the light to ascertain their potential role in viral clearance by the gonad to avoid vertical transmission.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Brain; Complement C3; Fish Diseases; Fish Proteins; Gene Expression; Immunity, Innate; Male; Muramidase; Nodaviridae; RNA Virus Infections; Sea Bream; Testis; Vibrio

Pseudorhabdosynochus jeanloui n. sp. (Monogenoidea, Diplectanidae) is described from specimens collected from the gills of the Pacific creolefish, Paranthias colonus (Perciformes, Serranidae) from a fish market in Chorrillos, Lima, Peru. The new species is differentiated from other members of the genus by the structure of its sclerotized vagina, which has two spherical chambers of similar diameter. This is the first Pseudorhabdosynochus species described from the Pacific coast of America, the third species of the genus reported from South America and the first described from a member of Paranthias.

Topics: Animals; Bass; Cestode Infections; Female; Fish Diseases; Gills; Helminthiasis, Animal; Male; Pacific Ocean; Peru; Platyhelminths; Vagina

Infection with nervous necrosis virus (NNV) causes viral nervous necrosis, which inflicts serious economic losses in marine fish cultivation. Virus-like particles (VLPs) are protein complexes consisting of recombinant virus capsid proteins, whose shapes are similar to native virions. VLPs are considered a novel vaccine platform because they are not infectious and have the ability to induce neutralizing antibodies efficiently. However, there have been few studies of protective immune responses employing virus challenge following immunization with NNV VLPs, and this is important for evaluating the utility of the vaccine. In the present study, we produced red-spotted grouper (Epinephelus akaara) NNV (RGNNV) VLPs in Saccharomyces cerevisiae and investigated protective immune responses in convict grouper (Epinephelus septemfasciatus) following intraperitoneal injection and oral immunization with the RGNNV VLPs. The parenterally administered VLPs elicited neutralizing antibody with high efficacy, and provided the fish with full protection against RGNNV challenge: 100% of the immunized fish survived compared with only 37% of the control fish receiving phosphate-buffered saline. RGNNV VLPs administered orally provoked neutralizing antibody systemically and conferred protective immunity against virus challenge: however only 57% of the fish survived. Our results demonstrate that RGNNV VLP produced in yeast has great potential as vaccine in fish.

Topics: Animals; Antibodies, Neutralizing; Bass; Capsid Proteins; Fish Diseases; Nodaviridae; RNA Virus Infections; Saccharomyces cerevisiae; Vaccination; Vaccines, Virus-Like Particle

The susceptibility of juvenile European sea bass and Senegalese sole to three VNNV isolates (a reassortant RGNNV/SJNNV, as well as the parental RGNNV and SJNNV genotypes) has been evaluated by challenges using two inoculation ways (bath and intramuscular injection). The results demonstrate that these two fish species are susceptible to all the VNNV isolates tested. In European sea bass, RGNNV caused the highest cumulative mortality, reaching maximum values of viral RNA and titres. Although the SJNNV isolate did not provoke mortality or clinical signs of disease in this fish species, viral production in survivor fish was determined; on the other hand the reassortant isolate did cause mortality and clinical signs of disease, although less evident than those recorded after RGNNV infection. These results suggest that the changes suffered by the SJNNV RNA2 segment of the reassortant isolate, compared to the parental SJNNV, may have involved host-specificity and/or virulence determinants for European sea bass. Regarding Senegalese sole, although the three isolates caused 100% mortality, the reassortant strain provoked the most acute symptoms, and more quickly, especially in the bath challenge. This was also the isolate showing less difference between the number of RNA copies and viral titre, reaching the highest titres of infective viral particles in nervous tissue of infected animals. The RGNNV isolate produced the lowest values of infective viral particles. All these results suggest that the RGNNV and the reassortant isolates are the most suited for infecting European sea bass and Senegalese sole, respectively.

Topics: Animals; Bass; Disease Susceptibility; Fish Diseases; Flatfishes; Genotype; Nodaviridae; RNA Virus Infections; RNA, Viral; Viral Load

Lateral flow paper biosensors are an attractive analytical platform for detection of human and veterinary disease pathogens because they are optimal for accurate, rapid and sensitive analysis in research laboratory setups, as well as field analysis. Since diseases of viral etiology have been wreaking havoc in aquaculture industry, as well as the environment, the present study aims at the development of a gold nanoparticle-based biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification. The PCR products were mixed with a specific oligonucleotide probe and applied next to oligonucleotide conjugated Au NPs. A red test line was formed when nodavirus product was present. The visual detection of the RT-PCR product was completed within 20 min. Following optimization, the biosensor was able to visually detect 270 pg of nodavirus initial total RNA. The present study describes a simple, accurate, robust and low cost method for nodavirus detection in biological samples. Apart contribution on basic research, the proposed biosensor offers great potential for commercial kit development for use on the site of fish culture by fish farmers. This fact will have great impact on environmental safety and disease monitoring without time consuming and costly procedures.

Topics: Animals; Bass; Biosensing Techniques; Fish Diseases; Gold; Nanoparticles; Nodaviridae; Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sensitivity and Specificity

In this study, we investigated the responsiveness of 15 Vibrio anguillarum strains to three host factors (mucin, bile salts and cholesterol).. Three virulence-related phenotypes were investigated in this respect, i.e. motility, biofilm formation and exopolysaccharide production. Almost all V. anguillarum strains showed a significantly increased motility in the presence of either of the three host factors. Only five of the strains showed increased biofilm formation in the presence of host factors and only three strains showed increased exopolysaccharide production in the presence of the host factors.. There were no significant correlations between the three putatively virulence-linked phenotypes (in the absence of host factors) and virulence to sea bass larvae. There was no correlation between responsiveness to the host factors (percentage increase in motility, biofilm formation or exopolysaccharide production in the presence of the three host factors) and virulence to sea bass larvae. However, the responses of these virulence-related phenotypes upon the addition of either of the three host factors were significantly correlated with each other. This result suggests that the mechanisms by which V. anguillarum responds to these three host factors is linked.. Although the mechanism by which V. anguillarum responds to the host factors mucin, bile salts and cholesterol seems to be linked, there is no correlation between host factor responsiveness and virulence towards sea bass larvae. This emphasizes that one should be careful when extrapolating results obtained for one particular strain to reach general conclusions on a species of pathogenic bacteria.

Topics: Animals; Bass; Bile Acids and Salts; Cholesterol; Fish Diseases; Host-Pathogen Interactions; Larva; Mucins; Phenotype; Vibrio; Vibrio Infections; Virulence

Effective parasite management can be achieved through strategically timed treatments that break the life cycle. We examined the effects of temperature (2 °C increments from 22 to 34 °C) and salinity (0, 11, 22, 35, 40‰) on the life cycle (embryonation period, hatching success, oncomiracidia (larvae) longevity, infection success, and time to sexual maturity) of Neobenedenia sp. (Monogenea: Capsalidae), a harmful ectoparasite of farmed marine fishes. Experiments were conducted in controlled conditions in the laboratory. The life cycle was faster in warm, high saline conditions compared to cooler conditions (10-13 days between 26-32 °C, 40‰; 15-16 days between 22-24 °C at 40‰). Warm seawater and high saline conditions (24-32 °C, 35-40‰) improved egg hatching success, reduced time to sexual maturity, and resulted in parasites reaching sexual maturity at a larger size (at 30-32 °C) compared to cooler conditions (22 °C). In contrast, cool, hypersaline conditions (22 °C, 40‰) increased oncomiracidia longevity and infection success. Linear and quantile regression models were used to construct an interactive, online parasite management interface to enable strategic treatment of parasites in aquaculture corresponding to observed temperature and salinity variation on farms in the tropics. It was recommended that farmers treat their stock more frequently during summer (27-31 °C) when parasites can complete their life cycle more quickly. Nevertheless, farmers should be aware of the potential for increased Neobenedenia sp. infections during winter months (21-26 °C) due to increased infection success.

Topics: Animals; Aquaculture; Bass; Cestode Infections; Female; Fish Diseases; Larva; Life Cycle Stages; Ovum; Platyhelminths; Salinity; Seasons; Seawater; Sodium Chloride; Temperature

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFκB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny--an aspect that is worth investigating in aquatic animals.

Topics: Animals; Bass; Brain; Fish Diseases; Fish Proteins; Gonads; Immunity, Innate; Infectious Disease Transmission, Vertical; Interferon Regulatory Factor-3; Interferons; Nodaviridae; RNA Virus Infections; Sea Bream; Signal Transduction

Vibrio alginolyticus containing the highly toxic extracellular product is one of the most serious threats to grouper survival and its minimum lethal dose is approximately 500 CFU/g fish body weight in grouper. To study the toxic effects of V. alginolyticus on the immune system in teleost, Calmodulin (CaM), an important molecular indicator gene, was cloned from the orange-spotted grouper (Epinephelus coioides). The full-length Ec-CaM consisted of a 5'-UTR of 103 bp, an ORF of 450 bp and a 3'-UTR of 104 bp. The Ec-CaM gene encoded a protein of 149 amino acids with an estimated molecular mass of 16.4 kDa and a predicted isoelectric point of 3.93. The deduced amino acid sequence showed that Ec-CaM contained four highly conserved EF-hand domains known to be critical for the function of CaM. Ec-CaM was widely expressed and the highest expression level was observed in liver. Following V. alginolyticus challenge, a sharp increase level of respiratory burst activity and apoptosis ratio were observed. Further analyses of CaM expression and p53 expression in liver, kidney and spleen by qRT-PCR demonstrated that the up-regulated expression of CaM and p53 were observed in the vibrio challenge group. Western blotting analysis confirmed that the Ec-CaM protein was strongly induced in liver at 12 h post-injection, while a sharp increase of p53 protein expression was observed at 24 h post-injection. These results showed CaM expression serving as a potential molecular indicator may help to assess the toxicological effects of V. alginolyticus on the ROS generation and apoptotic process in grouper.

Topics: Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Bass; Calmodulin; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Molecular Sequence Data; Organ Specificity; Respiratory Burst; RNA, Messenger; Sequence Alignment; Tumor Suppressor Protein p53; Vibrio alginolyticus; Vibrio Infections

A survey of largemouth (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu) parasite communities in Oneida Lake, New York, was conducted in the summer of 2012 and compared to an earlier survey conducted by Van Cleave and Mueller during the summers of 1929 to 1931. The component helminth communities between surveys were 31% similar in composition for largemouth and 28% similar for smallmouth bass. Between species, the component helminth communities were considerably more similar in the present survey (71%) than in the survey conducted by Van Cleave and Mueller (47%). Seven species reported by Van Cleave and Mueller were present in this survey and 21 species are new records for the bass of Oneida Lake. Van Cleave and Mueller did not report prevalence values for several taxa (Monogenea, Copepoda, Myxozoa, and a Trichodina sp.) that were important for separation of parasite infracommunities in species space for both bass species. These parasites represented 28% of all species found in the current survey and may be ecologically important. Several species of parasites exhibited differences in prevalence between surveys. Two species (Rhipidocotyle papillosa and Crepidostomum cornutum) were absent from this survey but were reported as common in the 1929-1931 survey and almost certainly represent extirpations that coincide with the loss of their native bivalve hosts from Oneida Lake. Other differences in the parasite communities may also be explained by the ecological disturbances in Oneida Lake over the past 81 yr. The changes in bass parasite communities between surveys emphasize the importance of recognizing the historical nature of parasite communities, especially in ecosystems with a history of large-scale changes. Most importantly our findings suggest that, similar to trends observed in free-living freshwater biotic communities, anthropogenic ecosystem disturbances may homogenize fish parasite communities.

Topics: Animals; Bass; Ecosystem; Fish Diseases; Lakes; New York; Parasites; Parasitic Diseases, Animal; Time Factors

Nervous necrosis virus (NNV) is a non-enveloped virus with 2 segmented positive-sense single-stranded RNAs. NNV-induced mass mortality has occurred worldwide in many species of cultured marine fish and resulted in substantial economic losses. In our previous study, we cloned the gene of voltage-dependent anion selective channel protein 2 (GVDAC2), and the NNV RNA2 expression level decreased in GVDAC2-knockdown GF-1 cells 24 h after infection. Here, we investigated the role of GVDAC2 in the NNV infection in GF-1 cells. NNV infection did not considerably affect GVDAC2 gene expression. After performing immunostaining, we detected GVDAC2 at the mitochondria and GVDAC2 was colocalized with NNV-RNA-dependent RNA polymerase. However, these 2 proteins did not interact with each other in immunoprecipitation assay. The cellular ATP level in GVDAC2-downregulated cells was lower than that in control cells, and NNV-induced apoptosis was delayed in GVDAC2-siRNA-transfected cells. Therefore, we suggest that GVDAC2 is required for NNV infection for maintaining the cellular ATP level and has positive impact on virus-induced apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Bass; Down-Regulation; Fish Diseases; Fish Proteins; Nodaviridae; RNA Virus Infections; RNA, Small Interfering; Voltage-Dependent Anion Channel 2

The giant seaperch iridovirus (GSIV) induces host cell apoptosis by a poorly-understood process. In this study, GSIV is shown to upregulate the pro-apoptotic death genes Bax and Bak at the middle replication stage, and factors in the grouper fin cell line (GF-1) are shown to modulate this process. Studying the mechanism of cell death, we found that upregulated, de novo-synthesized Bax and Bak proteins formed heterodimers. This up-regulation process correlated with mitochondrial membrane potential (MMP) loss, increased caspase-3 activity, and increased apoptotic cell death. All effects were diminished by treatment of infected GF-1 cells with the protein synthesis inhibitor cycloheximide. Interestingly, overexpression of the anti-apoptotic gene Bcl-xL also diminished GSIV-induced mitochondria-mediated cell death, increasing host cell viability and decreasing MMP loss at the early replication stage. Our data suggest that GSIV induces GF-1 apoptotic cell death through up-regulation of the pro-apoptotic genes Bax and Bak, which are regulated by Bcl-xL overexpression on mitochondria in GF-1 cells.

Topics: Animals; Apoptosis; Bass; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 3; Cell Line; DNA Virus Infections; Fish Diseases; Fish Proteins; Iridovirus; Membrane Potential, Mitochondrial; Up-Regulation

We report the development of a DNA vaccine pcMGNNV2 against nervous necrosis virus (NNV), a leading cause of mass mortality in grouper larvae. In addition, the modulatory effect of CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 agonist, on the DNA vaccine was evaluated. The DNA vaccine alone elicited the production of NNV-specific antibodies, indicating that the vaccine was capable of triggering adaptive humoral response. Furthermore, significant induction of TLR9, Mx and IL-1β was observed in the spleen on day 7 post-vaccination, supporting that the vaccine could trigger TLR9 signaling. The incorporation of CpG ODN at high dose did not significantly affect the level of NNV-specific antibodies, but was able to moderately enhance the expression of Mx and IL-1β on day 7, indicating its ability in modulating innate response. After challenge with NNV, the vaccine alone enhanced the survival rate in infected larvae at both 1 and 2 weeks post-vaccination. The combination of CpG ODN further increased the survival rate at week 1 but not week 2. Interestingly, at week 2 the ODN appeared to induce a Th1-like response, as indicated by upregulation of T-bet (a Th1 marker) and downregulation of GATA-3 (a Th2 marker). Thus, the results suggest that the boosted Th1 response by CpG ODN does not augment the protection efficacy of pcMGNNV2 vaccine. To our best knowledge, this is the first report of a successful DNA vaccine against NNV in grouper.

Topics: Adjuvants, Immunologic; Animals; Bass; Fish Diseases; Nodaviridae; Oligodeoxyribonucleotides; RNA Virus Infections; Vaccines, DNA; Viral Vaccines

Streptococcus iniae is a Gram-positive bacteria that causes invasive infections with severe septicemia and meningitis, producing high economic losses in marine and continental aquaculture. Head kidney leukocytes of European sea bass (Dicentrarchus labrax) were used to measure the differential innate immune response upon infection with S. iniae. The complete inhibition in the production of intracellular superoxide radicals and total peroxidase content was observed in infected cells. This study also elucidates changes in the relative expression of some immune-related genes. Interleukin 1β, tumor necrosis factor-α and interleukin-6 reached a peak of expression at 4-8 h post-infection, subsequently decreasing significantly up to 48 h post-infection. However, interleukin-10 and Mx protein increased over time, reaching the pick of expression at 48 h post-infection, whereas caspase-3 showed down regulation until 48 h post-infection. The in vivo study of immune related genes show the same kinetics of mRNAs expression as in vitro experience. The proinflammatory cytokines mRNA transcription levels peaked at an earlier time in vivo than in vitro system. Our findings indicate that there is a direct relationship between the dissemination of bacteria and the resulting infection-associated inhibition of respiratory burst, apoptosis, and the pro- and anti-inflammatory gene expression profiles.

Topics: Animals; Bass; Cytokines; Fish Diseases; Head Kidney; Immunity, Innate; Leukocytes; Peroxidases; Respiratory Burst; Streptococcal Infections; Streptococcus

TTRAP (TRAF and TNF receptor-associated protein) is latest identified cytosolic protein that serves as a negative regulator for TNF signaling pathway. In this study, a member of TNF superfamily, TTRAP gene (designed as EcTTRAP) was cloned from grouper, Epinephelus coioides. There was an Exo_endo_phos type domain in EcTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. EcTTRAP exhibited prominent endonuclease activity against the genome DNA from Escherichia coli, Vibrio vulnificus and E. coli JM109. Intracellular localization revealed that EcTTRAP expression distributed in both cytoplasm and nucleus. Real-time PCR analysis indicates that EcTTRAP is expressed in all selective grouper tissues, with the higher expression level in muscle, skin and gills. EcTTRAP was identified as a remarkably (P < 0.01) up-regulated protein responding to Singapore grouper iridovirus (SGIV) infection. Overexpression of EcTTRAP inhibited NF-κB activation, meanwhile the C terminal portion of the protein was found to be responsive domain for the inhibition. Stable transfection of FHM cells with EcTTRAP inhibited apoptosis induced by SGIV. Overexpression of EcTTRAP in grouper spleen (GS) cells inhibited the replication of SGIV. The present results provided new evidences for the potential roles of such molecule in E. coioides, and further confirmed the existence of TTRAP modulated TNF signaling pathway in grouper.

Topics: Amino Acid Sequence; Animals; Bacterial Physiological Phenomena; Bass; Cytokines; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Organ Specificity; Phylogeny; Ranavirus; Real-Time Polymerase Chain Reaction; Sequence Alignment; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

The black grouper Mycteroperca bonaci is a commercially important fish off the coast of Yucatan, Mexico. To investigate possible differences between parasite communities in two different environments, 60 fish were collected from two areas during 2010 and 2011 and examined for parasites. The fish were classified into two age groups, in each of which the parameters of parasitic infection - prevalence, abundance and intensity - were determined. Parasite faunas were further described at the infracommunity level. Using both univariate (PERMANOVA) and multivariate statistical methods, the values of richness, abundance, diversity and Brillouin evenness as well as the Index values of Bray-Curtis and Jaccard for similarity were calculated and compared. The results of these tests and of CAP discriminant analyses at the two sites showed the existence of two distinct parasite communities. The parasite taxa mainly responsible for the differences were the digeneans Dollfustrema sp., Prosorhynchus spp., Lepidapedoides epinepheli and Hamacreadium mutabile, and the nematode Philometra salgadoi. The potential for some of these parasites to be used as biological tags for stock identification of M. bonaci is discussed.

Topics: Animals; Bass; Biota; Fish Diseases; Mexico; Parasite Load; Parasites; Parasitic Diseases, Animal; Prevalence

The opecoelid Helicometrina nimia Linton, 1910 has been reported from numerous marine fishes along the Pacific and Atlantic coasts of the Americas. Along the Chilean coast, H. nimia is found in fishes belonging to at least 9 families. This surprisingly low host specificity of H. nimia raises question about the correct identification of specimens assigned to this species. Here we evaluate whether H. nimia specimens isolated from sympatric fish species in northern Chile but with different diets and found in different habitats (water column and demersal) are the same species. Our results demonstrate that specimens from the shallow benthic fish Labrisomus philippii (Steindachner) do not correspond to H. nimia but instead belong to a new species of Helicometrina. This species is described and distinguished from H. nimia using morphological descriptions and 2 molecular markers (the cytochrome c oxidase subunit 1 gene and the V4 region of the SSU rRNA gene). The new species Helicometrina labrisomi (Digenea: Opecoelidae), is found in the intestine of L. philippii (Steindachner, 1866) (Pisces: Labrisomidae), a shallow benthic fish that inhabits the northern coast of Chile. We also studied the related Helicometrina nimia Linton, 1910 from the benthopelagic fishes Paralabrax humeralis (Valenciennes, 1828) and Acanthistius pictus (Tschudi, 1846) (Serranidae). The new species differs from H. nimia by a combination of characters that include ovary shape, number of uterine loops, and position of the genital pore. Our results indicate that morphological characteristics, such as body size, extent of the vitellarium, shape of the testes, and cirrus sac size and extent, traditionally used in the taxonomy of Helicometrina are highly variable. In contrast, meristic and morphological characteristics, such as a lobed ovary, the number of uterine loops, dimensions of the pharynx, and the opening of the genital pore, are highly constant.

Topics: Animals; Bass; Chile; Diet; Ecosystem; Electron Transport Complex IV; Fish Diseases; Fishes; Haplotypes; Intestinal Diseases, Parasitic; Phylogeny; RNA, Ribosomal, 18S; Sequence Alignment; Trematoda; Trematode Infections

Sevenband grouper, Epinephelus septemfasciatus, is becoming an important aquaculture species in Korea. However, viral nervous necrosis disease is a large problem causing mass mortality in sevenband grouper aquaculture. Recombinant protein vaccines are one of the best methods to reduce these economic losses. However, the cell-based expression method mainly produces inclusion bodies and requires additional procedures. In this study, we expressed a recombinant viral coat protein of sevenband grouper nervous necrosis virus (NNV) using a cell-free protein synthesis system. The purified recombinant NNV coat protein (rNNV-CP) was injected into sevenband grouper at different doses followed by a NNV challenge. Nonimmunized fish in the first trial (20 μg/fish) began to die 5 days post-challenge and reached 70% cumulative mortality. In contrast, immunized fish also starting dying 5 days postchallenge but lower cumulative mortality (10%) was observed. Cumulative morality in the second trial with different doses (20, 4, and 0.8 μg/fish) was 10%, 40%, and 50%, respectively. These results suggest that rNNV-CP can effectively immunize sevenband grouper depending on the dose administered. This study provides a new approach to develop a recombinant vaccine against NNV infection for sevenband grouper.

Topics: Animals; Bass; Capsid Proteins; Fish Diseases; Korea; Nodaviridae; Recombinant Proteins; Survival Analysis; Treatment Outcome; Vaccines, Synthetic; Viral Vaccines; Virus Diseases

Aquareoviruses are serious pathogens of aquatic animals. Here, genome characterization and functional gene analysis of a novel aquareovirus, largemouth bass Micropterus salmoides reovirus (MsReV), was described. It comprises 11 dsRNA segments (S1-S11) covering 24,024 bp, and encodes 12 putative proteins including the inclusion forming-related protein NS87 and the fusion-associated small transmembrane (FAST) protein NS22. The function of NS22 was confirmed by expression in fish cells. Subsequently, MsReV was compared with two representative aquareoviruses, saltwater fish turbot Scophthalmus maximus reovirus (SMReV) and freshwater fish grass carp reovirus strain 109 (GCReV-109). MsReV NS87 and NS22 genes have the same structure and function with those of SMReV, whereas GCReV-109 is either missing the coiled-coil region in NS79 or the gene-encoding NS22. Significant similarities are also revealed among equivalent genome segments between MsReV and SMReV, but a difference is found between MsReV and GCReV-109. Furthermore, phylogenetic analysis showed that 13 aquareoviruses could be divided into freshwater and saline environments subgroups, and MsReV was closely related to SMReV in saline environments. Consequently, these viruses from hosts in saline environments have more genomic structural similarities than the viruses from hosts in freshwater. This is the first study of the relationships between aquareovirus genomic structure and their host environments.

Topics: Animals; Bass; Cluster Analysis; Fish Diseases; Gene Order; Genetic Variation; Genome, Viral; Molecular Sequence Data; Phylogeny; Reoviridae; RNA, Viral; Sequence Analysis, DNA; Sequence Homology; Synteny; Viral Proteins

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Topics: Animals; Bass; Fish Diseases; Iridoviridae; Phylogeny; Polymerase Chain Reaction

IRF10 gene was cloned in orange spotted grouper, Epinephelus coioides, and its expression was examined following poly(I:C) stimulation and bacterial infection. The cDNA sequence of grouper IRF10 contains an open reading frame of 1197 bp, flanked by 99 bp 5'-untranslated region and 480 bp 3'- untranslated region. Multiple alignments showed that the grouper IRF10 has a highly conserved DNA binding domain in the N terminus with characteristic motif containing five tryptophan residues. Quantitative real-time PCR analysis revealed that the expression of IRF10 was responsive to both poly(I:C) stimulation and Vibrio parahemolyticus infection, with a higher increase to poly(I:C), indicating an important role of IRF10 in host immune response during infection. A phyletic distribution of IRF members was also examined in vertebrates, and IRF10 was found in most lineages of vertebrates, not in modern primates and rodents. It is suggested that the first divergence of IRF members might have occurred before the evolutionary split of vertebrate and cephalochordates, producing ancestors of IRF (1/2/11) and IRF (4/8/9/10)[(3/7) (5/6)], and that the second and/or third divergence of IRF members occurred following the split, thus leading to the subsets of the IRF family in vertebrates.

Topics: Amino Acid Sequence; Animals; Avian Proteins; Bass; Cloning, Molecular; Evolution, Molecular; Fish Diseases; Fish Proteins; Gene Expression Regulation; Interferon Regulatory Factors; Molecular Sequence Data; Phylogeny; Poly I-C; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment; Vertebrates; Vibrio; Vibrio Infections

Teleost fish rely heavily on their innate immunity for an adequate response against pathogens and environmental challenges, with the production of antimicrobial peptides being one of their first lines of defense. Among those is hepcidin, a small cysteine-rich antimicrobial peptide that is also the key regulator of iron metabolism. Although most mammals possess a single hepcidin gene, with a dual role in both iron metabolism regulation and antimicrobial response, many teleost fish present multiple copies of hepcidin, most likely because of genome duplications and positive Darwinian selection, suggesting that different hepcidins may perform different functions. To study the roles of hepcidin in teleost fish, we have isolated and characterized several genes in the European sea bass (Dicentrarchus labrax) and evaluated variations in their expression levels in response to different experimental conditions. Although several hepcidin genes were found, after phylogenetic analysis they could be clustered in two groups: hamp1-like, with a single isoform similar to mammalian hepcidins, and hamp2-like, with several isoforms. Under experimental conditions, hamp1 was upregulated in response to iron overload and infection and downregulated during anemia and hypoxic conditions. Hamp2 did not respond to either iron overload or anemia but was highly upregulated during infection and hypoxia. In addition, Hamp2 synthetic peptides exhibited a clear antimicrobial activity against several bacterial strains in vitro. In conclusion, teleost fish that present two hepcidin types show a degree of subfunctionalization of its functions, with hamp1 more involved in the regulation of iron metabolism and hamp2 mostly performing an antimicrobial role.

Topics: Amino Acid Sequence; Anemia; Animals; Anti-Infective Agents; Base Sequence; Bass; Fish Diseases; Gene Expression Regulation; Hepcidins; Hypoxia; Immunity, Innate; Iron; Molecular Sequence Data; Protein Isoforms; Sequence Alignment

Stimulator of interferon genes (STING, also known as MITA, ERIS, MPYS or TMEM173) has been identified as a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the detailed role of STING during fish iridovirus infection still remained largely unknown. Here, the STING homolog from grouper Epinephelus coioides (EcSTING) was cloned and its effects on IFN response and antiviral activity were investigated. The full-length EcSTING cDNA was composed of 1590 bp and encoded a polypeptide of 409 amino acids with 80% identity to STING homolog from large yellow croaker. Amino acid alignment analysis indicated that EcSTING contained 4 predicated transmembrane motifs (TMs) in the N terminal, and a C-terminal domain (CTD) which consisted of a dimerization domain (DD), c-di-GMP-binding domain (CBD) and a C-terminal tail (CTT). Expression profile analysis revealed that EcSTING was abundant in gill, spleen, brain, skin, and liver. Upon different stimuli in vivo, the EcSTING transcript was dramatically up-regulated after challenging with Singapore grouper iridovirus (SGIV), lipopolysaccharide (LPS) and polyinosin-polycytidylic acid (poly I:C). Reporter gene assay showed that EcSTING activated ISRE, zebrafish type I IFN and type III IFN promoter in vitro. Mutant analysis showed that IFN promoter activity was mostly mediated by the phosphorylation sites at serine residue S379 and S387. Moreover, EcSTING induced type I and III IFN promoter activity could be impaired by overexpression of EcIRF3-DN or EcIRF7-DN, suggesting that EcSTING mediated IFN response in IRF3/IRF7 dependent manner. In addition, the cytopathic effect (CPE) progression of SGIV infection and viral protein synthesis was significantly inhibited by overexpression of EcSTING, and the inhibitory effect was abolished in serine residue S379 and S387 mutant transfected cells. Together, our results demonstrated that EcSTING might be an important regulator of grouper innate immune response against iridovirus infection.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Lipopolysaccharides; Organ Specificity; Poly I-C; Ranavirus; RNA, Messenger; Sequence Alignment

Although the skin is one of the main defense barriers of fish to date, very little is known about the immune implications and the properties of the numerous substances present in skin cells. In the present study, terminal carbohydrate composition and some components of the skin immunity (total IgM level, and several enzymatic and bacteriostatic activities) present on aqueous and organic epidermal extracts of European sea bass (Dicentrarchus labrax) were determined. Most of the parameters measured followed a protein concentration dose-response. Curiously, both skin extracts have similar levels of total IgM. However, aqueous extracts showed higher presence of some terminal carbohydrates, alkaline phosphatase and esterase activities and lower proteases and ceruloplasmin activities than epidermal organic extracts. Regarding the bacteriostatic activity, the growth of all the bacterial strains tested was reduced when cultivated in presence of organic extracts, being the observed reduction correlated to the protein concentration present in the extract sample. On the contrary, skin aqueous extracts have no significant effect on bacterial growth or even allow bacteria to overgrow, suggesting that the bacteria could use the extracts as a nutrient source. The results are discussed and compared with the same activities studied on fish skin mucus in order to understand their possible implications on mucosal immunity.

Topics: Animals; Anti-Bacterial Agents; Bass; Carbohydrate Metabolism; Epidermis; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunity, Mucosal; Immunoglobulin M; Shewanella putrefaciens; Vibrionaceae

CCR6 have been demonstrated playing an important role in immune cells homing to mucosal tissues, mediating antigen presentation and immune response in mammals. CCR6 in lower vertebrate leukocyte homing has not yet been revealed. Cryptocaryon irritans is believed to be a good pathogen model for skin and gill mucosal immunity. In this study, we identified two CCR6s and their three possible ligands CCL20 like cDNA sequences, designated as grouper EcCCR6A, EcCCR6B, EcCCL20L1, EcCCL20L2 and EcCCL20L3. It is interesting to find that EcCCR6A has a longer second extracellular loop than EcCCR6B, which is more similar to mammalian CCR6. Tissue distribution analysis showed that EcCCR6A pronouncedly dominates in gill and brain while EcCCR6B dominates in head kidney, trunk kidney and thymus. Three chemokine ligands have their own distinct expression pattern in health grouper tissues. EcCCL20L1 dominates in spleen and head kidney, EcCCL20L2 dominates in gill and thymus, whereas EcCCL20L3 dominates in skin and brain. The expression patterns of these chemokines and chemokine receptors were detected in C. irritans infected grouper and the results showed that EcCCR6A, EcCCR6B and EcCCL20L1 were significantly up-regulated in the skin of C. irritans infected fish, which indicated these two chemokine receptors and their ligand may play important role in immune cells' homing to skin mucosal immune tissues under pathogen caused inflammation.

Topics: Amino Acid Sequence; Animals; Bass; Chemokine CCL20; Ciliophora; Ciliophora Infections; Fish Diseases; Fish Proteins; Immunity, Mucosal; Ligands; Organ Specificity; Phylogeny; Receptors, CCR6; Sequence Alignment

Toll-interacting protein (Tollip) is one of the important regulatory proteins of Toll-like receptor (TLR) signaling pathways. In previous studies, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip were studied. However, the response of Tollip to virus infection has not been characterized from grouper. In the present paper, the Tollip homolog (EtTollip) from grouper (Epinephelus tauvina) was cloned and its immune response to Singapore grouper iridovirus (SGIV) was investigated. EtTollip shares significant similarities to other mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenging with SGIV, the expression levels of EtTollip were altered in the spleen and head kidney of grouper. EtTollip mainly aggregated in the cytoplasm in a condensed state and was also distributed on the membranes of GS cells. EtTollip significantly inhibited the activities of NF-κB and IFN-β luciferase reporter when transfected into grouper spleen (GS) cells. SGIV can increase the activities of NF-κB and IFN-β luciferase reporter, especially to IFN-β. When transfected EtTollip with EcMyd88, the activity of NF-κB was increased, while transfected EtTollip with EcIRF3, the activity of IFN-β was significantly increased. Over-expressed EtTollip inhibited the transcription of SGIV genes significantly in GS cells, and silencing of EtTollip with siRNA led to increase of SGIV genes loads. Taken together, the results provide new insights in to the importance of Tollip as evolutionarily conserved molecule for grouper innate immunity against virus infection.

Topics: Amino Acid Sequence; Animals; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Intracellular Signaling Peptides and Proteins; Ranavirus; RNA, Messenger; Sequence Alignment

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.

Topics: Animals; Bass; Fish Diseases; Male; Nodaviridae; Reproduction; RNA Virus Infections; Sea Bream; Testis; Virus Replication

Recent years have seen a global and rapid resurgence of fungal diseases with direct impact on biodiversity and local extinctions of amphibian, coral, or bat populations. Despite similar evidence of population extinction in European fish populations and the associated risk of food aquaculture due to the emerging rosette agent Sphaerothecum destruens, an emerging infectious eukaryotic intracellular pathogen on the fungal-animal boundary, our understanding of current threats remained limited. Long-term monitoring of population decline for the 8-year post-introduction of the fungal pathogen was coupled with seasonal molecular analyses of the 18S rDNA and histological work of native fish species organs. A phylogenetic relationship between the existing EU and US strains using the ribosomal internal transcribed spacer sequences was also carried out. Here, we provide evidence that this emerging parasite has now been introduced via Pseudorasbora parva to sea bass farms, an industry that represents over 400 M€€ annually in a Mediterranean region that is already economically vulnerable. We also provide for the first time evidence linking S. destruens to disease and severe declines in International Union for Conservation of Nature threatened European endemic freshwater fishes (i.e. 80% to 90 % mortalities). Our findings are thus of major economic and conservation importance.

Topics: Animals; Aquaculture; Base Sequence; Bass; Biodiversity; Cyprinidae; Europe; Fish Diseases; Introduced Species; Mesomycetozoea; Molecular Sequence Data; Phylogeny; Prevalence; Seasons; Sequence Analysis, DNA

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV-type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.

Topics: Animals; Bass; Fish Diseases; Genotype; Molecular Sequence Data; Nodaviridae; Phylogeny; RNA Virus Infections; RNA, Viral; Sequence Analysis, DNA; Virulence

Garlic, Allium sativum L., extract administered as a therapeutic bath was shown to have antiparasitic properties towards Neobenedenia sp. (MacCallum) (Platyhelminthes: Monogenea) infecting farmed barramundi, Lates calcarifer (Bloch). The effect of garlic extract (active component allicin) immersion on Neobenedenia sp. egg development, hatching success, oncomiracidia (larvae) longevity, infection success and juvenile Neobenedenia survival was examined and compared with freshwater and formalin immersion. Garlic extract was found to significantly impede hatching success (5% ± 5%) and oncomiracidia longevity (<2 h) at allicin concentrations of 15.2 μL L(-1) , while eggs in the seawater control had >95% hatching success and mean oncomiracidia longevity of 37 ± 3 h. At much lower allicin concentrations (0.76 and 1.52 μL L(-1)), garlic extract also significantly reduced Neobenedenia infection success of L. calcarifer to 25% ± 4% and 11% ± 4%, respectively, compared with 55% ± 7% in the seawater control. Juvenile Neobenedenia attached to host fish proved to be highly resistant to allicin with 96% surviving 1-h immersion in 10 mL L(-1) (15.2 μL L(-1) allicin) of garlic extract. Allicin-containing garlic extracts show potential for development as a therapy to manage monogenean infections in intensive aquaculture with the greatest impact at the egg and larval stages.

Topics: Animals; Bass; Disulfides; Ectoparasitic Infestations; Fish Diseases; Fisheries; Garlic; Helminthiasis, Animal; Immersion; Ovum; Phytotherapy; Plant Extracts; Platyhelminths; Sulfinic Acids

This study was conducted to investigate the effect of katuk (Sauropus androgynus L. Merr) on growth, non-specific immune and diseases resistance against Vibrio alginolyticus in grouper (Epinephelus coioides). Grouper juveniles (mean weight 10.97 ± 1.99 g, and length 9.67 ± 0.33 cm) were separated into four groups and cultivated in 100-L tank. Each group was fed with diets containing 0, 1.0, 2.5 and 5.0 g/kg herbs diet twice daily. Fish were sampled for non-specific immune parameters at 0, 1, 2, 4, 7, 14 and 30 days. Results showed that fish received S. androgynus at 1.0 and 2.5 g/kg diets affected the growth and non-specific immune responses. Weight gain, specific growth rate, respiratory burst activity, phagocytosis and reactive oxygen species significantly increased in fish administered with 1.0 and 2.5 g/kg S. androgynus diets. The mortality rate after V. alginolyticus challenge decreased in fish fed with 1.0 g/kg S. androgynus extract. Thus, this study indicated that administration of grouper with S. androgynus supplemented diets can affect the growth performances, diseases resistance and enhances non-specific immune responses.

Topics: Animal Feed; Animals; Bass; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Immunity, Innate; Magnoliopsida; Plant Extracts; Vibrio alginolyticus; Vibrio Infections

Topics: Algeria; Animals; Bass; Fish Diseases; Fisheries; Molecular Sequence Data; Necrosis; Nodaviridae; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; Species Specificity; Viral Proteins

Leukocyte cell-derived chemotaxin-2 (LECT2) is an important protein of the innate immune system for the defense against bacterial infection. We cloned and characterized the LECT2 gene from Asian seabass (Lates calcarifer). Its complete cDNA consisted of an open reading frame of 459 bp encoding a protein of 152 amino acids. The genomic DNA sequence of this gene consists of four exons and three introns. Quantitative real-time PCR revealed that the LECT2 gene was expressed predominantly in liver while its expression was moderate in spleen and heart, and weak in other tissues. The LECT2 transcript was up-regulated in the kidney, spleen and liver in response to a challenge with a pathogenic bacterium Vibrio harveyi. In addition, we identified three single nucleotide polymorphisms (SNPs) in the LECT2 gene, and found significant associations between these polymorphisms and resistance to the big belly disease. These results suggest that the LECT2 gene play an important role in resistance to bacterial pathogens in fish. The SNP markers in the gene associated with the resistance to bacterial pathogens may facilitate selecting Asian seabass resistant to bacterial diseases.

Topics: Animals; Base Sequence; Bass; Cloning, Molecular; Disease Resistance; DNA Primers; DNA, Complementary; Fish Diseases; Gene Components; Gene Expression Profiling; Intracellular Signaling Peptides and Proteins; Liver; Molecular Sequence Data; Polymorphism, Single Nucleotide; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Spleen; Vibrio; Vibrio Infections

The authors describe the prevalence and severity of intersex in the form of testicular oocytes in largemouth bass (Micropterus salmoides) collected over a 5-yr period from a variety of surface waters on the Delmarva Peninsula, USA, a region dominated by poultry production and agricultural land use. During a survey from 2005 to 2007 of approximately 200 male specimens representing 6 fish and 2 frog species collected from numerous small-order streams on Delmarva, intersex was observed in only largemouth bass (system-wide prevalence 17%). During 2008 and 2009, testicular oocytes were encountered in male largemouth bass from 6 lakes and 1 large river system, with prevalence ranging from 33% to 88% (weighted arithmetic mean, 57%). The prevalence of testicular oocytes in largemouth bass from Delmarva lakes was comparable to the highest levels reported in a national US Geological Survey reconnaissance of this species, which also occurred in regions of the Atlantic coastal plain with intensive row-crop and animal agriculture. To the authors' knowledge, the present study represents the first report in the peer-reviewed scientific literature of testicular oocytes in fish on the Delmarva Peninsula.

Topics: Agriculture; Animals; Bass; Delaware; Disorders of Sex Development; Female; Fish Diseases; Lakes; Male; Maryland; Oocytes; Rivers; Testis

Mycobacteriosis, a chronic bacterial disease of fishes, is prevalent in adult striped bass from Chesapeake Bay (USA). Although environmental factors may play a role in disease expression, the interaction between the disease and environmental stress remains unexplored. We therefore examined the individual and interactive effects of elevated temperature, hypoxia, and mycobacteriosis on the metabolism of wild-caught adult striped bass from Chesapeake Bay using respirometry. Because the spleen is the primary target organ of mycobacteriosis in striped bass, we hypothesized that the disease interferes with the ability of fish to increase their hematocrit in the face of increasing oxygen demands. We determined standard metabolic rate (SMR), maximum metabolic rate under normoxia (MMRN), critical oxygen saturation (S(crit)), and MMR under hypoxia (3 mg O(2) l-1: MMR(H)) for healthy and visibly diseased fish (i.e. exhibiting skin lesions indicative of mycobacteriosis). Measurements were taken at a temperature within the preferred thermal range (20°C) and at an elevated temperature (28°C) considered stressful to striped bass. In addition, we calculated aerobic scope (AS(N) = MMR(N) - SMR, AS(H) = MMR(H) - SMR) and factorial scope (FS(N) = MMR(N) SMR-1, FS(H) = MMR(H) SMR-1). SMR increased with increasing temperature, and hypoxia reduced MMR, AS, and FS. Mycobacteriosis alone did not affect either MMR(N) or MMR(H). However, elevated temperature affected the ability of diseased striped bass to tolerate hypoxia (S(crit)). Overall, our data indicate that striped bass performance under hypoxia is impaired, and that elevated water temperatures, hypoxia, and severe mycobacteriosis together reduce aerobic scope more than any of these stressors acting alone. We conclude that the scope for activity of diseased striped bass in warm hypoxic waters is significantly compromised.

Topics: Animals; Bass; Energy Metabolism; Fish Diseases; Mycobacterium Infections; Oxygen; Sodium-Potassium-Exchanging ATPase; Temperature

Unicapsula seriolae (Myxozoa; Multivalvulida) was found in the trunk muscle of Malabar grouper Epinephelus malabaricus caught off Wakayama Prefecture, Japan. Numerous filamentous or sesamoid brown to black lesions were observed in the skeletal muscle. Histopathological observation indicated that the lesions were myxosporean plasmodia encapsulated by a fibrous layer, accompanied by melanin deposition. Spores having one large and two rudimentary polar capsules were subspherical in shape and 6.6 × 6.9 μm in size. Scanning electron microscopy revealed that spores were composed of three spore valves. Morphological characteristics were consistent with U. seriolae, which is reported to cause myoliquefaction in yellowtail kingfish Seriola lalandi in Australia. Molecular analysis of the SSU and LSU rDNA supported identification of the species as U. seriolae. This is the first report of Unicapsula in Japan.

Topics: Animals; Bass; Fish Diseases; Japan; Microscopy, Electron, Scanning; Molecular Sequence Data; Muscle, Skeletal; Myxozoa; Parasitic Diseases, Animal; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 18S; RNA, Ribosomal, 28S; Seafood; Sequence Analysis, DNA

Vibrio anguillarum is the major cause of haemorrhagic septicaemia, vibriosis, which is a severe disease affecting marine fish. In this work, it was found that the mortality of gnotobiotic sea bass larvae challenged with V. anguillarum was dependent on the number of dead fish in the vials at the moment of challenge. Based on this finding, the effect of dead hosts (homogenised sea bass larvae or brine shrimp) on the virulence of V. anguillarum towards sea bass larvae was further investigated. Addition of homogenised hosts led to significantly increased larval mortality of challenged larvae, and this was observed for 3 different V. anguillarum strains, i.e. 43, NB 10 and HI 610. In contrast, the addition of similar levels of tryptone had no effect on mortality. In line with this, the motility of all 3 V. anguillarum strains was significantly increased by the addition of homogenised hosts but not by tryptone. These results suggest that dead hosts increase infectivity of V. anguillarum, not merely by offering nutrients to the bacteria, but also by increasing virulence-associated activities such as motility.

Topics: Animals; Bass; Fish Diseases; Host-Pathogen Interactions; Larva; Vibrio; Vibrio Infections

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6-7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cell Line; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Interferons; Molecular Sequence Data; Myxovirus Resistance Proteins; Nodaviridae; Organ Specificity; Phylogeny; Promoter Regions, Genetic; RNA Virus Infections; Sequence Analysis, DNA

Hybrid striped bass (HSB) and white bass (WB) were evaluated for their susceptibility to Flavobacterium columnare, the causative agent of columnaris disease, in 3 fundamental studies. In the first experiment, we determined whether columnaris disease could be developed by experimental challenge in HSB. This challenge consisted of 3 levels of F. columnare (10, 30, and 60 ml volumes) determined to be 2.25 × 10(7), 6.75 × 10(7), and 1.35 × 10(8) CFU ml(-1), respectively. Each treatment group exhibited significantly different survival rates: 0, 3.3, and 13.3% in the 60, 30, and 10 ml groups, respectively. In Expt 2, using the 30 ml dose, both HSB and WB had a 0% survival rate, with WB taking significantly longer to reach 100% mortality. In Expt 3, using the 10 ml dose, no HSB survived, whereas 33% of WB survived (p < 0.0001). Compared to controls, HSB treated with 10 ml showed extensive gill damage at 24 h, which could have contributed to the higher mortality observed in HSB; in contrast, WB gills showed noticeably less damage. From these series of experiments, it is clear that HSB are more sensitive to F. columnare, having lower survival and more extensive histological damage compared to WB following challenge.

Topics: Animals; Bass; Crosses, Genetic; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Genetic Predisposition to Disease; Gills

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aa-116aa), two zinc fingers of TRAF-type (159aa-210aa and 212aa-269aa), one coiled-coil region (370aa-394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aa-526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosine-polycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid · Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that Et-TRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Molecular Sequence Data; Phylogeny; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

Betanodaviruses are the causative agents of viral nervous necrosis and affect a broad range of fish species worldwide. Their bi-segmented genome is composed of the RNA1 and the RNA2 molecules encoding the viral polymerase and the coat protein, respectively. In southern Europe the presence of the RGNNV and the SJNNV genotypes, and the RGNNV/SJNNV and RGNNV/SJNNV reassortants has been documented. Several studies have reported a correlation between water temperature and disease onset. To explore the replication efficiency of betanodaviruses with different genomes in relation to temperature and to understand the role of genetic reassortment on viral phenotype, RGNNV, SJNNV, RGNNV/SJNNV and RGNNV/SJNNV field isolates were fully sequenced, and growth curves generated in vitro at four different temperatures (15, 20, 25, 30 °C) were developed for each isolate. The data obtained, corroborated by statistical analysis, demonstrated that viral titres of diverse betanodavirus genotypes varied significantly in relation to the incubation temperature of the culture. In particular, at 30 °C betanodaviruses under investigation presented different phenotypes, and viruses containing the RNA1 of the RGNNV genotype showed the best replication efficiency. Laboratory results demonstrated that viruses clustering within the same genotype based on the polymerase gene, possess similar growth kinetics in response to temperature, thus highlighting the key role of RNA1 in controlling viral replication at different environmental conditions. The results generated might have practical implications for the inference of viral phenotype according to genetic features and may contribute to a better understanding of betanodavirus ecology.

Topics: Animals; Bass; Fish Diseases; Flatfishes; Genotype; Molecular Sequence Data; Nodaviridae; Phenotype; Phylogeny; Reassortant Viruses; RNA Virus Infections; RNA, Viral; Sequence Analysis, RNA; Temperature; Virus Replication

Vaccination is an important strategy in the protection of aquaculture species from major diseases. However, we still do not have a good understanding of the mechanisms underlying vaccine-induced disease resistance. This is further complicated by the presence of several lymphoid organs that play different roles when mounting an immune response. In this study, we attempt to elucidate some of these mechanisms using a microarray-based approach. Asian seabass (Lates calcarifer) were vaccinated against Streptococcus iniae and the transcriptomic changes within the spleen and head kidney at one and seven days post-vaccination were profiled. We subsequently challenged the seabass at three weeks post-vaccination with live S. iniae and similarly profiled the transcriptomes of the two organs after the challenge. We found that vaccination induced an early, but transient transcriptomic change in the spleens and a delayed response in the head kidneys, which became more similar to one another compared to un-vaccinated ones. When challenged with the pathogen, the spleen, but not the head kidneys, responded transcriptomically at 25-29 hours post-challenge. A unique set of genes, in particular those involved in the activation of NF-κB signaling, was up-regulated in the vaccinated spleens upon pathogen challenge but not in the un-vaccinated spleens. A semi-quantitative PCR detection of S. iniae using metagenomic DNA extracted from the water containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. This result indicated that vaccination not only led to a successful immune defense against the infection, but also reduced the chances for horizontal transmission of the pathogen. In conclusion, we have provided a transcriptomic analysis of how the teleost spleen and head kidneys responded to vaccination and subsequent infection. The different responses from the two organs are suggestive of their unique roles in establishing a vaccine-induced disease resistance.

Topics: Animals; Bass; Fish Diseases; Kidney; Spleen; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Transcriptome; Vaccination

Influence of temperature on the susceptibility of fish against virus infection has been studied for a decade. Recent reports have been shown the effects of rearing temperatures on the fish immune system against virus infection. However, the roles of temperature in regulation of type I interferon (IFN) system has not yet been investigated. Thus, the effects of temperature on type I IFN response were investigated in this study using poly (I:C) injection in sevenband grouper and Mx gene was used as a marker for type I IFN expression. Quantitative real-time PCR (qPCR) result showed that Mx expression profiles were moderately different between temperatures. The highly up-regulated Mx transcripts at 3 h post injection (hpi) were observed in high temperatures (25 °C and 30 °C) but not in low temperatures (15 °C and 20 °C). Meanwhile, low temperatures (15 °C and 20 °C) could detect the highly up-regulated Mx transcripts at 24 hpi. Expression of Mx transcripts was also observed at 72 hpi at 15 °C. Poly (I:C)-injected fish were challenged with RGNNV after 72 and 168 hpi. At 72 hpi, 100% of fish survived at all temperatures, whereas 95% survival rate was observed at 168 hpi at 25 °C during 14 days of observation. To further verify the duration period of an antiviral state at different temperatures, qPCR and endpoint dilution assay were used to quantify the number of virus in fish challenged with RGNNV. The reduction of viral copy numbers and viral titers could be observed at 72 and 168 hpi. However, high viral copy numbers and viral titers could be detected at 168 hpi at 30 °C. These results demonstrate that temperatures influenced on the Mx expression profiles and the duration period of an antiviral state efficiently interfered with virus replication at different temperatures.

Topics: Animals; Bass; Fish Diseases; Fish Proteins; Gene Expression Regulation; Genetic Markers; Interferon Type I; Myxovirus Resistance Proteins; Nodaviridae; Poly I-C; Real-Time Polymerase Chain Reaction; RNA Virus Infections; Temperature; Transcriptome

Nervous necrosis virus (NNV) has caused mass mortality in many mariculture fish species. Bath vaccination of inactivated NNV and oral immunization of recombinant NNV coat protein are reported to protect grouper larvae against NNV infection. However, the information of immune gene expression in grouper larvae (Epinephelus coioides) after bath and oral immunizations is still limited. In this study, grouper larvae were respectively bath- and orally immunized with binary ethylenimine (BEI)-inactivated NNV, and the expression levels of immune genes were analyzed. Significant gene expressions of IL-1β, Mx, MHC-I, MHC-II, CD8α, IgM and IgT were observed in bath- and orally immunized fish 1-4 weeks post immunization (wpi). Particularly, the up-regulation of IL-1β and Mx gene expression lasted for 4 weeks. The IgT gene expression in gill was only induced by bath immunization, while that in gut was only stimulated by oral immunization. Both immunizations elicited MHC-I and CD8α gene expression relative to cellular immunity. Furthermore, NNV RNA genome, which was detected in inactivated NNV, could induce Mx gene expression in grouper brain (GB) cells, indicating that NNV RNA genome could be recognized by pathogen-recognition receptors (PRRs). In summary, bath and oral vaccinations with BEI-inactivated NNV triggered the gene expression of not only humoral immunity but also cellular immunity.

Topics: Administration, Oral; Animals; Bass; Fish Diseases; Gene Expression Regulation; Gene Flow; Immunity, Mucosal; Immunization; Nodaviridae; Polymerase Chain Reaction; RNA Virus Infections; Vaccines, Inactivated; Viral Vaccines

Vaccination is the most effective means of preventing infectious diseases; however, few vaccines are effective against Streptococcus iniae (S. iniae) in grouper. This work presents an efficacious and safe vaccine against S. iniae infections in the grouper Epinephelus coioides. The vaccine candidate was the S. iniae GSI-310 strain. The vaccination was administered by intraperitoneal injection, and consisted of formalin-inactivated antigens combined with an AS-F or ISA763A adjuvant. Peripheral blood samples were collected for RT-qPCR and phagocytosis and agglutination assays. Our results indicated that immunoglobulin M (igm) was maximally expressed in the two vaccinated groups at 3 months post-secondary vaccination (PSV). A significant upregulation of mRNA expression for interleukin-1β (il-1β) and tumor necrosis factor-α (tnf-α) was also observed in fish treated with antigens combined with ISA763A, which peaked at 3 months PSV. In fish treated with antigens combined with AS-F, il-1β and tnf-α expression peaked at 14 days post-primary vaccination (PPV). Phagocytic activity and index increased significantly in the two vaccinated groups. Furthermore, fish in the two vaccinated groups exhibited significantly elevated agglutination titers compared to fish in the control group, in which almost no agglutination reaction was detected. In the efficacy test, the vaccinated and control groupers were treated with S. iniae at 1, 3, and 6 months PSV. The relative percentage survival (RPS) values of antigens with AS-F and antigens with ISA763A were both 100% at 1 and 3 months PSV; at 6 months PSV, the RPS values for these groups were 100% and 97.7%, respectively. Furthermore, the level of protection observed in the field trial closely resembled that achieved on a laboratory scale. Therefore, the proposed vaccine mixed with AS-F or ISA763A improved immune responses and provided safe and long-lasting protection in farmed groupers.

Topics: Agglutination Tests; Animals; Antibodies, Bacterial; Bass; Fish Diseases; Immunization; Immunoglobulin M; Injections, Intraperitoneal; Interleukin-1beta; Leukocytes, Mononuclear; Phagocytosis; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Survival Analysis; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

Abstract We investigated the optimum dilution of nervous necrosis virus (NNV) for use as antigens to detect antibodies by an enzyme-linked immunosorbent assay (ELISA) in the Sevenband Grouper Epinephelus septemfasciatus. The ELISA values for a standardized suspension of antigens diluted with L-15 medium containing 1% fetal bovine serum decreased gradually with the dilution of the antigens, whereas those for the antigens diluted with distilled water (DW) initially increased with the dilution of the antigens, peaked at a 320-fold dilution, and then decreased thereafter. Additional studies revealed that binding of NNV antigens to ELISA wells was inhibited by fetal bovine serum and other substances in the L-15 medium. Sera obtained from Sevenband Grouper vaccinated with live NNV vaccine and survivors from natural NNV-infection were subjected to antibody detection by ELISA. All of the sera were positive by ELISA when the standardized suspension was diluted 320-fold, whereas sera from five out of the six survivors and two out of the six vaccinated fish were negative or weakly positive by ELISA using NNV antigens diluted 10-fold. We therefore concluded that cultured NNV solutions prepared in cell culture media may need to be diluted with distilled water for use in ELISA. Received January 28, 2014; accepted April 16, 2014.

Topics: Animals; Antibodies; Antibodies, Viral; Bass; Culture Media; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Nodaviridae; RNA Virus Infections

Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

Topics: Animals; Bass; Cell Line; Disease Susceptibility; DNA Virus Infections; Fish Diseases; Perciformes

Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

Topics: Animals; Apoptosis; Bass; Caspases; DNA Virus Infections; Fish Diseases; Flow Cytometry; In Vitro Techniques; MAP Kinase Signaling System; Microscopy, Fluorescence; Phosphatidylinositol 3-Kinases; Ranavirus; Reactive Oxygen Species; Virus Replication

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Topics: Animals; Bass; Fish Diseases; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Head Kidney; Hepcidins; HSP70 Heat-Shock Proteins; Immunohistochemistry; Interleukins; Listonella; Photobacterium; Random Allocation; Real-Time Polymerase Chain Reaction; RNA; Spleen; Thymus Gland; Tumor Necrosis Factor-alpha

Sixty Epinephelus areolatus were examined for metazoan fish parasites in Indonesia, off Segara Anakan lagoon, Java and in Balinese waters. The study revealed 21 different parasite species, and 14 new host and locality records. The anisakid nematodes Anisakis typica and, for the first time in Indonesia, Anisakis sp. HC-2005 were identified by using molecular methods. Ecological parameters were calculated for both sites off the anthropogenically influenced Segara Anakan lagoon and the relatively undisturbed reference site at the southern Balinese coast. The fish from Segara Anakan demonstrated a significantly higher enzymatic activity (Hepatosomatic index) and a significantly reduced number of heteroxenous gut helminths (e.g. the digenean Didymodiclinus sp., the nematode Raphidascaris sp. and the acanthocephalan Serrasentis sagittifer). Other regional differences for E. areolatus included ecto-/endoparasite ratio, endoparasite diversity, the parasite species composition and prevalence of infection of the respective parasite species. We applied the stargraph method to visualize observed regional differences using grouper parasites as biological indicators for the sampled coastal ecosystems at both sampling sites.

Topics: Animals; Bass; Biodiversity; Fish Diseases; Indonesia; Molecular Diagnostic Techniques; Parasites; Parasitic Diseases, Animal; Parasitology

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bacterial Toxins; Bass; Fish Diseases; Host-Pathogen Interactions; Leukocytes; Metalloproteases; Photobacterium; Recombinant Proteins; Transcription Factor RelA; Virulence Factors

Four different endohelminth parasite taxa were found in the viscera of the blacktail comber Serranus atricauda Günther, 1874 caught in the Madeira Archipelago. Nematodes were the dominant group, represented by 2 different taxa, Hysterothylacium spp. Ward & Magath, 1917 and Procamallanus (Spirocamallanus) halitrophus Fusco & Overstreet, 1978 comb. n. Plerocerci of the trypanorhynch Pseudogrillotia epinepheli (synonym: Grillotia epinepheli) Scholz, Garippa & Scala, 1993, and cystacanths of the acanthocephalan Bolbosoma vasculosum Rudolphi, 1819 were found in the visceral cavity. New host records for P. (S.) halitrophus and P. epinepheli and the extension of the geographic distribution of these 2 parasite species provide evidence of parasite transference between the Madeira Archipelago, the Mediterranean and the Gulf of Mexico. The paucity of the parasite fauna of blacktail comber reflect a combination of fish host selective feeding on particular dietary items and its territorial behaviour.

Topics: Animals; Atlantic Ocean; Bass; DNA, Intergenic; Female; Fish Diseases; Helminthiasis, Animal; Helminths; Larva; Male; Phylogeny

Nervous necrosis viruses (NNVs) cause mass mortality of marine fish, leading to large economic losses for aquaculturists. A promising vaccine candidate for preventing NNV infection is the NNV virus-like particle (VLP), which is a structure resulting from assembly of recombinant NNV capsid protein. NNV capsid proteins have been expressed in insect cells and the Escherichia coli expression system, and purified by non-scalable protocols such as ultracentrifugation on sucrose and cesium chloride density gradients. In this study, we expressed red-spotted grouper NNV (RGNNV) capsid proteins in Saccharomyces cerevisiae (S. cerevisiae) and developed a chromatography-based method with potential for large-scale vaccine production. The RGNNV capsid protein was successfully purified by a single-step of heparin chromatography. Transmission electron microscopy (TEM) confirmed the high quality of the purified RGNNV capsid protein: it was in the form of VLPs with mean diameters of 25nm, in homogeneous suspension without any aggregation. Moreover, the RGNNV capsid protein elicited anti-RGNNV capsid protein antibodies in mice. We suggest that RGNNV capsid protein expressed in S. cerevisiae and purified by heparin chromatography, is of sufficient quality for use as a vaccine.

Topics: Animals; Bass; Capsid; Capsid Proteins; Cloning, Molecular; Female; Fish Diseases; Immunoglobulin G; Mice; Nodaviridae; Recombinant Proteins; RNA Virus Infections; Saccharomyces cerevisiae; Vaccines, Virus-Like Particle; Virion

Mx proteins are key components of the antiviral state triggered by interferon type I in response to viral infections. In this study, two different Mx genes have been identified in European sea bass (Dicentrarchus labrax), and their sequences were cloned and characterized. MxA cDNA consists of 1881 bp coding for a putative 626 aminoacids protein, while MxB cDNA has 1920 bp and results in a protein with 639 residues. Their corresponding genomic sequences contain 3538 bp and 5326 bp, respectively, and both present 12 exons and 11 introns. The expression patterns of the two Mx genes after an in vivo challenge with the viral nervous necrosis virus (VNNV), a serious pathogen in farmed European sea bass, have been characterized by real-time PCR. The results showed interesting differences in the transcription profile of both Mx, thus suggesting a differential role for each Mx isoform in the immune response of European sea bass to VNNV, and most likely in the general viral response of this species.

Topics: Amino Acid Sequence; Animals; Bass; Fish Diseases; Fish Proteins; GTP-Binding Proteins; Molecular Sequence Data; Myxovirus Resistance Proteins; Nodaviridae; RNA Virus Infections

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0 g and 2.0 g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers. Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus. Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls.

Topics: Animal Feed; Animals; Aquaculture; Bass; beta-Glucans; Complement Pathway, Alternative; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Gas Chromatography-Mass Spectrometry; Immunity, Innate; Muramidase; Phagocytosis; Reishi; Respiratory Burst; Trametes; Vibrio alginolyticus; Vibrio Infections

The M-CSF/M-CSFR system plays a central role in the cell survival, proliferation, differentiation and maturation of the monocyte/macrophage lineage. In present study, we cloned the sequence of the M-CSFR cDNA from the orange-spotted grouper (Epinephelus coioides). Sequence analysis reveals that ten cysteines in the extracellular immunoglobulin-like (Ig-like) domains of EcM-CSFR are conserved in fish and mammals, its nine possible N-glycosylation sites are conserved in fish but not mammals, 7 of 8 identified mammal M-CSFR intracellular autophosphorylation tyrosine sites was found in EcM-CSFR. Real-time PCR showed that the constitutive expression level of EcM-CSFR was the highest in the spleen, less in the gill, kidney, head kidney and liver, least in the blood, skin, gut and thymus. A rabbit anti-EcM-CSFR polyclonal antibody against the recombinant EcM-CSFR extracellular domain was developed and it was efficient in labeling the monocytes and macrophages isolated from the head kidney. Immunochemistry analysis showed that M-CSFR(+) cells located in all tested paraffin-embedded tissues and M-CSFR(+) cell centres with the characteristic of melano-macrophage centres(MMCs) was found in the spleen, head kidney, kidney, gut and liver. All these results indicate the widespread distribution of macrophages in grouper tissues and its importance in fish immune system. In Crytocaryon irritans infected grouper, EcM-CSFR was transient up-regulated and rapidly down-regulated in skin, gill, head kidney and spleen. The possible activation mechanism of macrophage via EcM-CSFR signal transduction in the fish anti-C. irritans infection was discussed.

Topics: Amino Acid Sequence; Animals; Antibodies, Protozoan; Bass; Ciliophora; Ciliophora Infections; DNA, Complementary; Escherichia coli; Fish Diseases; Macrophage Colony-Stimulating Factor; Macrophages; Molecular Sequence Data; Monocytes; Organ Specificity; Rabbits; Recombinant Proteins; RNA, Messenger; Sequence Alignment

We examined four species of Plectropomus Oken, 1817 (Serranidae: Epinephelinae), Plectropomus areolatus (Rüppell), Plectropomus laevis (Lacepède), Plectropomus leopardus (Lacepède) and Plectropomus maculatus (Bloch) from sites off Heron Island and Lizard Island on the Great Barrier Reef, Australia (GBR), and the Gambier Islands, French Polynesia. Three new species of Neidhartia Nagaty, 1937, five new species of Prosorhynchus Odhner, 1905, and one previously described species, Prosorhynchus freitasi Nagaty, 1937, are characterised. The three species of Neidhartia, Neidhartia haywardi n. sp., Neidhartia plectropomi n. sp. and Neidhartia tyleri n. sp. are readily distinguishable morphologically. Two of the six species of Prosorhynchus (Prosorhynchus lesteri n. sp. and Prosorhynchus wrightae n. sp.) are easily distinguished from their other congeners by morphology but the other four species (P. freitasi, Prosorhynchus heronensis n. sp., Prosorhynchus munozae n. sp. and Prosorhynchus plectropomi n. sp.) are generally similar in morphology and were only distinguished initially by comparing their ITS2 rRNA sequences. Three additional taxa, one from the GBR and two from French Polynesia, were recognised as distinct on the basis that their ITS2 rRNA sequences differed from those of the new taxa described here; these species remain unnamed for the present. Inter-specific divergence observed within these genera in the ITS2 rRNA ranged from 10 to 42 base pairs (4-16 %) for species of Neidhartia and 2-57 base pairs (3-21 %) for species of Prosorhynchus. Inter-generic divergences were 42-55 base pairs (17-21 %). No intraspecific variation in the ITS2 rRNA region was observed for any of the six species for which multiple sequence replicates were obtained. Phylogenetic analysis of 12 operational taxa from Plectropomus together with sequences of three other species from epinepheline serranids demonstrated that Neidhartia and Prosorhynchus were reciprocally monophyletic with the exception that P. wrightae n. sp. fell either within or basal to the Neidhartia species. The richness of bucephalids in species of Plectropomus appears to be exceptional within the Serranidae relative to that observed in other serranid genera in the tropical Indo-West Pacific.

Topics: Animals; Bass; Cluster Analysis; DNA, Helminth; DNA, Ribosomal Spacer; Fish Diseases; Microscopy; Molecular Sequence Data; Pacific Islands; Phylogeny; Sequence Analysis, DNA; Trematoda; Trematode Infections

Streptococcosis is a common cause of pathology and mortality in fishes resulting in significant economic losses for the aquaculture industry. One etiologic agent of the disease, Streptococcus parauberis, has been associated with fish mortalities in Spain and Korea. Here we report the first identification of S. parauberis in wild finfish in Chesapeake Bay, USA. Gram-positive cocci were isolated from the spleens of striped bass, Morone saxatilis, and identified via species-specific primers and 16S rRNA gene sequencing. Biochemical characterization and antibiotic susceptibility tests were used to compare local isolates to isolates infecting aquacultured fishes and dairy cattle. This is also the first report of a plasmid in S. parauberis from any host.

Topics: Animals; Animals, Wild; Bass; Cattle; Fish Diseases; North America; Spleen; Streptococcal Infections; Streptococcus

Laribacter hongkongensis is a Gram-negative, facultative anaerobic, motile, S-shaped, urease-positive bacillus associated with invasive infections in liver cirrhosis patients and community-acquired gastroenteritis. Most cases of L hongkongensis infections occur in eastern countries. Information is lacking on the usefulness of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria important in eastern countries. Using the Bruker database extended with 21 L hongkongensis reference strains, all 240 L hongkongensis isolates recovered from patients, fish, frogs and water were correctly identified, with 224 (93.3%) strains having top match scores ≥2.0. Notably, the strain of Chromobacterium violaceum was not reliably identified although it is included in the database. MALDI-TOF MS is useful for the accurate routine identification of L hongkongensis after adding reference L hongkongensis main spectra to the database. The number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

Topics: Animals; Bass; Carps; Community-Acquired Infections; Databases, Factual; Fish Diseases; Fresh Water; Gastroenteritis; Humans; Liver Cirrhosis; Neisseriaceae; Neisseriaceae Infections; Ranidae; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Water Microbiology; Water Supply

Impulsive pile driving sound can cause injury to fishes, but no studies to date have examined whether such injuries include damage to sensory hair cells in the ear. Possible effects on hair cells were tested using a specially designed wave tube to expose two species, hybrid striped bass (white bass Morone chrysops × striped bass Morone saxatilis) and Mozambique tilapia (Oreochromis mossambicus), to pile driving sounds. Fish were exposed to 960 pile driving strikes at one of three treatment levels: 216, 213, or 210dB re 1 μPa(2)·s cumulative Sound Exposure Level. Both hybrid striped bass and tilapia exhibited barotraumas such as swim bladder ruptures, herniations, and hematomas to several organs. Hybrid striped bass exposed to the highest sound level had significant numbers of damaged hair cells, while no damage was found when fish were exposed at lower sound levels. Considerable hair cell damage was found in only one out of 11 tilapia specimens exposed at the highest sound level. Results suggest that impulsive sounds such as from pile driving may have a more significant effect on the swim bladders and surrounding organs than on the inner ears of fishes, at least at the sound exposure levels used in this study.

Topics: Animals; Bass; Construction Industry; Ear, Inner; Environmental Exposure; Fish Diseases; Hair Cells, Auditory; Hearing Loss, Noise-Induced; Noise; Oceans and Seas; Tilapia

In April 2011, 40% mortality of Largemouth Bass Micropterus salmoides juveniles occurred at a farm of Zhongshan City, Guangdong Province, China. Infected fish became lethargic, exhibited corkscrew and irregular swimming, and developed a distended abdomen and crooked body. Fish began to die within 2 d after the appearance of clinical signs. In order to analyze the pathogeny and diagnose the disease earlier, observation of clinical signs, cell infection, titer calculation, electron microscopy, immersion infection assay for fish, and nucleotide sequence analysis were carried out. Fathead minnow (FHM) cell cultures, inoculated with filtrate of liver and spleen homogenates from the diseased fish, developed the obvious cytopathic effect 46 h after inoculation in the primary culture and 24 h at the first passage. Typical rhabdovirus particles, 115-143 nm in length and 62-78 nm in diameter, were observed in infected FHM cells by direct transmission electron microscopy. The isolated virus produced a titer of 10(7.15) TCID50/mL. Immersion-Fish infected with the virus had similar clinical signs and 80% mortality with 10(2.5) LD50/mL. The data indicated that the rhabdovirus was the lethal pathogeny of the current disease. Based on nucleoprotein-gene nucleotide sequence multiple alignment analysis, the newly isolated virus is a strain of Siniperca chuatsi rhabdovirus (SCRV) under family Rhabdoviridae, which was initially isolated from Mandarin Fish Siniperca chuatsi. Up to the present, at least four virus strains have been isolated from diseased Largemouth Bass, which have had different clinical signs. Comparison of the clinical signs can help in an early diagnosis of the disease.

Topics: Animals; Aquaculture; Bass; China; Fish Diseases; Phylogeny; Polymerase Chain Reaction; Rhabdoviridae; Rhabdoviridae Infections

Vibrio anguillarum is the main causative agent of vibriosis in cultured sea bass. Unfortunately, available vaccines against this disease do not achieve the desired protection. In this study, to accomplish uptake, processing, and presentation of luminal antigens, a commercial sea bass oral vaccine against V. anguillarum was improved with the addition of recombinant fish-self tumor necrosis factor α (rTNFα), as adjuvant. To explore mechanisms, systemic and local responses were analyzed through serum specific IgM titers, gene expression, lymphocytes spatial distribution in the gut, and in vitro functional assays. We found along the trial, over expressed transcripts of genes encoding cytokines and antimicrobial molecules at the gut of rTNFα supplied group. Orally immunized fish with vaccine alone confer protection against V. anguillarum challenge throughout a short time period. In contrast, adjuvant-treated group significantly extended the response. In both cases, achieved protection was independent of serum IgM. Yet, IgT transcripts were found to increase in the gut of rTNFα-treated fish. More importantly, fish treated with rTNFα showed a dramatic change of their T lymphocytes distribution and localization in gut mucosal tissue, suggesting specific antigen recognition and further intraepithelial T lymphocytes (IEL) activation. To determine the mechanism behind IEL infiltration, we characterized the constitutive and activated pattern of chemokines in sea bass hematopoietic tissues, identifying for the first time in fish gut, an intimate relation between the chemokine ligand/receptor CCL25/CCR9. Ex-vivo, chemotaxis analyses confirmed these findings. Together, our results demonstrate that improved oral vaccines targeting key cytokines may provide a means to selectively modulate fish immune defence.

Topics: Animals; Aquaculture; Bacterial Vaccines; Bass; Chemokines, CC; Fish Diseases; Fish Proteins; Immunity, Innate; Real-Time Polymerase Chain Reaction; Receptors, CCR; Recombinant Proteins; Tumor Necrosis Factor-alpha; Vibrio; Vibrio Infections

In vitro studies have confirmed the inhibitory effect of the azol-derivative ketoconazole (KZ) on the growth of Ichthyophonus, an important pathogen causing epizootics in wild and cultured fish. We evaluated the effect of KZ in vivo in European sea bass Dicentrarchus labrax experimentally infected with the same Ichthyophonus isolate. Liposomes were used to vehiculate different doses of KZ to increase the effect on Ichthyophonus and lower the toxicity of the drug, and KZ toxicity was assessed in cultured sea bass juveniles. We also studied the effect of liposome-vehiculated KZ included in medicated food on ichthyophoniasis. KZ causes clear toxic effects in D. labrax juveniles at doses >80 mg kg-1, apparent in the reduced survival of fish and histological alterations to livers, kidneys and spleens. Fish injected with Ichthyophonus and treated with KZ dosages of ≤80 mg kg-1 d-1 presented lower ichthyophoniasis prevalence, fewer organs infected per fish, and fewer spores in the affected organs than the untreated fish. KZ seems to delay the onset of infection, but cannot stop further progression once established. However, this behaviour is not clearly reflected in the biometric and haematological data collected from these fish. We hypothesise that KZ's delaying effect would increase, if lower infective doses (more similar to natural situations) were used. The drug administration vehicle (liposomes vs. emulsions) did not affect the results. Our data confirm the potential utility of KZ in treating ichthyophoniasis and reveal its low toxicity for sea bass. Nevertheless, the optimal dose and appropriate application protocol remain to be determined.

Topics: Animals; Antifungal Agents; Bass; Dose-Response Relationship, Drug; Fish Diseases; Ketoconazole; Mesomycetozoea; Mesomycetozoea Infections

In 2007-2008, 162 samples (72 marine water samples, 90 swabs from the gills and skin) were collected from three European seabass fish farms in Eastern Adriatic. The aim of study was to determine the occurrence of Vibrio, to identify the isolated strains and to investigate their antimicrobial resistance. The comparison of the results obtained in spring and autumn periods indicated a higher Vibrio concentration in spring samplings. The greatest prevalence of Vibrio alginolyticus was on gills than on skin, whereas statistically significant differences were obtained between sampling periods with maximal prevalence in spring. Vibrio isolates from the marine water and from swabs of European seabass were analysed by DNA sequencing of partial 16S rDNA and gyrB genes and identified as V. alginolyticus. Isolates were highly susceptible to flumequine, chloramphenicol and oxytetracycline.

Topics: Animals; Bass; Drug Resistance, Bacterial; Environmental Monitoring; Fish Diseases; Mediterranean Sea; Prevalence; Vibrio alginolyticus; Vibrio Infections

Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50) and Asp(67)) and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu(71), Asp(84), Asp(95)) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

Topics: Alleles; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Base Sequence; Bass; Disease Resistance; DNA, Complementary; Evolution, Molecular; Female; Fish Diseases; Gene Expression Profiling; Gene Frequency; Genotype; Male; Molecular Sequence Data; Muramidase; Phylogeny; Polymorphism, Single Nucleotide; Recombinant Proteins

Many bucephalid species, mainly of the subfamily Prosorhynchinae, have been described from epinepheline serranids (groupers) throughout the World's Oceans. In this paper eight named prosorhynchine species are described and/or illustrated from epinepheline fishes from New Caledonia. Neidhartia lochepintade n. sp. in Epinephelus chlorostigma differs from other Neidhartia spp. in various combinations of distinct body-size, rhynchus size, previtelline and pre-mouth distance, post-testicular distance, cirrus-sac reach and egg-size. Other species are: Neidhartia haywardi Bott, Miller & Cribb, 2013 in Plectropomus leopardus; Neidhartia tyleri Bott, Miller & Cribb, 2013 in Plectropomus leopardus and Plectropomus laevis; Prosorhynchus freitasi Nagaty, 1937 in Plectropomus leopardus and Plectropomus laevis; Prosorhynchus robertsthomsoni Bott & Cribb, 2009 in Cephalopholis argus; Prosorhynchus longisaccatus Durio & Manter, 1968 in Cephalopholis urodeta, Epinephelus areolatus, Epinephelus cyanopodus and Epinephelus maculatus. Prosorhynchus luzonicus Velasquez, 1959 and Prosorhynchus sp. B. in Epinephelus coioides; Prosorhynchus serrani Durio & Manter, 1968 in Variola albimarginata and Variola louti; Prosorhynchus sp. A in Epinephelus morrhua; Prosorhynchus sp. immature in Epinephelus coeruleopunctatus. The new combination Neidhartia longivesicula (Bilqees, Khalil, Khan, Perveen & Muti-ur-Rehman, 2009) (Syn. Prosorhynchus longivesicula) is formed. Evidence from this paper and earlier molecular studies indicates that there are numerous morphologically similar prosorhynchine species in serranids, most of which show a high degree of host-specificity.

Topics: Animals; Bass; Fish Diseases; New Caledonia; Prevalence; Seawater; Trematoda; Trematode Infections

Topics: Animals; Aquaculture; Bass; Fish Diseases; Germ-Free Life; Larva; Vibrio; Vibrio Infections; Virulence

Topics: Animal Feed; Animals; Bass; Fish Diseases; Fisheries; Larva; Nodaviridae; RNA Virus Infections; Specific Pathogen-Free Organisms; Viral Vaccines

The genus Vibrio is characterized by a large number of species and some of them are human pathogens causing gastrointestinal and wound infections through the ingestion or manipulation of contaminated fishes and shellfish including Vibrio parahaemolyticus and Vibrio alginolyticus. In this study, we reported the phenotypic and molecular characterization of 9 V. parahaemolyticus and 27 V. alginolyticus strains isolated from outbreaks affecting cultured Gilthead sea bream (Sparus aurata L.) and Sea bass (Dicentrarchus labrax) along the Tunisian coast from 2008 to 2009. All isolates were tested for the presence of DNase, caseinase, protease, lipase, amylase, gelatinase, hemolytic activity and antibacterial resistance to different drugs. Randomly amplified polymorphic DNA was used to examine the genetic relatedness among the V. parahaemolyticus and V. alginolyticus strains.

Topics: Animals; Bass; Disease Outbreaks; DNA Fingerprinting; Fish Diseases; Phenotype; Random Amplified Polymorphic DNA Technique; Sea Bream; Tunisia; Vibrio alginolyticus; Vibrio Infections; Vibrio parahaemolyticus

Streptococcus iniae causes invasive infections in fresh and saltwater fish and occasional zoonoses. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. A potential target for serologically cross-protective vaccines is the M-like protein SiMA, an essential virulence factor in S. iniae that is highly conserved amongst virulent strains. The present study determined how SiMA is regulated and investigated potential as a cross-protective vaccine for fish. Electrophoretic mobility shift suggested that SiMA is regulated by the multigene regulator Mgx via a binding site in the -35 region of the simA promoter. Moreover, expression of simA and mgx was highly correlated, with the highest level of simA and mgx expression during exponential growth under iron limitation (20-fold increase in relative expression compared to growth in Todd-Hewitt broth). Based on these results, a vaccination and challenge experiment was conducted in barramundi (Lates calcarifer) to determine whether SiMA is protective against S. iniae infection and cross-protective against a different capsular serotype. The challenge resulted in 60% mortality in control fish. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection, whereas bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Moreover, recombinant SiMA protein was not protective against the challenge strain in spite of eliciting specific antibody response in vaccinated fish. Specific antibody did not increase oxidative activity or phagocytosis by barramundi macrophages. Indeed incubating S. iniae with antisera significantly reduced phagocytosis. Lack of specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous vaccine suggests that other immune parameters result in protection of challenged fish.

Topics: Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Bass; Cross Protection; Fish Diseases; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Vaccination; Virulence Factors

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Topics: Animals; Bass; Enterocytes; Fish Diseases; Germ-Free Life; Gram-Negative Bacterial Infections; Intestines; Larva; Listonella

Topics: Adjuvants, Immunologic; Animals; Bass; Brain; Disease Resistance; Fish Diseases; Immunization; Nodaviridae; Poly I-C; RNA Virus Infections; Viral Load

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.

Topics: Amino Acid Sequence; Animals; Bacteria; Bacterial Infections; Bass; Cloning, Molecular; DNA Virus Infections; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Molecular Sequence Data; Organ Specificity; Phylogeny; Polymerase Chain Reaction; Ranavirus; RNA, Messenger; Saccharomyces cerevisiae; Sequence Alignment; Sequence Analysis, DNA; Serum Amyloid A Protein

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Topics: Animals; Antigens, Bacterial; Bacterial Vaccines; Bass; DNA, Bacterial; Fish Diseases; Molecular Sequence Data; Pasteurella Infections; Photobacterium; Sequence Analysis, DNA; Vaccines, Synthetic

The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95 dph; booster: 165 dph) or by immersion (priming: 95 dph; booster: 165 dph) and subsequent i.p. injection (booster: 233 dph). Serum specific IgM and numbers of IgM(+) cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-β transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Head Kidney; Immersion; Immunoglobulin M; Injections, Intraperitoneal; Listonella; Lymphocytes; Spleen; Vaccination

Viral nervous necrosis (VNN) is a serious viral disease affecting farmed sea bass (Dicentrarchus labrax). Only scarce molecular data are available on the disease-causing betanodavirus populations in Tunisia. Therefore, we carried out the first molecular survey of betanodaviruses in farmed sea bass and sea bream (Sparus aurata) along the Tunisian coasts. Among 81 samples from five farms, 20 tested positive with RT-PCR, not only in clinical cases but also in asymptomatic fish before and during outbreaks. Positive fish were found in all farms, except in one farm investigated in the south of Tunisia. Sequencing the fragments of both genomic components (RNA1 and RNA2) in 16 isolates revealed that the Tunisian viruses were related to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. Furthermore, the newly sequenced isolates were generally highly related to one another suggesting a recent common ancestor. They also showed high identities with other isolates obtained from wild fishes in the Mediterranean, but were slightly more divergent from strains recently obtained from farmed fishes in the Mediterranean. The poor genetic diversity of the viral population along the Tunisian coasts is striking. One hypothesis is that it is the result of the maintenance of a homogenous genetic pool among infected wild fish, groupers for instance and subsequent dissemination to farmed fish over the seasons.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Genotype; Molecular Epidemiology; Molecular Sequence Data; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sea Bream; Sequence Analysis, DNA; Tunisia

Gravid females of the little-known nematode species Philometra rubra ( Leidy, 1856 ) (Philometridae) are described from specimens from the abdominal cavity of the striped bass, Morone saxatilis (Walbaum), in South Carolina and Canada. The specimens were studied with the use of light and scanning electron microscopy. This species is mainly characterized by the distribution and different sizes of cephalic papillae from the external circle, which is a unique feature compared to other philometrids. Other characteristic features are the shape of the posterior end of body, size and location of caudal projections, and the presence of a well-developed anterior esophageal inflation. The morphology of the male of P. rubra and the life cycle of this nematode are still unknown.

Topics: Abdominal Cavity; Animals; Bass; Dracunculoidea; Female; Fish Diseases; Lakes; New Brunswick; Prevalence; Quebec; Rivers; South Carolina; Spirurida Infections

Betanodaviruses are the causative agents of Viral Encephalopathy and Retinopathy (VER). To date, more than 50 species have proved to be susceptible and among them, those found in genus Epinephelus are highly represented. Clinical disease outbreaks are generally characterized by typical nervous signs and significant mortalities mainly associated with aquaculture activities, although some concerns for the impact of this infection in wild fish have been raised. In this study, the authors present the first documented report describing an outbreak of VER in wild species in the Mediterranean basin.. In late summer--early winter 2011 (September-December), significant mortalities affecting wild Dusky grouper (Epinephelus marginatus), Golden grouper (Epinephelus costae) and European sea bass (Dicentrarchus labrax) were reported in the municipality of Santa Maria di Leuca (Northern Ionian Sea, Italy). The affected fish showed an abnormal swimming behavior and swollen abdomens. During this epizootic, five moribund fish showing clear neurological signs were captured and underwent laboratory investigations. Analytical results confirmed the diagnosis of VER in all the specimens. Genetic characterization classified all betanodavirus isolates as belonging to the RGNNV genotype, revealing a close genetic relationship with viral sequences obtained from diseased farmed fish reared in the same area in previous years.. The close relationship of the viral sequences between the isolates collected in wild affected fish and those isolated during clinical disease outbreaks in farmed fish in the same area in previous years suggests a persistent circulation of betanodaviruses and transmission between wild and farmed stocks. Further investigations are necessary to assess the risk of viral transmission between wild and farmed fish populations, particularly in marine protected areas where endangered species are present.

Topics: Animals; Animals, Wild; Bass; Disease Outbreaks; Endangered Species; Fish Diseases; Italy; Mediterranean Sea; Nodaviridae; Phylogeny; Real-Time Polymerase Chain Reaction; RNA Virus Infections

Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 10(3) at day 1 postinfection to 10(8) TCID50 ml(-1) at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines.

Topics: Animals; Bass; Cell Line; Fish Diseases; Fishes; Immunoblotting; India; Nodaviridae; Real-Time Polymerase Chain Reaction; RNA Virus Infections; Virus Replication

A novel Gram-stain-negative rod-shaped gliding bacterial strain, designated 35/09(T), was isolated from diseased European sea bass (Dicentrarchus labrax L.) in Spain. Colonies were pale-yellow-pigmented with uneven edges and did not adhere to the agar. The DNA G+C content of the isolate was 31.3 mol%. 16S rRNA gene sequence analysis indicated affiliation to the genus Tenacibaculum (family Flavobacteriaceae, phylum 'Bacteroidetes'). Sequence similarities between the isolate and type strains of other members of the genus were 93.1-97.3 %. The major fatty acids (>5 % of the total fatty acids) were iso-C(15 : 0) (24.8 %), iso-C(15 : 0) 3-OH (18.0 %), anteiso-C(15 : 0) (8.1 %), C(15 : 1)ω6c (6.9 %) and iso-C(15 : 1) (6.2 %). Genotypic and phenotypic data indicate that strain 35/09(T) should be classified as a representative of a novel species in the genus Tenacibaculum, for which the name Tenacibaculum dicentrarchi sp. nov. is proposed; the type strain is 35/09(T) ( = CECT 7612(T) = NCIMB 14598(T)).

Topics: Animals; Bacterial Typing Techniques; Base Composition; Bass; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Fish Diseases; Flavobacteriaceae Infections; Genes, rRNA; Genotype; Molecular Sequence Data; Phenotype; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Spain; Tenacibaculum

A high prevalence of intersex or testicular oocytes (TO) in male smallmouth bass within the Potomac River drainage has raised concerns as to the health of the river. Studies were conducted to document biomarker responses both temporally and spatially to better understand the influence of normal physiological cycles, as well as water quality and land-use influences. Smallmouth bass were collected over a 2-year period from three tributaries of the Potomac River: the Shenandoah River, the South Branch Potomac and Conococheague Creek, and an out-of-basin reference site on the Gauley River. The prevalence of TO varied seasonally with the lowest prevalence observed in July, post-spawn. Reproductive maturity and/or lack of spawning the previous spring, as well as land-use practices such as application of manure and pesticides, may influence the seasonal observations. Annual, seasonal, and site differences were also observed in the percentage of males with measurable concentrations of plasma vitellogenin, mean concentration of plasma vitellogenin in females, and plasma concentrations of 17β-estradiol and testosterone in both sexes. Bass collected in the South Branch Potomac (moderate to high prevalence of TO) had less sperm per testes mass with a lower percentage of those sperm being motile when compared to those from the Gauley River (low prevalence of TO). An inverse relationship was noted between TO severity and sperm motility. An association between TO severity and wastewater treatment plant flow, percent of agriculture, total number of animal feeding operations, the number of poultry houses, and animal density within the catchment was observed.

Topics: Agriculture; Animals; Bass; Biomarkers; Disorders of Sex Development; Endocrine Disruptors; Environmental Monitoring; Epidemiological Monitoring; Female; Fish Diseases; Male; Pesticides; Reproduction; Rivers; Seasons; Testis; Vitellogenins; Water Pollutants, Chemical

This study assessed the anisakid nematode distribution pattern in the fish collected from coasts of Mediterranean Sea, Egypt, during the period September 2010-April 2011. Two hundred thirty out of 300 (76.7%) Dicentrarchus labrax (European seabass) marine fishes belonging to family Moronidae were dissected and found to be infected with larva three nematodes. The larvae had been studied by light and scanning electron microscopy. The present work represents the first record of the presence of the parasite in this fish in the Mediterranean Sea. The concentrations of some heavy metals (Pb, Zn, Fe, Cd, Cu, Mn, Ni) in parasites as well as in tissues of fish were measured. The presented results showed that the nematode parasites are able to accumulate heavy metals in their tissues and in some cases that they are able to accumulate large amounts of heavy metals in a higher amount than host tissues. This demonstrated their sustainability as bioindicators of environmental pollution by removing heavy metals and help in the survival of fish.

Topics: Animals; Anisakiasis; Anisakis; Bass; Egypt; Environmental Monitoring; Fish Diseases; Larva; Mediterranean Sea; Metals, Heavy; Microscopy, Electron, Scanning; Water Pollutants; Water Pollution

The Asian seabass is euryhaline, therefore it is interesting to describe the infestation and survival of caligids at varying salinity on the host. In this study, two different brackish water culture systems with monoculture and polyculture practices were investigated for the occurrence of Caligus spp. on Lates calcarifer. Polyculture practices mainly consisted of snapper (Lutjanus spp.), grouper (Epinephelus spp.) and seabass (L. calcarifer), while the monoculture was stocked with only seabass. A total of 777 Caligus spp. specimens were isolated from the sampling in 2009, consisting of three species; Caligus chiastos, Caligus epidemicus and Caligus rotundigenitalis. In 2011, the total specimen was increased to 3110 and two additional species were found; Caligus punctatus and one unknown species (Caligus sp.). A 98.6% of the total examination was represented by C. epidemicus. Constant presence of C. epidemicus was observed throughout the study, regardless the differences in between culturing practices and systems. This species was able to survive within wide salinity range, from 5 to 28 ppt. The other isolated species (C. chiastos, C. punctatus, C. rotundigenitalis and Caligus sp.) were only found infesting in polyculture cages with the salinity ranging from 25 to 28 ppt. Despite accounts for less than 2% of the total specimens, these species may able to produce a challenge for L. calcarifer polyculture farming activity due to their capability for host switching. The present study revealed the potential risk for cross-species transmission in polyculture practices.

Topics: Animals; Bass; Copepoda; Ectoparasitic Infestations; Fish Diseases; Fisheries; Malaysia; Salinity

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.

Topics: Animals; Aquaculture; Bass; Enterococcaceae; Fish Diseases; Intestinal Mucosa; Probiotics; Sea Bream; Vibrio; Vibrio Infections

Pearsonellum lemusi n. sp. (Digenea: Aporocotylidae) infects the blood vascular system of the gag grouper, Mycteroperca microlepis (Perciformes: Serranidae), in the north central Gulf of Mexico, approximately 80 km south of Dauphin Island, Alabama (29°34'09″N, 88°22'16″W). The new species can be most easily differentiated from its only congeners Pearsonellum corventum Overstreet and Køie, 1989 (type species) and Pearsonellum pygmaeus Nolan and Cribb, 2004 , both of which infect Australian serranids, by the combination of having a large adult body (3,237 × 570 µm), a cecal intersection comprising an elongated medial channel, anterior ceca >10% of total body length, ovary narrower than testis, and pre-ovarian uterus not looping between testis and ovary. The embryonated eggs of the new species infect gill epithelium, are spheroid, and measure 25-30 µm in diameter. Sympatric Gulf of Mexico serranids were negative for aporocotylid infections: coney, Cephalopholis fulva (n  =  1); Nassau grouper, Epinephelus striatus (3); red grouper, Epinephelus morio (32); yellowedge grouper, Epinephelus flavolimbatus (1); rock hind, Epinephelus adscensionis (1); red hind, Epinephelus guttatus (2); Warsaw grouper, Epinephelus nigritus (3); graysby, Cephalopholis cruentata (1); black grouper, Mycteroperca bonaci (1), and tattler, Serranus phoebe (2). The new species is the first aporocotylid described from a serranid outside of the southwestern Pacific Ocean. The diagnosis of Pearsonellum Overstreet and Køie, 1989 is herein emended to include anterior sucker having concentric rows of spines anterior to mouth, pharynx absent, esophagus length <1/2 total body length, vas deferens connecting with cirrus sac anteromedially, ovary occupying posterior 1/4-1/3 of body, primary vitelline duct dextral, and oviducal seminal receptacle extending posteriad in parallel with lateral body margin, not transverse nor constricted anteriorly or posteriorly by sharp bends or kinks.

Topics: Alabama; Animals; Bass; Fish Diseases; Microscopy, Interference; Seawater; Trematoda; Trematode Infections

We report the binding kinetics of fish-infected grouper nervous necrosis viruses (NNV) and selected antimicrobial peptides (AMPs) by nanomechanical detection. AMPs, the vital member in an innate immunity, are promising candidates in the fight against pathogens due to their broad range of antimicrobial activity and low toxicity. Grouper NNV primarily cause mass mortality of many marine cultured fish species, and two selected AMPs in this study were found to inhibit viruses by agglutinating its virions to form aggregates. The binding activity of NNVs with functionalized AMPs onto a sensing microcantilever yielded induced surface stresses, indicating high binding strength of molecular interaction. The binding affinity and kinetic rate constants of molecular recognition events calculated for NNV-AMP(TH1-5) compared to NNV-AMP(cSALF) were found to be 2.1-fold and 4.43-fold, respectively, indicating TH1-5 effectively bind with NNV more than cSALF. Moreover, a microscopic X-ray photoelectron spectroscopy technique was employed for further validation of pre- and post-NNV binding onto peptides-functionalized sensing surface. An increase in the spectrum and intensity of the P 2p and N 1s elements for the post-NNV binding was clearly shown to ensure the existence of phosphate groups and nitrogen-containing ring structures of specific NNV-TH1-5 interaction. Therefore, the microcantilever biosensing technique provides a potential and useful screening of AMPs for affinity to NNVs.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Biosensing Techniques; Equipment Design; Equipment Failure Analysis; Fish Diseases; Micro-Electrical-Mechanical Systems; Nodaviridae; Protein Binding; Protein Interaction Mapping; Reproducibility of Results; Sensitivity and Specificity; Viral Tropism; Virology; Virus Diseases

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii .

Topics: Animals; Base Sequence; Bass; DNA, Ribosomal; Fish Diseases; Gills; Molecular Sequence Data; Multigene Family; Myxobolus; Parasitic Diseases, Animal; Phylogeny; Polymerase Chain Reaction; Prevalence; Rivers; RNA, Ribosomal, 18S; Seasons; Sequence Analysis, DNA; Spores; Virginia; West Virginia

This is the first pathological description of 'scale drop syndrome' (SDS) in Asian seabass, Lates calcarifer Bloch. Cumulative mortality was estimated at 40-50%. The vasculitis in all major organs including the skin and associated tissue necrosis was distinctive. The dermis overlying scale beds was often necrotic and associated with scale loss. Necrosis of splenic ellipsoids, renal glomeruli and choroid rete glands of eye were further hallmarks of a disease with systemic vascular involvement. The brain was not spared vascular damage, and the resulting multifocal encephalomalacia probably accounts for the spiral swimming behaviour in some affected fish. Other lesions included accentuated hepatic lobulation and gastric gland necrosis. Nuclear chromatin margination and karyolysis in hepatocytes, renal tubular epithelium and gastric and intestinal epithelium suggest specific targeting of cells. Basophilic cytoplasmic inclusions were present in spleen, kidney, liver, heart and choroid rete, but they were not prominent. Using transmission electron microscopy, two morphological forms of virions were observed: single- and double-enveloped hexagonal virions. Based on size and morphology, these virions resemble iridovirus or herpesvirus. The cause of SDS is unknown, but the pathological changes, especially the vasculitis, suggest an infectious aetiology, possibly viral.

Topics: Animals; Asia; Bass; Fish Diseases; Microscopy, Electron, Transmission; Syndrome; Virion

The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 10(7) CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1-4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1-4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum.

Topics: Animals; Bass; Blood Proteins; Dietary Supplements; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Leukocytes; Listonella; Muramidase; Peroxidase; Phagocytosis; Reactive Nitrogen Species; Reactive Oxygen Species; Time Factors

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), one of the most serious diseases in over 30 species of cultured marine fishes worldwide. Although several kinds of NNV vaccines have been developed, none of these vaccines have been yet marketed. Here, we demonstrate the potentiality of a live NNV vaccine for sevenband grouper Epinephelus septemfasciatus at a low rearing temperature (17°C). Moreover, we investigated the kinetics of NNV infectivity titer in fish reared at low and optimum temperatures (17°C and 26°C) for VNN onset to determine why sevenband grouper reared at 17°C survive NNV infection. In pathogenicity tests of NNV, fish mortality was reduced by decreasing the fish rearing temperature, and no mortality was observed in fish reared at 17°C regardless of the infection method. During fish acclimation to the optimum temperature of VNN onset (26°C), increased mortalities were observed in the survivors from the 1st NNV-infection. Little or no mortality was observed in the 2nd NNV-infection. Thus, it was demonstrated that the survivors from the 1st NNV-infection mounted a specific protective immune response against NNV. Especially, in the fish infected with NNV by immersion at 17°C, only two out of 30 fish died until the end of the 2nd infection (total survival rate: 93.3%), suggesting a positive potentiality for a live NNV vaccine. In the analysis of NNV kinetics in the fish reared at 26°C, NNV rapidly multiplied up to ≥ 10(9)TCID(50)g(-1) before fish began to die, and the critical level of NNV was around 10(10)TCID(50)g(-1). Probability of NNV multiplication reduced by decreasing the inoculated NNV dose, but NNV multiplication rate was independent of the NNV dose. The threshold of NNV for fish mounting a protective immune response was around >10(4)TCID(50)g(-1). Against this, in the fish reared at 17°C, NNV slowly multiplied in comparison with that in fish at 26°C. NNV titer in the peak was at 10(7.1 ± 1.4)TCID(50)g(-1), which was far behind the critical level of NNV but still greatly above the threshold level (10(4)TCID(50)g(-1)). Thus, it was demonstrated that the multiplication rate of NNV in vivo was strongly correlated to NNV virulence and fish mortality, and down-regulation of NNV multiplication in fish reared at 17°C enabled control of VNN onset for development of a live NNV vaccine.

Topics: Animals; Bass; Fish Diseases; Nodaviridae; RNA Virus Infections; Survival Analysis; Temperature; Vaccines, Attenuated; Viral Vaccines

A nodavirus isolated from red-spotted grouper (Epinephelus akaara) larvae in China has been subjected to genome analysis. The full-length genome sequences of RNA1 and RNA2 were determined, and the 5'-non-coding region (NCR) and 3'NCR sequences were determined by 5' rapid amplification of cDNA ends (RACE) and 3'RACE. RNA1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 110.74 kDa. RNA2 is 1,433 nt long and contains a 338-amino-acid major ORF encoding coat protein with a calculated molecular mass of 37.059 kDa. Multiple alignment and phylogenetic analysis clearly supported including this virus in the species Redspotted grouper nervous necrosis virus, genus Betanodavirus, family Nodaviridae.

Topics: Animals; Bass; China; Cluster Analysis; Fish Diseases; Genome, Viral; Molecular Sequence Data; Molecular Weight; Nodaviridae; Open Reading Frames; Phylogeny; RNA Virus Infections; RNA, Viral; Sequence Analysis, DNA; Viral Proteins

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Topics: Air Sacs; Animals; Bass; Enterocytes; Esophagus; Fish Diseases; Germ-Free Life; Green Fluorescent Proteins; Host-Pathogen Interactions; Larva; Vibrio; Vibrio Infections

Nervous necrosis virus (NNV) is the cause of viral nervous disease, which is a serious constraint on production for grouper aquaculture. Real-time PCR is commonly used to detect and quantify NNV, has the disadvantages of being expensive and technically demanding. In this study, an immunomagnetic reduction (IMR) assay was developed as a rapid and cost-effective alternative to real-time PCR. This method used magnetic nanoparticles conjugated with antibodies specific for viral surface antigens to detect NNV in grouper tissue samples. The association of NNV with the antibody-conjugated magnetic particles resulted in a reduction in magnetic signal, which was strongly correlated with the concentration of NNV, as determined by real-time PCR. Grouper larvae were prepared for testing using a viral extraction buffer which provided a rapid, 15-min method of extracting viral antigens and had an extraction efficiency of higher than 80%. In addition, this study proposes using magnetic nanoparticles as labeling markers and as an assaying reagent for NNV. The magnetic nanoparticles are functionalized with antibodies against the viral surface of NNV and are able to associate specifically with NNV. The reduction of the magnetic signals comes from the association between magnetic particles and NNV, and relates to the concentration of NNV. The results show that the detected concentrations of NNV are highly correlated to those detected by real-time PCR.

Topics: Animals; Antibodies, Viral; Bass; Fish Diseases; Immunomagnetic Separation; Nodaviridae; Rabbits; Sensitivity and Specificity; Viral Load

Leucocyte cell-derived chemotaxin 2 (LECT2) was first identified as a chemotactic factor and has been subsequently proven to be a multifunctional protein that mediates the regulation of liver regeneration, carcinogenesis and Natural killer T (NKT) cell homeostasis in mammals. In fish, it has been recently found to be critical for the inflammatory response to stimuli. However, the in vivo function of LECT2 in fish remains obscure. Base on the full-length cDNA of the Epinephelus akaraa LECT2 (EaLECT2) gene we previously isolated, we sought to analyze its genomic structure and context. The genomic DNA of the EaLECT2 gene spans 2866bp from the transcription start site to the termination codon. As in most LECT2 genes in other vertebrates, the EaLECT2 genomic DNA contains four exons and three introns. An analysis of the promoter region revealed the presence of a TATA box and several putative transcription factor-binding sites. And transcriptional activity analysis suggested that most basal DNA regulatory elements required for EaLECT2 transcriptional activity might be contained within the 581bp region upstream of the transcription start codon. A real-time PCR analysis showed that the EaLECT2 expression levels were slightly increased in the head kidney, liver, gill and brain by bacterial challenge with Vibrio harveyi. Furthermore, the transcriptional level of the EaLECT2 gene in the liver was significantly up-regulated within 1h and reached its peak level at 12h post-stimulation. Higher levels of LECT2 expression were also observed in head kidney in challenged individuals.The expression pattern demonstrates the role of EaLECT2 in the immune response and its functions under other conditions. Additionally, we found that the recombinant EaLECT2 could be expressed as a soluble protein using a prokaryotic expression system with the expression vector pET32a(+) and the soluble protein was further proved to be the recombinant EaLECT2 with the rat antiserum against EaLECT2 we obtained. This work provides a unique basis for substantial work in future projects.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Chemotactic Factors; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gene Order; Genome; Leukocytes; Molecular Sequence Data; Promoter Regions, Genetic; Random Allocation; Vibrio Infections

The objective of the present study was to determine the effect of Se inclusion in high-DHA and vitamin E microdiets (5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight; 5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight supplemented with Se) in comparison with a control diet (1 g DHA/100 g dry weight and 150 mg vitamin E/100 g dry weight) on sea bass larval growth, survival, biochemical composition, malonaldehyde (MDA) content, muscle morphology and antioxidant enzymes (AOE), insulin-like growth factors (IGF) and myosin expression. For a given DHA and vitamin E dietary content, Se inclusion favoured larval total length and specific growth rate, and reduced the incidence of muscular lesions, MDA contents and AOE gene expression. In contrast, IGF gene expression was elevated in the 5/300 larvae, suggesting an increased muscle mitogenesis that was corroborated by the increase in mRNA copies of myosin heavy chain. The results of the present study denoted the beneficial effect of Se not only in preventing oxidative stress, as a glutathione peroxidase cofactor, but probably due to other as yet unknown physiological functions.

Topics: Animals; Aquaculture; Bass; Diet; Docosahexaenoic Acids; Fish Diseases; Glutathione Peroxidase; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Larva; Malondialdehyde; Microscopy, Electron, Transmission; Muscles; Myosin Heavy Chains; Oxidative Stress; RNA, Messenger; Selenium; Superoxide Dismutase; Vitamin E

The present study investigated the effect of 1.0% chitin and chitosan supplementation diets on haematology and immune response in Kelp grouper, Epinephelus bruneus against protozoan parasite, Philasterides dicentrarchi. The red blood cells (RBC), white blood cells (WBC), haemoglobin levels, lymphocytes, monocytes, and neutrophils significantly increased in kelp grouper fed with chitin or chitosan enriched diets against P. dicentrarchi. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and thrombocytes did not significantly change against pathogen. The phagocytic activity, respiratory burst activity, complement activity, antiprotease activity, and α2-macroglobulin were significantly enhanced in fish fed with 1% chitin and chitosan diet on weeks 2 and 4. The lysozyme activity, total protein, and myeloperoxidase activity significantly increased in fish fed with chitin or chitosan supplementation diet from weeks 1 to 4 against pathogen. The cumulative mortality was found low in fish fed with chitin and chitosan enriched diets than those of control against pathogen. The present study suggests that supplementation of 1.0% chitin or chitosan in diets positively enhances immune response and affords disease resistance in kelp grouper, E. bruneus against P. dicentrarchi infection.

Topics: alpha-Macroglobulins; Animals; Bass; Blood Cell Count; Blood Proteins; Chitin; Chitosan; Ciliophora Infections; Complement System Proteins; Diet; Erythrocyte Indices; Fish Diseases; Fisheries; Hemoglobins; Macrophages; Muramidase; Oligohymenophorea; Peroxidase; Phagocytosis; Protease Inhibitors; Respiratory Burst

The efficacy of poly D,L-lactide-co-glycolic acid (PLGA)-liposome (L) encapsulated oligodeoxynucleotides with unmethylated deoxycytidyl-deoxyguanosine motifs (CpG-ODNs) on innate and adaptive immune response and disease resistance in kelp grouper (Epinephelus bruneus) against Vibrio alginolyticus at weeks 1, 2, and 4 is reported. The superoxide dismutase (SOD), respiratory burst, and lysozyme activities significantly increased in E. bruneus when immunized with ODN, PLGA+ODN, L+ODN, and PLGA+L+ODN on weeks 2 and 4. The serum complement activity was significantly enhanced with L+ODN and PLGA+L+ODN on week 1 while it increased with PLGA+ODN, L+ODN, and PLGA+L+ODN on weeks 2 and 4. The antibody titre consistently was increased with PLGA or L encapsulated with ODN (PLGA+ODN, L+ODN, and PLGA+L+ODN) from weeks 1 to 4. The cumulative mortality was 20% each in PLGA+ODN administered groups and 15% each in ODN, L+ODN, and PLGA+L+ODN groups during a period of 30 days. The present study suggests that PLGA-liposome encapsulated ODN has the potential to modulate the immune system and can serve as a useful tool for further design of immunoprophylatic nano drug formulations against bacterial diseases.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bass; Fish Diseases; Immunity, Innate; Lactic Acid; Liposomes; Muramidase; Oligodeoxyribonucleotides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Respiratory Burst; Superoxide Dismutase; Vibrio alginolyticus; Vibrio Infections

The CD83 cell surface marker is an important and intriguing component of immune system. It is considered the best marker for mature human dendritic cells, but it is also important for thymic development of T cells, and it also plays a role as a regulator of peripheral B-cell function and homeostasis. A CD83-like molecule was identified in sea bass (Dicentrarchus labrax) by EST sequencing of a thymus cDNA library; the CD83 cDNA is composed of 816 bp and the mature CD83 peptide consists of 195 amino acids, with a putative signal peptide of 18 amino acids and two possible N-glycosylation sites. The comparison of sea bass CD83 sequence with its homologues in other fish species and mammals shows some differences, with two cysteine residues conserved from fish to mammals and a high variability both in the total number of cysteines and in mature CD83 sequence polypeptide length. Basal transcripts levels of CD83 mRNA are highest in liver, followed by thymus. The in vitro treatment of head kidney leukocytes with LPS resulted in a down-regulation on CD83 mRNA leves both after 4 and 24 h, whereas with poly I:C an up-regulation after 4h followed by a down-regulation at 24 h was observed. An in vivo infection of sea bass juveniles with nodavirus induced an increase of CD83 expression on head kidney leukocytes both after 6 and 24 h and a decrease after 72 h. On the other hand, an in vivo infection with Photobacterium damselae bacteria induced a decrease of CD83 transcript levels after 6 and 24 h and an increase after 72 h. These findings suggest in sea bass CD83 expression could be modulated by viral and bacterial immune response.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Antigens, CD; Bass; CD83 Antigen; Cells, Cultured; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunoglobulins; Leukocytes; Lipopolysaccharides; Liver; Membrane Glycoproteins; Molecular Sequence Data; Nodaviridae; Photobacterium; Poly I-C; RNA Virus Infections; Sequence Alignment; Sequence Homology, Amino Acid; Thymus Gland

We investigate the efficacy of poly D,L-lactide-co-glycolic acid (PLGA)-encapsulated vaccine on innate and adaptive immune response in kelp grouper (Epinephelus bruneus) against Uronema marinum at weeks 1, 2, and 4. The respiratory burst (RB) activity, complement activity, and α2-macroglobulin were significantly enhanced in fish immunization with vaccine on week 4 whereas vaccine and PLGA-encapsulated vaccine from weeks 1 to 4. The serum lysozyme activity, antiprotease activity, and antibody level were significantly enhanced in fish immunized with vaccine and PLGA-encapsulated vaccine on weeks 2 and 4. The cumulative mortality was low in PLGA-encapsulated vaccine with 20% whereas high in PLGA and vaccine with 40% and 30%. The results from the present study suggest that PLGA-encapsulated vaccine is useful for further design of immunoprophylatic nano formulation against scuticociliatosis.

Topics: alpha-Macroglobulins; Animals; Antibodies; Bass; Biocompatible Materials; Ciliophora Infections; Complement System Proteins; Drug Carriers; Fish Diseases; Lactic Acid; Microspheres; Muramidase; Oligohymenophorea; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protease Inhibitors; Respiratory Burst; Vaccines

Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.

Topics: Animals; Bass; Cloning, Molecular; DNA Virus Infections; Fish Diseases; Fish Proteins; Gene Expression Profiling; Iridovirus; Phylogeny; Thioredoxins

The immunomodulatory effect of Siegesbeckia glabrescens extract-supplementation diets on innate immune response and disease resistance of kelp grouper, Epinephelus bruneus against Vibrio parahaemolyticus at weeks 1, 2, and 4 is reported. The serum lysozyme activity was significantly enhanced with any enriched diet from weeks 1-4 when compared to control diet (0%). The alternative complement haemolytic activities significantly were enhanced with all enriched diets on weeks 2 and 4 whereas the cellular reactive oxygen species (ROS) was significantly enhanced only with 1.0% and 2.0% diets. The reactive nitrogen intermediate (RNI) value was significantly enhanced with any enriched diet on weeks 2 and 4, but on first week it did not differ from control. The myeloperoxidase (MPO) production significantly increased with 1.0% and 2.0% diets from second week onwards; with other enriched diets the increase manifested on fourth week; but during first week it did not vary from that of the control with any enriched diet. The protection in terms of cumulative mortality was the least being 25% and 20% when fed with 1.0% and 2.0% diets. The present results indicate that feeding kelp grouper with S. glabrescens extract enriched diet at 1.0% and 2.0% levels significantly enhance the immunological parameters, increase the disease resistance and minimize the cumulative mortality in E. bruneus against V. parahaemolyticus.

Topics: Animals; Asteraceae; Bass; Complement Pathway, Alternative; Dietary Supplements; Disease Resistance; Fish Diseases; Muramidase; Peroxidase; Plant Extracts; Reactive Nitrogen Species; Reactive Oxygen Species; Time Factors; Vibrio Infections; Vibrio parahaemolyticus

Topics: Animals; Aquaculture; Bass; Fish Diseases; Flavobacteriaceae Infections; Greece; Phylogeny; Sea Bream; Tenacibaculum

During investigations of young-of-the year smallmouth bass ( Micropterus dolomieu ) mortalities in the Susquehanna River, Pennsylvania, U.S.A. and affected tributaries, raised areas were noted in the muscle in the vicinity of the caudal peduncle. The raised areas were caused by plasmodia of a myxozoan parasite. Spores found within plasmodia were similar to those of Myxobolus inornatus previously described from the caudal peduncle of fingerling largemouth bass ( Micropterus salmoides ) in Montana. Here, M. inornatus is redescribed based on histologic comparisons and spore measurements. The addition of spore photographs, line drawings, a voucher specimen, and partial small-subunit ribosomal (rSSU) DNA gene sequence are new in this study. This is also the first description of M. inornatus from smallmouth bass. The plasmodia of M. inornatus were grossly observed at the base of the caudal and dorsal fins and were 280.3 ± 33.5 (range 77.1-920.3) μm long and 320.6 ± 41.0 (range 74.85-898.4) μm wide. In some instances, plasmodia of M. inornatus were large enough to rupture the epidermis or were associated with misaligned vertebrae. The slightly pyriform spores were 11.3 ± 0.2 (range 8.6-17.4) μm in length and 8.6 ± 0.2 (range 7.1-13.7) μm wide with an iodinophilous vacuole and a sutural ridge with 8 to 10 sutural folds. The SSU rDNA gene sequence places M. inornatus in a sister group with Myxobolus osburni .

Topics: Animals; Base Sequence; Bass; DNA, Ribosomal; Fish Diseases; Molecular Sequence Data; Muscle, Skeletal; Myxobolus; Parasitic Diseases, Animal; Pennsylvania; Phylogeny; Rivers; Sequence Analysis, DNA; Spores; Subcutaneous Tissue

European sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5×10(7) CFUmL(-1) for 1h. Fish either received no treatment or oral treatment with Artemia franciscana (Kellog) nauplii enriched with oxolinic acid, or bath treatments with oxolinic acid. Medication commenced 1day following challenge and was performed on days 1, 3 and 5 post-challenge at a dosage of 20mgL(-1) for 2h for bath treatments, while two doses each of 750 nauplii per fish were administered daily for five consecutive days in oral treatments. Cumulative mortality reached 96% for the unmedicated challenged group, 32% in the group receiving bath treatments and 17% in the group receiving medicated nauplii. Pharmacokinetic parameters of oxolinic acid were calculated in sea bass larvae, for both treatments. Steady-state concentrations of oxolinic acid of 48.0 and 75.2μgg(-1) were achieved for bath treatment and oral treatment, respectively, while the elimination half-life was calculated to be 25.1h for bath treatment and 21.7h for oral treatment.

Topics: Animals; Anti-Bacterial Agents; Artemia; Bass; Fish Diseases; Immersion; Oxolinic Acid; Vibrio Infections

The effects of dietary mannan oligosaccharides (MOS; 4 g kg(-1) ; Bio-Mos, Alltech Inc, USA) in diets for European sea bass, Dicentrarchus labrax (L.), juveniles in relation to disease and stress resistance, combining intestinal infection with Vibrio anguillarum and stress challenge by confinement, were assessed in this study. After 8 weeks of MOS supplementation, fish were exposed to a pathogen challenge test against V. anguillarum by direct gut inoculation combined with a confinement stressor panel. Cumulative mortality of fish fed MOS caused by anally inoculated V. anguillarum decreased from 66% to 12.5% and from 54.1% to 25% in infected and infected + stressed fish, respectively, compared to fish fed control diet. Results for European sea bass revealed a positive effect of MOS dietary inclusion on disease resistance, in terms of cumulative mortality, against gut inoculated V. anguillarum, as well as reduced effects of stress on microbiota diversity. Both of these findings, together with the enhanced innate immune response and the higher gut mucus production and density of eosinophil granulocytes in gut mucosa obtained in previous studies after MOS supplementation (Torrecillas et al. 2007, 2011a,b) suggest that general reinforcement of the innate immune system, and particularly of the intestinal barrier efficiency, is the main defence mechanism of European sea bass fed MOS against pathogenic microorganisms.

Topics: Animals; Bass; Dietary Supplements; Fish Diseases; Hydrocortisone; Intestines; Mannans; Stress, Physiological; Survival Analysis; Vibrio; Vibrio Infections

In the present study, out of 200, 120 (60%) Plectropomus maculates fish were found to be naturally infected with Kudoa sp. The infection was intensive and appeared as clusters of ovoid to ellipsoidal plasmodia being restricted to the cardiac muscles. More than 100 plasmodia were counted per infected heart and measured 1.53 ± 0.2 (1.2-2.5) × 0.65 ± 0.2 (0.63-0.80) mm. On the basis of spore morphology, the parasite was identified as Kudoa sp. The spore measures 4.8 ± 0.3 (4.7-6.8) × .0 ± 0.3 (4.6-6.5) μm. The four polar capsules were pyrifom in shape measuring 1.4 ± 0.2 (1.3-3.5) × 1.2 ± 0.2 (1.1-2.2) μm. Transmission electron microscopy showed that the plasmodia were bordered by a single membrane which invaginates into pinocytotic canals. Adjacent to the plasmodial wall, the generative cells and the early pansporoblasts were located peripherally. The developmental stages characterizing sporogenesis, capsulogenisis, and valvogenesis of the present parasites were ultrastructurally studied.

Topics: Animals; Bass; Fish Diseases; Heart; Indian Ocean; Microscopy, Electron, Transmission; Myocardium; Myxozoa; Parasite Load; Parasitic Diseases, Animal; Spores, Protozoan

Cultured juvenile white seabass Atractoscion nobilis (WSB) can suffer from intraocular emphysemas and exophthalmia in the hatchery environment. To identify the cause, two size-groups of WSB were exposed to five gas saturation levels, ranging from 98% to 122% total gas pressure (TGP), over a 96-h exposure period in 18 degrees C and 23 degrees C seawater. Histological examination revealed that the gross and subgross lesions associated with gas supersaturation included corneal and orbital emphysema, along with subretinal, optic nerve, and iridial hemorrhage. Corneal emphysema was the most prominent gross lesion, with the severity and prevalence increasing between size-groups and water temperatures as TGP increased. Following the same pattern was orbital emphysema, which affected more than 93% of the fish examined and caused hemorrhage in the subretinal space, around the optic nerve, in the iris, or a combination thereof. Iridial hemorrhage occurred in 91% of the fish examined and decreased significantly with fish size. The prevalence and severity of hemorrhage in the subretinal space increased significantly with TGP and fish size but not with temperature. Optic nerve hemorrhage was absent in small fish exposed at 18 degrees C but increased significantly with temperature and fish size. The reverse was true for the large fish.

Topics: Air Pressure; Animals; Bass; Body Size; Embolism, Air; Emphysema; Eye Diseases; Fish Diseases; Hemorrhage; Optic Nerve

The European sea bass (Dicentrarchus labrax) is, along with the gilthead sea bream (Sparus aurata), one of the most extensively cultured species in European aquaculture productions. Massive mortalities may be caused by bacterial or viral infections in intensive aquaculture production. Evaluation of the efficacy of an oral vaccine against Vibrio anguillarum (Aquavac Vibrio Oral) in sea bass revealed specific immune gene expression profiles in the gut as well as protection of fish. In the present study, we performed RNA SEQ in two different tissues: the hind gut and the head kidney. For each tissue, one control sample (where a sample presents a pool of four to five individuals) and one sample after oral vaccine against V. anguillarum were submitted to 454 next-generation sequencing. In total, 269,043 sequences were obtained, 143,007 for head kidney and 125,036 for gut. The read lengths ranged from 40 to 706 bp with an average length of 348 bp. The total number of clustered sequences for head kidney is accounting to 49,089 (∼34 %) and for gut to 71,676 (~57 %). Differential expression was detected for 496 transcripts in head kidney and for 336 in gut. The results not only enrich the present collection of expressed sequence tag sequences including rare transcripts like leukocyte immune-type receptors, cullin, or supervillin but also show the efficacy of oral vaccination against V. anguillarum.

Topics: Animals; Aquaculture; Bacterial Vaccines; Bass; Computational Biology; DNA Primers; DNA, Complementary; Fish Diseases; Gastrointestinal Tract; Gene Expression Profiling; Head Kidney; Sequence Analysis, RNA; Vibrio Infections

Two experiments were conducted to test the effectiveness of different therapeutants against a mixed infection of Aeromonas hydrophila and Flavobacterium columnare in sunshine bass. Experiment 1 evaluated copper sulphate, florfenicol-medicated feed and potassium permanganate (KMnO(4) ) against a natural mixed infection. Experiment 2 further evaluated copper sulphate as a treatment to control an experimental mixed infection. In experiment 1, naturally infected untreated fish had the lowest final survival per cent, at 71%, while florfenicol-medicated feed at 15mgkg(-1) body weight for 10days or copper sulphate at 2.1mgL(-1) (1% of the total alkalinity) for 24h produced the highest final survivals, at 90% and 88%, respectively. The final survival of the naturally infected fish administered florfenicol-medicated feed was significantly different (P<0.1) from the untreated fish. The survival curves for the florfenicol and the copper sulphate at 2.1mgL(-1) were significantly improved from the untreated fish. In experiment 2, fish were challenged by waterborne exposure to A. hydrophila and F. columnare and either not treated or treated with copper sulphate at 2.1mgL(-1) . At the end of experiment 2, the per cent survival of the challenged fish treated with copper sulphate (99%) was significantly higher (P<0.05) than the non-treated (61%). The results illustrate clear benefit of florfenicol and copper sulphate against a mixed infection of A. hydrophila and F. columnare.

Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bass; Coinfection; Copper Sulfate; Female; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gram-Negative Bacterial Infections; Male; Potassium Permanganate; Treatment Outcome

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including β-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.

Topics: Animals; Aquaculture; Bass; DNA Virus Infections; Fish Diseases; Iridovirus; Vaccines, Inactivated; Viral Vaccines

Viral nervous necrosis (VNN) virus produces great mortalities in fish having susceptible and reservoir species between the most important marine aquaculture species. Cell-mediated cytotoxicity (CMC) is considered, towards the interferon (IFN), the most important mechanism of the immune response to fight against viral infections but it has been very scarcely evaluated. We aimed to evaluate the effects of VNNV infection in the reservoir gilthead seabream (Sparus aurata) and susceptible European sea bass (Dicentrarchus labrax). Firstly, after experimental infection we found mortalities in the sea bass (55%) but no in the seabream. Moreover, VNN virus replicates in the brain of both species as it was reflected by the high up-regulation of the Mx gene expression. Interestingly, the head-kidney leucocyte cell-mediated cytotoxic activity was significantly increased in both species reaching highest activity at 7 days: 3.65- and 2.7-fold increase in seabream and sea bass, respectively. This is supported by the significant up-regulation of the non-specific cytotoxic cell receptor (NCCRP-1) in the two fish species. By contrast, phagocytosis was unaffected in both species. The respiratory burst was increased in seabream 7 days post-infection whilst in sea bass this activity was significantly decreased at days 7 and 15. Our results demonstrate the significance of the CMC activity in both gilthead seabream and European sea bass against nodavirus infections but further studies are still needed to understand the role of cytotoxic cells in the antiviral immune response and the mechanisms involved in either reservoir or susceptible fish species.

Topics: Analysis of Variance; Animals; Bass; Brain; DNA Primers; Fish Diseases; Head Kidney; Immunity, Cellular; Leukocytes; Nodaviridae; Phagocytosis; Real-Time Polymerase Chain Reaction; Respiratory Burst; RNA Virus Infections; Sea Bream; Time Factors

Twenty Asian sea bass Lates calcarifer from a floating cage in Bt. Tambun, Penang were examined for the presence of parasitic gill copepod, Lernanthropus latis. The prevalence of L. latis was 100% with the intensity of infection ranging from 1 to 18 parasites per host or 3.75 of mean intensity. Female parasites having oblong cephalothorax and egg-strings were seen mainly on the entire gill of examined Asian sea bass. The infected gill of Asian sea bass was pale and had eccessive mucus production. Under light and scanning electron microscopies (SEM), L. latis was seen grasping or holding tightly to the gill filament using their antenna, maxilla and maxilliped. These structures are characteristically prehensile and uncinate for the parasite to attach onto the host tissue. The damage was clearly seen under SEM as the hooked end of the antenna was embedded into the gill filament. The parasite also has the mandible which is styliform with eight teeth on the inner margin. The pathological effects such as erosion, haemorrhages, hyperplasia and necrosis along the secondary lamellae of gill filaments were seen and more severe at the attachment site. The combined actions of the antenna, maxilla and maxilliped together with the mandible resulted in extensive damage as L. latis attached and fed on the host tissues.

Topics: Animals; Aquaculture; Bass; Copepoda; Female; Fish Diseases; Gills; Histological Techniques; Malaysia; Male; Microscopy, Electron, Scanning; Parasitic Diseases, Animal; Prevalence

The warm temperature acclimation related 65 kDa protein-2 (Wap65-2), a teleost plasma glycoprotein, plays an important role in immune regulation against bacterial infection. Here, for the first time we determined the full length cDNA sequence of the Japanese sea bass Wap65-2 gene (1 601 bp in length excluding the 3'-polyA tail). The sequence contains an open reading frame that encodes a protein of 436 amino acids with a molecular weight of 4.87×10(4). The predicted protein had a signal peptide in the N-terminal domain containing 19 residues. Sequence comparison and phylogenetic tree analysis showed that the Japanese sea bass Wap65-2 has a relatively high similarity to the Dicentrarchus labrax Wap65-2. In the healthy Japanese sea bass, Wap65-2 mRNA was expressed mainly in the liver and weakly in the heart and muscle. qRT-PCR results revealed that liver Wap65-2 transcripts were significantly increased after a Vibrio harveyi infection, and peaked 24 hour post injection (6.89 fold increase). The Japanese sea bass Wap65-2 protein was expressed in Escherichia coli and subsequently used for antiserum preparation. Western blot analysis showed that Wap65-2 was significantly increased in V. harveyi infected Japanese sea bass and reached a maximum of 5.33-fold increase at 36 h. In conclusion, the alteration of Japanese sea bass Wap65-2 expression was tightly associated with the progression of the V. harveyi bacterial infection.

Topics: Animals; Bass; Cloning, Molecular; Fish Diseases; Fish Proteins; Hemopexin; Molecular Sequence Data; Phylogeny; Vibrio; Vibrio Infections

An aquaculture research facility experienced high mortality rates in white bass Morone chrysops associated with a monogenean infestation of the gills, but not in striped bass Morone saxatilis in the same facility. All mortalities had pale gills. Monogeneans, identified as Gamacallum macroura (MacCallum and MacCallum 1913) Unnithan 1971, were found on the gills. Pale-gilled and healthy white bass were selected with no particular attention to condition for venipuncture and euthanasia for postmortem examination, including parasite counts from gills. The median packed cell volume (PCV) of fish with gill pallor was 12.5% (range 9-37%) while PVC of fish with more normal color was 30% (27-33%). Association between the PCV and gill pallor score was statistically significant, as was the association between PCV and the number of monogeneans found on the gills of each fish. Median estimated white blood cell count of fish with gill pallor, at 12.05 × 10(3/)μL (range 3.8-24.7), was significantly lower than of apparently healthy fish: 24.7 × 10(3)/μL (17.3-31.5). Histopathology of the gill arches of pale-gilled fish revealed multifocal moderate to severe branchitis, focal areas of dilated hyperplastic lamellae occluded by fibrin, and monogeneans attached to the lamellae. Fish that were apparently healthy had grossly similar histologic lesions, but at lower frequency and severity.

Topics: Animals; Bass; Fish Diseases; Platyhelminths; Trematode Infections

Vibrio harveyi is an important pathogen that causes vibriosis in various aquatic organisms. Here, we announce the draft genome sequence of V. harveyi strain ZJ0603, which was isolated from diseased Orange-spotted grouper (Epinephelus coioides) in Guangdong, China.

Topics: Animals; Bass; China; DNA, Bacterial; Fish Diseases; Genome, Bacterial; Molecular Sequence Data; Sequence Analysis, DNA; Vibrio; Vibrio Infections

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

Topics: Animal Fins; Animals; Antibodies, Viral; Antigens, Viral; Bass; Brain; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunohistochemistry; In Situ Hybridization; Kidney; Liver; Nodaviridae; Real-Time Polymerase Chain Reaction; Retina; RNA Virus Infections; Spleen

A sensitive and quantitative one step RT-qPCR method was developed to study Viral Nervous Necrosis (VNN) virus loads in sea bass Dicentrarchus labrax (L.) in hatcheries. After determining the limits of this new method, fin tissues were identified as an interesting new simple non-invasive sample source, which might be useful for screening D. labrax (L.) in hatcheries. We observed VNN virus strain V26 associated to D. labrax (L.) eggs and it's release in tank water during spawning suggesting both vertical transmission to the eggs and the possibility of horizontal transmission by contamination of tank water. VNN is widespread in water bodies and has the ability to infect a large number of fish species, with this in mind, this PCR technique may be used for the surveillance of various fish farms.

Topics: Animal Fins; Animals; Aquaculture; Base Sequence; Bass; Fish Diseases; Molecular Sequence Data; Ovum; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA Viruses; RNA, Viral; Sensitivity and Specificity

In order to protect DNA vaccine against degradation in alimentary tract of fish, poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating vaccine were prepared using W/O/W emulsification combined with spray drying technique in our laboratory. The characteristics of PLGA nanoparticles were described as follows: (1) shape, spherical; (2) size, <500 nm; (3) yield, ∼96.2%; loading percentage, ∼0.5%; encapsulation efficiency, ∼63.7%; supercoiled conformation percentage, ∼65%; (4) release dynamics, gradual release. In vitro transfection in SISK cells showed that PLGA nanoparticles could be utilized to transfect eukaryotes. After oral administration, FITC-labeled PLGA nanoparticles were detected in blood of fish, and RNA containing major capsid protein (MCP) gene information existed in various tissues of fish 10-90 days. In addition, the analysis of immune parameters in sera of treatment fish showed that: (1) infection rate of LCDV post-challenge, ∼16.7%; (2) prophenoloxidase, superoxide dismutase, respiratory burst, lysozyme and antibody levels, increased significantly (p<0.05); (3) activities of serum complement, changed a little (p>0.05). Pearson's correlation displayed that correlation of immune factors mentioned above (not including serum complement) were all positive for fish vaccinated. The data in this study suggested that PLGA nanoparticles were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by oral approach.

Topics: Animals; Bass; Cell Line; DNA Virus Infections; Fish Diseases; Flounder; Iridoviridae; Kidney; Lactic Acid; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Vaccines, DNA; Viral Vaccines

The purpose of this study was to determine whether gilthead sea bream and sea bass treated with combination of trimethoprim and sulfamethoxazole (TMP-SMX) differed in terms of physiological and innate immune biomarkers. Fish were exposed to TMP-SMX at 40 ppm concentration for 1 h as a prophylactic usage. Plasma cortisol, glucose, electrolytes (Ca, P, Na, K, Cl, and Mg) as well as plasma lysozyme activity, C-reactive protein (CRP), and ceruloplasmin (Cp) were measured soon after treatment and following 24 and 48 h in normal sea water for recovery. Treatment with TMP-SMX in both gilthead sea bream and sea bass led to an increase in plasma cortisol and glucose. Fluctuations in some electrolytes were found after treatment and during recovery period, however, the ratios of monovalent ions in treated sea bream were similar to control. Hematocrit values as well as plasma lysozyme activity in gilthead sea bream and sea bass were not affected by the treatment. CRP in gilthead sea bream and Cp in sea bass responded to the treatment with decreased levels. Both gilthead sea bream and sea bass displayed a rapid physiological stress response and sensitivity to TMP-SMX exposure, which requires more than 48-h period for regaining homeostasis.

Topics: Animals; Anti-Bacterial Agents; Bass; Biomarkers; Blood Glucose; Fish Diseases; Hydrocortisone; Immunity, Innate; Sea Bream; Stress, Physiological; Trimethoprim, Sulfamethoxazole Drug Combination

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.

Topics: Animals; Bass; DNA, Ribosomal; Fish Diseases; Kidney; Myxozoa; Parasitic Diseases, Animal; Perciformes; Phylogeny; Sequence Analysis, DNA; Species Specificity; Spores, Protozoan

Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) were subjected to either experimental infection with Photobacterium damselae subsp. piscicida or Nodavirus after a period of 2 weeks of crowding in which fish were subjected to a 5-fold increase in density (10-50 kg/m(3)). Samples were obtained before the crowding period (0 h or control) and at 24h and 72 h after crowding from both groups of infected fish. The Complement haemolytic activity and the expression of the C3 gene were evaluated in blood and liver samples respectively. The bacteriolytic and lysozyme activities were also assessed. The results showed that Complement haemolytic activity was reduced at 72 h with both bacteria and virus in high density Gilthead seabream, and a similar increase was observed at low density. Bacteriolytic activity under both bacterial and viral challenges for both species was increased at 24h, under low density. At high density, the bacterial challenge did not induce significant changes. C3 mRNA abundance was substantially increased after pathogen treatments in low density groups at 24h but no significant changes were detected at high densities. These results support the idea of the suppressor effect of stressors on the immune system since a reduction of Complement activity under virus and high density, or lack of response in C3 expression under high density were observed.

Topics: Animal Husbandry; Animals; Bass; Complement System Proteins; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; Liver; Nodaviridae; Photobacterium; RNA Virus Infections; Sea Bream; Stress, Physiological; Time Factors

Vibrio anguillarum is a pathogen that causes high mortality in marine and freshwater fish. The aim of this study was to develop a real-time PCR assay for identification and quantification of V. anguillarum in fish tissue. The assay was carried out using two target genes, 16SrDNA and toxR, to evaluate the influence of differences in the operon copy number in quantitative assessment, both in pure cultures of V. anguillarum serovar O1 (strain 975/I), as a reference, and in the liver and kidney of a sea bass (Dicentrarchus labrax) specimen. Real-time PCR analysis showed high specificity for both target genes, with a detection limit of approximately 1-10 bacterial cells per reaction in pure culture and 10/100 V. anguillarum cells per reaction in fish tissue, which corresponds to 2 × 10(2)/2 × 10(3) cells g(-1) fish tissue. Moreover, both genes showed high specificity but differing sensitivity due to the different operon copy number; as a result, it is possible to target the high copy number gene to improve sensitivity. Our results suggest that the protocol we tested can be used as a sensitive and specific molecular method for the detection of the fish pathogen V. anguillarum in fish tissue.

Topics: Animals; Bacterial Proteins; Bacteriological Techniques; Bass; DNA-Binding Proteins; Fish Diseases; Kidney; Liver; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sensitivity and Specificity; Transcription Factors; Vibrio; Vibrio Infections

The self-organizing map (SOM), which is widely used in economics and engineering applications, is a type of artificial neural network trained without supervision. SOM is used to represent multidimensional data in much lower dimensional spaces-usually in two dimensions (2D)-while preserving the topological properties of the input space. In this study, 2D maps were produced by using SOM to display the relationship between seasons, length, weight, and isopod infestation of goldblotch grouper (Epinephelus costae Staindahner, 1878). This is first study of gnathiid isopod praniza larvae infesting goldblotch grouper (E. costae Staindahner, 1878) in the northeast Mediterranean Sea (36°36' N-36°07' E, 35°52' N-36°25' E) in Iskenderun Bay. Fish were sampled monthly from Iskenderun Bay for a period of 12 months from 2006 May to 2007 April (Nt=331, Wt+/-SD (range)=392.92+/-72.76 g (169-927 g) TLt+/-SD (range)=30.85+/-3.88 cm)). Gnathia sp. was only extracted from the epithelium of the buccal cavity and internal side of the gills arch. The monthly patterns in infested fish samples (Np=109, Wp+/-SD (range)=349.25+/-182.79 g (169-853 g) TLt+/-SD (range)=26.05+/-12.68 cm (18.2-45.0 cm)) infestation rates (mean prevalence, P=28.12% (0-60) and mean intensity (MI+/-SD=23.69+/-14.78 (4-82), the relationship between length-weight and infested/non-infested fish were calculated. Although the gnathiid parasite high intensities were observed in fish, there was no significant effect on the growth and general health condition of infested fish.

Topics: Algorithms; Animals; Bass; Crustacea; Fish Diseases; Isopoda; Larva; Mediterranean Sea; Neural Networks, Computer

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg⁻¹ dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.

Topics: Animals; Bass; Cell Wall; Fish Diseases; Gastrointestinal Tract; Immune System; Mannans; Mucus; Muramidase; Oligosaccharides; Saccharomyces cerevisiae; Vibrio; Vibrio Infections

Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

Topics: Animal Structures; Animals; Aquaculture; Bass; Fish Diseases; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; Sensitivity and Specificity; Taiwan; Virology

Effect of diet enriched with green tea at 0, 0.01, 0.1 or 1.0% levels on immune responses such as non-specific humoral (lysozyme, antiprotease and complement) and cellular (myeloperoxidase content, production of reactive oxygen, and nitrogen species) and disease resistance on week 1, 2 or 4 in kelp grouper Epinephelus bruneus challenged with Vibrio carchariae (2.47 × 10(8) CFU ml(-1)) was quantified. At all doses green tea supplementation significantly enhanced the serum lysozyme activity from weeks 1 to 4. On the other hand, after week 2 the serum hemolytic complement activity, leucocyte myeloperoxidase content and reactive nitrogen species protection significantly increased in groups fed with 0.01 and 0.1% green tea supplementation diets. The serum antiprotease activity significantly increased in group fed with at 1.0% green tea from week 1 to 4. However, all diets except at 0.01% level resulted in a significant decrease in reactive oxygen species protection during the experimental period. Challenged groups fed with green tea enriched diet at 0.01 and 0.1% level had a higher relative percent survival than with 1.0% diet on week 1, 2 or 4. The results suggest that dietary administration of green tea supplementation at a concentration of 0.01 and 0.1% level positively enhances the non-specific humoral and cellular immune responses and disease resistance of kelp grouper E. bruneus to V. carchariae.

Topics: Animals; Bass; Diet; Enzymes; Fish Diseases; Immunity, Cellular; Immunity, Humoral; Immunity, Innate; Leukocytes; Reactive Nitrogen Species; Reactive Oxygen Species; Survival Analysis; Tea; Vibrio; Vibrio Infections

The antibiotic resistance patterns of aetiological agents responsible for vibriosis and pasteurellosis were studied to contribute to control the spread of these two bacterial diseases in Mediterranean fish farming. Strains of Photobacterium damsela ssp. piscicida, Vibrio fluvialis, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio metschnikovii, isolated from Italian aquaculture (fish, shellfish and crustaceans) sites, were assayed for their susceptibility to some antibacterial agents currently used in farming practices. Kirby Bauer and Minimum Inhibitory Concentration (M.I.C.) tests were performed. The bacterial strains showed resistance to ampicillin, carbenicillin, kanamycin, cefalothin, while they were sensitive to chloramphenicol, nitrofurantoin and tobramycin; the sulfadiazine-trimethoprim association was completely ineffective. Conversely, flumequine showed the lowest M.I.C. value (0.97 �g mL-1), suggesting its marked antibiotic effect. Considering that quinolone resistance can be transmitted only by selection of mutations and not by other genetic mechanisms, this study stresses the importance of a more responsible use of this antibacterial drug in aquaculture.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bacterial Typing Techniques; Bass; Crustacea; Drug Resistance, Bacterial; Fish Diseases; Italy; Microbial Sensitivity Tests; Mollusca; Pasteurella Infections; Photobacterium; Vibrio; Vibrio Infections

Six new and 1 previously described species of Pseudorhabdosynochus (Diplectanidae) are described and/or reported from the gill lamellae of 5 serranid (Perciformes) fish species from the Pacific waters in Guerrero State of Mexico and Panama City, Panama. These species are Pseudorhabdosynochus guerreroensis n. sp. from the Pacific mutton hamlet Alphestes inmaculatus Breder (type host), rivulated mutton hamlet Alphestes multiguttatus (Günther), and spotted grouper Epinephelus analogus Gill from Mexico; Pseudorhabdosynochus urceolus n. sp. from the Pacific graysby Cephalopholis panamensis (Steindachner) from Taboga Island in Panama; Pseudorhabdosynochus spirani n. sp. from the starry grouper Epinephelus labriformis (Jenyns) from Mexico and the Perlas Archipelago and Taboga Island in Panama; Pseudorhabdosynochus fulgidus n. sp. from E. labriformis from Mexico and the Perlas Archipelago and Taboga Island (type locality) in Panama; Pseudorhabdosynochus tabogaensis n. sp. from E. labriformis from Mexico and the Perlas Archipelago and Taboga Island (type locality) in Panama; Pseudorhabdosynochus anulus n. sp. from E. labriformis from Mexico and Taboga Island (type locality) in Panama; and Pseudorhabdosynochus amplidiscatum (Bravo-Hollis, 1954) Kritsky and Beverley-Burton, 1986 from E. analogus and E. labriformis from Mexico and the Perlas Archipelago and Taboga Island in Panama. All new species are mainly distinguished from other species of the genus by the shape and size of the sclerotized vagina and haptoral structures. The present specimens of Alphestes, Cephalopholis, and Epinephelus spp. represent new host records and Panama represents a new geographic record for species of Pseudorhabdosynochus. The apparent common feature supporting a close similarity of these diplectanids is a single, secondary ejaculatory bulb with thickened wall.

Topics: Animals; Bass; Fish Diseases; Gills; Mexico; Pacific Ocean; Panama; Platyhelminths; Trematode Infections

Cucullanus mycteropercae n. sp. is described from the intestine of the black grouper, Mycteroperca bonaci Poey, from the northern coast of Yucatán, México. The new species is readily distinguishable from other Cucullanus species because it possesses an ellipsoidal papilla-like structure situated medially on the anterior cloacal lip of males. Other differentiating characters include the variable position of postcloacal pair 8 in males, the subventral position of phasmids, a slightly ventrally hooked posterior end of gubernaculum, and the presence of a large, cylindroconical sclerotized tail end in both sexes. This is the fourth record of a marine cucullanid off the Yucatán Peninsula in México belonging to Cucullanus Müller, 1777.

Topics: Animals; Ascaridida; Ascaridida Infections; Bass; Female; Fish Diseases; Male; Microscopy, Confocal; Microscopy, Electron, Scanning

Widespread mouth ulcerations were observed in largemouth bass collected from eight inland lakes in the Lower Peninsula of Michigan during the summer months of 2002 and 2003. These ulcerations were associated with, and most likely caused by, leech parasitism. Through the use of morphological dichotomous keys, it was determined that all leeches collected are of one species: Myzobdella lugubris. Among the eight lakes examined, Lake Orion and Devils Lake had the highest prevalence of leech parasitism (34% and 29%, respectively) and mouth ulcerations (53% and 68%, respectively). Statistical analyses demonstrated that leech and ulcer prevalence varied significantly from one lake to the other. Additionally, it was determined that the relationship between the prevalence of ulcers and the prevalence of leech attachment is significant, indicating that leech parasitism is most likely the cause of ulceration. The ulcers exhibited deep hemorrhagic centers and raised irregular edges. Affected areas lost their epithelial lining and submucosa, with masses of bacteria colonizing the damaged tissues. Since largemouth bass is a popular global sportfish and critical to the food web of inland lakes, there are concerns that the presence of leeches, damaged buccal mucosa, and general unsightliness may negatively affect this important sportfishery.

Topics: Animals; Bass; Fish Diseases; Fresh Water; Leeches; Michigan; Mouth; Mouth Diseases; Mouth Mucosa; Prevalence; Tongue; Ulcer

Topics: Animals; Bass; Cestode Infections; Enteritis; Fish Diseases; Hepatitis, Animal; Male; Nematode Infections

Land-use alterations can have profound influences on faunal distributions, including host-parasite relationships. Yellow grub trematodes ( Clinostomum spp.) have complex life cycles involving 3 hosts: a snail, a fish or amphibian, and a bird. Here, we analyze the distribution, prevalence, intensity, abundance, and genetic diversity of encysting metacercariae of Clinostomum spp. in salamanders and fishes throughout an aquatic system that includes a natural Ozark stream and man-made ponds. We found Clinostomum sp. infecting permanently aquatic Oklahoma salamanders ( Eurycea tynerensis ; 56% prevalence) and larval grotto salamanders ( Eurycea spelaea ) immediately downstream from a man-made pond. However, Clinostomum sp. did not infect any salamanders in the spring that supplies this pond, or in sections farther downstream (~0.5 and 2 km). Metacercariae of Clinostomum sp. were present in ~90% of introduced largemouth bass ( Micropterus salmoides ) in the man-made pond adjunct to the stream. Morphological examination and phylogenetic analyses based on the mitochondrial gene cytochrome oxidase 1 ( Co1 ) and the nuclear ribosomal gene 18S show that fishes and salamanders at this site are primarily infected with Clinostomum marginatum . There is a relatively high degree of mitochondrial haplotype diversity in C. marginatum at this site but no consistent genetic difference between parasites in largemouth bass from the man-made pond and those in salamanders from the stream. Based on the microgeographic distribution and relationships of metacercariae of C. marginatum at this site, we hypothesize that the adjunct man-made pond has created an ecological situation that brings the cercariae of this parasite into contact with novel stream salamander hosts.

Topics: Animals; Bass; Female; Fish Diseases; Genetic Variation; Haplotypes; Male; Oklahoma; Phylogeny; Prevalence; Rivers; Trematoda; Trematode Infections; Urodela

The striped bass, Morone saxatilis (Walbaum), once represented an important resource for fisheries in the St Lawrence River (Quebec, Canada). A restoration programme, involving captive propagation, was implemented with the objective of restocking the population, which had disappeared in the late 1960s. An unusually high rate of mortality was observed during the winter of 2006 in captive-raised fingerlings that were originally collected from the Miramichi River (New Brunswick, Canada) the previous summer. Post-mortem examinations revealed extensive granulomatous and hyperplastic peritonitis associated with numerous nematodes of the genus Philometra. Given the severity of the lesions, high intensity of infection by Philometra sp. was presumed to be the primary factor in the unusual mortalities reported that winter. Observations suggest that this nematode, which was acquired in the wild, cannot establish itself in a captive environment, most likely because of the absence of the obligate intermediate host. Examination of archived specimens of striped bass showed that this parasite was probably present in the St Lawrence River population prior to its extirpation. Consequently, the introduction of infected fish into this ecosystem should not be a concern. Nevertheless, infection-related mortalities of fingerlings might affect dynamics of wild striped bass populations.

Topics: Animals; Bass; Dracunculoidea; Female; Fish Diseases; New Brunswick; Peritonitis; Spirurida Infections

Few drugs are approved by the United States Food and Drug Administration for treating parasite infections in minor species such as fish, due in part to the high cost of developing such drugs and to a relatively small market share for drug sponsors. Because in vivo effectiveness trials for antiparasitic drugs are costly, time consuming, and use many animals, a systematic in vitro screening approach to describe parasite motility could help find promising drug candidates. We evaluated the effects of 7 antiparasitics on the activity and survival of the endoparasitic monogenean Acolpenteron ureteroecetes (Dactylogyridae) collected from the posterior kidneys of juvenile largemouth bass Micropterus salmoides (Lacepede, 1802) (Centrarchidae) held in the laboratory. Tests were conducted in 12 well tissue culture plates; each well had 3 parasites, and we tested 3 concentrations and 1 control for each of the 7 antiparasitics. The parasites were observed immediately after adding the drug, at 1 to 3 h, and 17 to 26 h, and video recordings were made. Drug effects were recorded by documenting morbidity (reduced movement, tremors, contracted body, abnormal morphology) and mortality. A. ureteroecetes was strongly affected by the quinoline praziquantel, the imidazothiazide levamisole, and the organophosphates dichlorvos and trichlorfon. The parasites were moderately affected by the macrocyclic lactones ivermectin and emamectin, and generally unaffected by the benzimidazole mebendazole. Our study demonstrates the utility of characterizing in vitro responses with video microscopy to document responses of fish parasites for initial screens of drug effects on a fish monogenean.

Topics: Animals; Anthelmintics; Bass; Fish Diseases; Trematoda; Trematode Infections

Mammalian secreted protein acidic and rich in cysteine (SPARC) is the primary regulator of cell shape and cell adhesion to fibronectin. We, for the first time, report the complete sequencing of SPARC cDNA from orange-spotted grouper. Despite the difference in the lengths of the SPARC transcripts, all of the SPARC molecules encoded a signal peptide, follistain-like copper binding sequence (KGHK) domain, and extracellular domain. The grouper SPARC gene was differentially expressed in vivo and contributed differently to high-level expression of SPARC in muscle. Immunohistochemical staining demonstrated a decreased level of SPARC in nodavirus-infected grouper compared with healthy grouper. Comparative real-time polymerase chain reaction analyses of eye tissues of viral nervous necrosis grouper and healthy grouper were performed. Recombinant SPARC produced changes in grouper cell shape 24 h after treatment. The results provide new insight into the pathogenesis of nodavirus, and demonstrate an experimental rationale for SPARC characterization in nodavirus-infected grouper.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation; Molecular Sequence Data; Nodaviridae; Osteonectin; RNA Virus Infections; Sequence Alignment

This is the first report of an intestinal Eimeria infection in Asian seabass (Lates calcarifer) at the histopathological and ultrastructural levels. The Eimeria infection was often associated with severe pathology and significant mortality in the absence of other pathogens. This showed that it is an important disease of juvenile L. calcarifer in small scale nurseries in Vietnam. Heavy infection and high prevalence levels of the Eimeria infection are suspected to be linked to the low daily water exchange rates practised in these nurseries. Although systemic iridovirus infection was concurrently observed in some of the fish examined, it was not as consistently present in diseased fish as the Eimeria infection.

Topics: Animals; Aquaculture; Bass; Coccidiosis; Fish Diseases; Intestinal Diseases, Parasitic; Intestinal Mucosa; Vietnam

An intestinal Eimeria was previously reported as a significant pathogen of Asian seabass (Lates calcarifer) in nurseries in Vietnam. In the present study, both Eimeria and Cryptosporidium were detected by sequence analyses of fragments of the 18S rRNA gene amplified from these Vietnamese L. calcarifer tissues. Based on these analyses, the Eimeria from the Vietnamese L. calcarifer formed clades with the Eimeria detected in L. calcarifer tissues from Australia, but clustered separately from other known Eimeria and Goussia species. The Cryptosporidium detected in L. calcarifer from Vietnam clustered closest with C. parvum and C. hominis. In situ hybridization using DIG-labeled DNA probes generated from 18S PCR products on the Vietnamese L. calcarifer wax block tissues showed that this method could not be used to distinguish between Eimeria and Cryptosporidium, due to the conserved nature of the 18S locus. A previously published study on the morphology of parasite developmental stages and oocysts in the Vietnamese L. calcarifer tissues showed only an intestinal Eimeria infection. The Cryptosporidium could be present at very low levels undetectable by microscopy in intestines, or being ubiquitous, was a possible contaminant from feed or water. While molecular analysis is a very useful tool in the study of disease and identification of aetiological agents, this study reiterates the importance of demonstrating organisms in situ in tissues.

Topics: Animals; Aquaculture; Bass; Coccidiosis; Cryptosporidiosis; Cryptosporidium; Eimeria; Fish Diseases; Gene Expression Regulation; Phylogeny; RNA, Ribosomal, 18S; Vietnam

Striped bass Morone saxatilis were studied in order to characterize their immune responses over the short term following challenge with Mycobacterium marinum. The expression of immunity-related genes (IL-1beta, TNF-alpha, Nramp and TGF-beta) quickly increased following infection with M. marinum, but these genes were subsequently down-regulated despite the fact that bacterial counts remained high. The number of monocytes and neutrophils also initially increased at 1 d postinfection. This confirms the importance of these types of cells in initial inflammation and mycobacterial infection in striped bass. The phagocytic index of splenic leukocytes over these same time frames did not change significantly following infection. The discrete window in which inflammatory mechanisms were stimulated in striped bass may be related to the intracellular nature of this pathogen.

Topics: Animals; Bass; Fish Diseases; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Time Factors

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.

Topics: Animals; Bass; Brain; Eye; Fish Diseases; Genome, Viral; In Situ Hybridization; Nodaviridae; Real-Time Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sensitivity and Specificity; Viral Tropism; Virus Replication

The present study investigated the immunostimulatory effect of Korean mistletoe Viscum album extract (KM-E) on innate immune response in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi. Kelp grouper were divided into four groups of 25 each and fed with 0 (control), 0.5, 1.0, and 2.0% enriched diets with Korean mistletoe extract (KM-E). After feeding for 30 days, the fish were injected intraperitoneally (i.p.) with 100 μl of P. dicentrarchi (4.2 × 10(7)ciliates/ml) to study the immune responses at weeks 1, 2, and 4. The respiratory burst activity did not significantly enhance when fed with 0.5% and 1.0% supplementation diets on week 1 when compared to control diet. On weeks 2 and 4, the respiratory burst activity significantly increased with 1.0% and 2.0% diets. The phagocytic activity significantly enhanced with 1.0% and 2.0% diets, but not with 0.5% diet at any time. When fed with 1.0% KM-E-diet the lysozyme activity did not significantly vary at any week whereas with 1.0% and 2.0% diets it was significantly enhanced. The total protein level significantly increased with 1.0% and 2.0% KM-E-diets from weeks 1 to 4 as compared to control. The present study suggests that 1.0% or 2.0% KM-E-supplementation diet positively enhances the innate immune response in E. bruneus against P. dicentrarchi infection.

Topics: Animals; Antibodies, Protozoan; Bass; Ciliophora Infections; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Immunity, Innate; Injections, Intraperitoneal; Muramidase; Oligohymenophorea; Plant Extracts; Respiratory Burst; Time Factors; Viscum album

Kelp grouper, Epinephelus bruneus, fed for 30 days with 0% (control), 0.1%, 1.0%, and 2.0% of Styrax japonica supplementation diets, led to reductions in mortality after being challenged with a bacterium (Vibrio harveyi) and a ciliate protozoan (Uronema marinum). The enriched diets significantly increased the survival rate as compared to the controls. The phagocytic and respiratory activities were significantly increased in kelp groupers given 1.0% and 2.0% enriched diets. The complement activity, lysozyme activity, serum bactericidal activity, and total protein level significantly increased with any enriched diet against the pathogens; however antiprotease activity and myeloperoxidase levels significantly increased only with 1.0% and 2.0% enriched diets while the α2-macroglobulin level was significantly enhanced with 1.0% enriched diet. The study suggests that incorporation of S. japonica at 1.0% and 2.0% level in the diet significantly enhances the immune responses in the kelp grouper E. bruneus against V. harveyi and U. marinum.

Topics: Animal Feed; Animals; Bass; Blood Bactericidal Activity; Ciliophora Infections; Fish Diseases; Macrophages; Oligohymenophorea; Phagocytosis; Phytotherapy; Plant Extracts; Respiratory Burst; Styrax; Vibrio Infections

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.

Topics: Animals; Bass; Fish Diseases; Mycobacterium; Mycobacterium Infections; Phylogeny

This study, reports the identification and analysis of ferritin M chain, from kelp grouper, Epinephelus bruneus (EbFerM); it comprises 1004 base pair (bp), including 528bp open reading frame (ORF) which encodes 176 amino acid (aa) residues; the calculated molecular weight is 20kDa. The 5'-untranslated region (UTR) possesses 476bp proceeded by a putative Iron Regulatory Element (IRE). Pair wise alignments showed that EbFerM shared 94% identity with that of Larimichthys crocea and Sciaenops. It is expressed in abundance in liver, spleen, and kidney when challenged with Vibrio anguillarum, lipopolysaccharide (LPS), or poly I:C.

Topics: Animals; Base Sequence; Bass; DNA, Complementary; Ferritins; Fish Diseases; Gene Expression Profiling; Lipopolysaccharides; Open Reading Frames; Organ Specificity; Phylogeny; Poly I-C; Sequence Alignment; Vibrio; Vibrio Infections

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune-related genes in antiparasitic immune responses in fish, we monitored the expression change of IL-8, COX-2, C-type lectin and transferrin in local and systemic immune organs of orange-spotted grouper post-C. irritans infection. IL-8 expression was up-regulated during the course of infection in the skin, while COX-2 and transferrin expression was up-regulated in the gill. COX-2 expression was significantly down-regulated in the spleen (0·7-5% of its control) and head kidney (0·5-4% of its control) post-primary infection. Transferrin expression was also down-regulated in the spleen and head kidney from 6 h to 5 days post-primary infection. However, C-type lectin expression was up-regulated in all tested organs post-infection, with the exception of day 7 in the spleen post-primary infection where the expression level was slightly down-regulated (44% of its control). These results suggest that these four immune-related genes play an important role in grouper anti-C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Cyclooxygenase 2; Fish Diseases; Gene Expression Profiling; Interleukin-8; Lectins, C-Type; Spleen; Time Factors; Transferrin

A tumour diagnosed as multicentric infiltrative lipoma affecting a single farmed seabass is described. The fish had 3 masses on the lateral side of its back, deforming the skin surface. The masses showed a tendency to invade the underlying musculature. Histologically, the neoplasm consisted of differentiated adipocytes. Biochemically, the neoplastic tissue showed lower values of monounsaturated fatty acids and higher values of polyunsaturated fatty acids compared with adjacent normal muscular tissue, particularly of the n3 series, such as eicosapentanoic (C20:5n3) and docosahexanoic (C22:6n3) acids. Data obtained suggest a metabolic disturbance in the lipid component of the muscular tissue metabolic pathway, which could be the starting point to promote lipoma formation. This is the first report of lipoma in Mediterranean seabass.

Topics: Animals; Bass; Fatty Acids; Fish Diseases; Lipoma

We examined male shoal bass Micropterus cataractae from the Flint River, Georgia, to determine the prevalence of intersex. During March and April 2010, we sampled 61 shoal bass from six sites along the Flint River. Testes were examined histologically and classified as intersex if the presence of oocytes was noted. Using a severity index, we compared samples collected on different dates and from different locations according to age and testis weight. No significant variations were noted among any of the groupings. Further investigation is needed to determine whether the intersex condition in shoal bass is severe enough to warrant concern and whether it is a natural phenomenon.

Topics: Animals; Bass; Disorders of Sex Development; Fish Diseases; Georgia; Male; Rivers; Testis

A public aquarium with a 4-mo history of occasional fish mortalities submitted for necropsy an adult female largemouth bass (Micropterus salmoides) that died unexpectedly. Gross necropsy revealed that the pericardial cavity was markedly distended with partially coagulated blood. Examination of the heart revealed multiple nodular masses in the area of the atrium and two small perforations on the surface of one of the nodular masses. Histopathologic exam of the atrium revealed severe fibrinonecrotic endocarditis and transmural myocarditis with intralesional bacteria. A pure culture of Edwardsiella tarda was obtained from culture of posterior kidney and spleen. An area of stagnant water that may serve as the source of E. tarda was identified, and steps to rectify this problem were taken. Low-level supersaturation was also a significant stressor; the source of the supersaturation was not identified. To our knowledge, this is the first report of cardiac tamponade in a largemouth bass.

Topics: Animals; Bass; Cardiac Tamponade; Edwardsiella tarda; Enterobacteriaceae Infections; Female; Fish Diseases

Three Haliotrema spp. are reported from the Vietnamese grouper. Morphological and morphometric characters show minor deviations from original descriptions of H. cromileptis Young, 1968 and H. epinepheli Young, 1968 . The third encountered species ( Haliotrema sp.) appears to be new to science. Genetically, H. cromileptis clusters with Bravohollisia , Pseudohaliotrema , and Haliotrema . The group is well supported by partial large subunit rDNA (LSU), complete small subunit rDNA (SSU), and partial SSU + ITS1 rDNA analyses. Ingroup phylogenetic relationships are not well resolved. Haliotrema cromileptis , H. fleti Young, 1968 , and Pseudohaliotrema sphincteroporus Yamaguti, 1953 are closely related to a monophyletic group of 5 Haliotrema spp. characterized by bell- or horn-shaped bases of the male copulatory organ (MCO), which contains an accessory piece. Based on SSU rDNA, H. cromileptis is a sister species to P. sphincteroporus and, together, they form a clade to 3 other Haliotrema spp. characterized by a bell-shape based MCO with an accessory piece. Data analysis conducted on partial SSU + ITS1 rDNA confirms the close phylogenetic relationship of H. cromileptis , H. fleti , H. chenhsintaoi Zhang, 2001 (possessing a horn-shaped base of the MCO), and Bravohollisia rosetta Lin, 1995. However, because major differences in diagnostic characters exist, this genetic relationship needs further elucidation.

Topics: Animals; Bass; Bayes Theorem; DNA, Protozoan; DNA, Ribosomal; Fish Diseases; Fisheries; Phylogeny; Platyhelminths; Sequence Analysis; Trematode Infections; Vietnam

The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Deltapdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Deltapdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen.

Topics: Amidohydrolases; Amino Acid Sequence; Animals; Bacterial Adhesion; Bacterial Proteins; Bacteriolysis; Bass; Blood Bactericidal Activity; Cell Line; DNA Transposable Elements; Epithelial Cells; Fish Diseases; Gene Targeting; Genes, Bacterial; Genome, Bacterial; Molecular Sequence Data; Muramidase; Mutagenesis; Reactive Oxygen Species; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Streptococcal Infections; Streptococcus; Virulence; Virulence Factors

Betanodaviruses are one of the serious pathogens in nervous necrosis viral (NNV) disease that brings about mortality in the larval stage of grouper (Epinephelus coioides). In this study, the efficacy of pretreatment, co-treatment, and posttreatment with the antimicrobial epinecidin-1 and hepcidin 1-5 peptides against a betanodavirus was evaluated by intraperitoneal inoculation in grouper. The results showed that co-treatment of epinecidin-1 or hepcidin 1-5 with the virus was effective in promoting a significant decrease in grouper mortality. Re-challenge with virus again after 30 day in co-treated grouper groups showed high survival suggesting that epinecidin-1 and hepcidin 1-5 enhanced fish survival. However, grouper inoculated with NNV and then inoculated with epinecidin-1 8 h later showed significantly different survival from the group inoculated with virus alone, suggesting that epinecidin-1 can be used as a drug to rescue infected grouper. Infection after pretreatment, co-treatment, and posttreatment with epinecidin-1 or hepcidin 1-5 was verified by RT-PCR which showed downregulation of Mx2 and Mx3 gene expressions. All these data strongly suggest that epinecidin-1 and hepcidin 1-5 are effective peptides for protecting grouper larvae by reducing NNV infection.

Topics: alpha-Defensins; Animals; Antimicrobial Cationic Peptides; Bass; Down-Regulation; Fish Diseases; Fish Proteins; Gene Expression Regulation; GTP-Binding Proteins; Hepcidins; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open-reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA-dependent RNA polymerase was obtained.

Topics: Animals; Base Composition; Bass; Capsid Proteins; Fish Diseases; Molecular Sequence Data; Nodaviridae; Phylogeny; RNA Virus Infections; RNA-Dependent RNA Polymerase; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Tunisia

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

Topics: Animals; Antibodies, Viral; Bass; Cell Line; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Lymphocytes; Nodaviridae; Polymerase Chain Reaction; RNA Virus Infections

Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured fish at larval stage. Strong evidence of vertical transmission of NNV has been reported in the carrier broodstock of striped jack and sea bass. An effective immunization program was developed and monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at 10(9)TCID(50)kg(-1), and the enhanced 2-fold neutralization activity could sustain up to 17 months post-vaccination (mpv). An immunization program was applied in the broodstocks of grouper (Epinephelus tukula) with body weight of 35-60kg. The levels of NNV-specific antibodies detected, from 1 to 5 mpi, in the homogenates of the eggs from the vaccinated broodfish were elevated than that from non-vaccinated fish. By nested RT-PCR, NNV was detectable in the eggs from the non-vaccinated fish at Month 5, but not in the eggs from vaccinated fish. It is therefore suggested that vaccination will be a potentially practical measure to reduce the risk of vertical transmission of NNV of grouper broodfish under stress of repeated spawning.

Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Bass; Eggs; Fish Diseases; Immunologic Memory; Infectious Disease Transmission, Vertical; Injections, Intramuscular; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Time Factors; Vaccines, Inactivated; Viral Vaccines

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Gills; Immunohistochemistry; Mast Cells; Trematoda; Trematode Infections

Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins.. ESTs were generated from a cDNA library of C. irritans tomonts isolated from infected Asian sea bass, Lates calcarifer. Clustering of the 5356 ESTs produced 2659 unique transcripts (UTs) containing 1989 singletons and 670 consensi. BLAST analysis showed that 74% of the UTs had significant similarity (E-value < 10-5) to sequences that are currently available in the GenBank database, with more than 15% of the significant hits showing unknown function. Forty percent of the UTs had significant similarity to ciliates from the genera Tetrahymena and Paramecium. Comparative gene family analysis with related taxa showed that many protein families are conserved among the protozoans. Based on gene ontology annotation, functional groups were successfully assigned to 790 UTs. Genes encoding excretory/secretory proteins and membrane and membrane-associated proteins were identified because these proteins often function as antigens and are good antibody targets. A total of 481 UTs were classified as encoding membrane proteins, 54 were classified as encoding for membrane-bound proteins, and 155 were found to contain excretory/secretory protein-coding sequences. Amino acid repeat-containing proteins and GPI-anchored proteins were also identified as potential candidates for the development of diagnostic and control strategies for C. irritans.. We successfully discovered and examined a large portion of the previously unexplored C. irritans transcriptome and identified potential genes for the development and validation of diagnostic and control strategies for cryptocaryonosis.

Topics: Animals; Bass; Ciliophora; Cluster Analysis; Comparative Genomic Hybridization; Expressed Sequence Tags; Fish Diseases; Gene Expression Profiling; Gene Library; Genes, Protozoan; Membrane Proteins; Multigene Family; Phylogeny; RNA, Protozoan; Sequence Analysis, DNA; Sequence Analysis, Protein

In this work, a survey of sea bass, Dicentrarchus labrax, for amoebae and scuticociliatidia infections was carried out to evaluate their effects on the aquaculture of this fish species. The study was conducted in two different fish farms, one using seawater and the other brackish water. Infection with parasitic amoebae was found to be fairly high (prevalence: 43-73%), being more frequent in sea bass from the brackish water system. Although it was never found to cause outbreaks of disease or mortality in the surveyed fish, amoebic gill disease (AGD) histopathological signs, i.e., hyperplasia, secondary lamellae fusion and cavity formation (interlamellar vesicles), were observed in fish manifesting no macroscopic lesions. Furthermore, some evidence was found that amoebae affects the fish's general state of health and growth rate. These results indicate that cautious and detailed surveys to detect this sort of infection, and thus carefully plan its control, are fully justified. Compared with amoebic infection, the prevalence of scuticociliatosis was found to be low (7-13%). No outbreaks of disease or mortality were ever recorded, even when scuticociliatidia was present in turbot raised in the same water system, leading to serious outbreaks of disease and mortalities in that species. This suggests that sea bass is far more resistant than turbot to such infections, and if this is the case, the former fish may be a good farming alternative when scuticociliatidia is present.

Topics: Amebiasis; Amoeba; Animals; Aquaculture; Bass; Fish Diseases; Gills; Portugal; Prevalence; Seawater; Statistics, Nonparametric; Virulence

Culture of Asian seabass, Lates calcarifer (Bloch) is a popular aquaculture activity in Malaysia. This fish is in high demand and fetches a good price in the local market. The seed for this fish is commercially produced by induced spawning in hatcheries. However, the seed supply is affected by frequent mass mortality of larvae aged between 15 and 60 dph. The clinical signs shown by the affected larvae include lethargy, loss of appetite, uncoordinated swimming, unusual spiral movement pattern and dark coloration. Histological examination of brain and eye of the affected specimens revealed extensive cell vacuolation in larvae aged 15-25 dph. Partial nucleotide sequence of the nervous necrosis virus coat protein gene of the affected larvae showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia and Australia. This study provides scientific evidence based on molecular technique that many episodes of mass mortality in seabass larvae in Sabah is associated with the viral nervous necrosis. Because no effective treatment has been reported for this infection, stringent biosecurity measures must be adopted for exclusion of the pathogen from the culture system.

Topics: Animals; Aquaculture; Base Sequence; Bass; Brain; Capsid Proteins; Cloning, Molecular; Fish Diseases; Larva; Malaysia; Molecular Sequence Data; Nodaviridae; Phylogeny; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; Sequence Alignment

In this article, we examined the influences of the polycultured potential hosts on monogenean seasonality and the possible linkage between the infestations on different species of hosts and host phylogeny. The seasonality of Diplectanum grouperi , the dominant species, on wild versus cultured groupers, Epinephelus spp., was analyzed in Daya Bay, South China Sea, between April 2008 and January 2009. The prevalence, mean intensity, abundance, and variance/mean ratio were calculated for each species of host under polycultured and wild conditions. Except for the overall prevalence, which was slightly higher in autumn than that in summer under wild conditions, the mean intensity and variance were highest in summer, decreasing slightly in autumn to lowest levels in winter or spring. The infection level (prevalence, mean intensity, and abundance) was correlated with changes in water temperature during the sampling period, with a peak in summer, with the exception of E. awoara in autumn under wild conditions. The prevalence, intensity, and mean intensity of D. grouperi on Epinephelus spp. in the wild were much lower than those in experimental (mixed species) culture ponds. The correlation between the molecular phylogeny of 5 species of Epinephelus and the dendrogram based on the susceptibility to D. grouperi was not significant, which infers that variable susceptibilities of these Epinephelus species cannot be revealed by phylogenetic relationships determined from mitochondrial 16S rDNA and Cyt b gene sequences.

Topics: Animals; Animals, Wild; Bass; China; Cytochromes b; DNA, Ribosomal; Fish Diseases; Fisheries; Gills; Host-Parasite Interactions; Molecular Sequence Data; Phylogeny; Platyhelminths; RNA, Ribosomal, 16S; Seasons; Sequence Alignment; Trematode Infections

Vibriosis caused by the pathogenic bacterium Vibrio (Listonella) anguillarum leads to serious losses in European sea bass, Dicentrarchus labrax. Because of its pleiotropic activity in controlling immune and inflammatory responses against various pathogens, interleukin-1beta (IL-1beta) is an attractive candidate for resistance to bacterial vibriosis. Four polymorphisms c.76 + 52C>T, c.76 + 157A>G, c.76 + 215A>and c76 + 310A>G of IL1B were genotyped in progeny of four families of wild sea bass captured in geographically distinct regions of the Black Sea and Sea of Azov and challenged with V. anguillarum. In the transmission disequilibrium test, the TGGG haplotype of IL1B showed significant overtransmission from parents to surviving progeny, thereby suggesting an association with higher resistance to V. anguillarum infection (Odds Ratio 0.38, P < 10(-7)). Using a luciferase reporter assay, we found a 1.4-fold increase in transcription activity of the protective IL1B TGGG variant compared to the susceptible CAAA variant of IL1B. The higher transcriptional activity of IL1B TGGG may arise from the functional effects of c.76 + 157A>G and c.76 + 215A>G polymorphisms disrupting potential binding sites for glucocorticoid receptor and YY1, both are negative transcription regulators.

Topics: Animals; Bass; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Genetic Variation; Genotype; Immunity, Innate; Interleukin-1beta; Vibrio; Vibrio Infections

Topics: Abdominal Cavity; Animals; Bass; Fish Diseases; Fisheries; Gills; Italy; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum

Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE) are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish.. RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution.. This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host-pathogen interactions and evolutionary history of immunogenetics from fish to mammals.

Topics: Adaptive Immunity; Animals; Bass; Female; Fish Diseases; Gene Expression Profiling; Host-Pathogen Interactions; Immunity, Innate; Male; Sequence Analysis, DNA; Vibrio; Vibrio Infections

The shaping forces of parasite community structure still is the main subject in the ecological parasitology whilst community predictability and repeatability showed that hardly a generally applicable role is ever going to be assessed. Defining and describing parasite communities can be very useful from the epizootiological point, in order to help in the assessment of the medical and economical impact of certain parasitosis, moreover when hosts are economically valuable species. Since parasite assemblages in reared European sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) in Adriatic cage systems can play an important role in the economic feasibility of the rearing process, we evaluated their character through assessing diversity indices, nestedness of parasite communities and their differences in respect to season and composition, as well as fish growth. We observed colonization of a new monogenean species (Furnestinia echeneis) and general impoverishment of parasites populations over time in the Adriatic-reared fish parasite assemblages. Parasite assemblages differed significantly between seasons for both fish species, while species richness, evenness, diversity indices and nestedness of parasitic communities in the sea bream showed to be significantly higher compared to those in the sea bass. Such characteristics define parasite communities of both Adriatic-reared fish as species poor although structured and ordered assemblages.

Topics: Analysis of Variance; Animals; Bass; Biodiversity; Fish Diseases; Fisheries; Oceans and Seas; Parasites; Parasitic Diseases, Animal; Prevalence; Sea Bream; Seasons; Temperature

A new nematode species, Philometra diplectri n. sp. (Philometridae), is described from male and female specimens found in unidentified tissues of head and anterior trunk (males) and subcutaneously in the mouth and under the operculum (females) of sand perch, Diplectrum formosum (Linnaeus) (Serranidae, Perciformes), from the northern Gulf of Mexico off Florida (Florida Middle Grounds). Based on light and scanning electron microscopy examination, the new species differs from other congeners parasitizing the subcutaneous tissues, fins, tissues of the buccal cavity, and gill covers or gill arches of marine and brackish-water fishes, mainly in having 8 conspicuously large cephalic papillae of the external circle, the absence of caudal projections, and the shape and small size of the anterior inflation of the esophagus in gravid females, and in possessing 5 pairs of caudal papillae and spicules 66-78 µm long in males. Philometra diplectri is the first known species of this genus whose gravid females are parasitic in the head tissues of serranid fishes.

Topics: Animals; Bass; Dracunculoidea; Female; Fish Diseases; Florida; Male; Microscopy, Electron, Scanning; Prevalence; Seawater; Spirurida Infections

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio sp. Fish disease will be controlled by proper vaccination trials and maintenance of fish form. Pathogenicity for Asian seabass (Lates calcarifer) against V anguillarum results in necrosis and haemorrhagic areas near the base of fins, exopthalmia and ulcers on the skin surface. Around 50, 100, 200 microl of formalin killed bacterial cells were injected (Intraperitoneal) to three different size (5-10, 20-30, 35-50 g) of seabass fishes respectively and control sere as saline were maintained separately. The Relative Percentage Survival (RPS) for vaccinated fishes was 60, 75, and 62.5 respectively and the vaccinations for 20-30 g fishes stay alive. These results stated that the vaccination for fishes with 20-30 g size may fabricate good immune response.

Topics: Animals; Bass; Fish Diseases; Immunization; Vibrio

Abstract: Over a 7-year period, parasites have been collected from 28 species of groupers (Serranidae, Epinephelinae) in the waters off New Caledonia. Host-parasite and parasite-host lists are provided, with a total of 337 host-parasite combinations, including 146 parasite identifications at the species level. Results are included for isopods (5 species), copepods (19), monogeneans (56), digeneans (28), cestodes (12), and nematodes (12). When results are restricted to those 14 fish species for which more than five specimens were examined and to parasites identified at the species level, 109 host-parasite combinations were recorded, with 63 different species, of which monogeneans account for half (32 species), and an average of 4.5 parasite species per fish species. Digenean records were compared for 16 fish species shared with the study of Cribb et al. (2002); based on a total of 90 parasite records identified at the species level, New Caledonia has 17 new records and only seven species were already known from other locations. We hypothesize that the present results represent only a small part of the actual biodiversity, and we predict a biodiversity of 10 different parasite species and 30 host-parasite combinations per serranid. A comparison with a study on Heron Island (Queensland, Australia) by Lester and Sewell (1989) was attempted: of the four species of fish in common and in a total of 91 host-parasite combinations, only six parasites identified at the species level were shared. This suggests strongly that insufficient sampling impairs proper biogeographical or ecological comparisons. Probably only 3% of the parasite species of coral reef fish are already known in New Caledonia.

Topics: Animals; Bass; Biodiversity; Copepoda; Coral Reefs; Fish Diseases; Helminths; Invertebrates; Isopoda; New Caledonia; Parasitic Diseases, Animal

The cercaria of Bucephalus minimus infects the digestive gland and gonads of its first intermediate host, the edible cockle, Cerastoderma edule. Light microscopy (LM) and scanning electron microscopy (SEM) of the cercaria showed a tail formed by a central stem, with 2 long contractile arms presenting distinct morphological surfaces. The encysted metacercaria naturally infected the flathead grey mullet, Mugil cephalus. The cysts found in the heart, liver, and spleen were shown to be identical by the internal transcribed spacer (ITS 1) sequence and morphological features and were associated with encapsulation, recruitment of cell infiltrates, and presence of melanomacrophages and adipose tissue. To establish the life cycle, we compared the ITS1 sequence in an adult from the known definitive host, Dicentrarchus labrax; encysted metacercariae from the liver, heart, and spleen of M. cephalus; and a cercaria from C. edule. With this comparison, we determined that they had a 100% similarity. Therefore, the ITSI sequence data clearly indicate that these 3 parasitic stages belong to the same species, i.e., B. minimus.

Topics: Animals; Base Sequence; Bass; Cardiidae; DNA, Helminth; Fish Diseases; Heart; Life Cycle Stages; Liver; Microscopy, Electron, Scanning; Molecular Sequence Data; Myocardium; Portugal; Prevalence; Sequence Alignment; Sequence Analysis, DNA; Smegmamorpha; Spleen; Trematoda; Trematode Infections

The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an "all-alpha" domain that shows an "up-down bundle" architecture made of six helices (ABB'CDE). The two cysteine residues present in the sequence (i.e. Cys(23) and Cys(126)) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and vertebrates more generally.

Topics: Animals; Bass; Cell Line; Cloning, Molecular; Fish Diseases; Fish Proteins; Gene Expression; Interferon Type I; Interferon-gamma; Introns; Open Reading Frames; Protein Sorting Signals; Protein Structure, Tertiary; Virus Diseases

Viruses belonging to the Nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. In the present study, a total of 69 farmed Tunisian sea bass (Dicentrarchus labrax) and 24 sea bream (Sparus aurata) samples were tested monthly for the detection of betanodavirus. The virus was identified in both species using indirect immunofluorescence assays (IFAT) and RT-PCR. In addition sequence analysis of part of the coat protein gene indicated that both species were infected by highly related, but distinct, strains belonging to the RGNNV genotype. The sequence of the coat protein gene of several strains was identical but up to 9 different sequences were detected in a single farm. In addition, viral sequences obtained from fish that were held at lower temperature (<20 degrees C) were distinct from the rest of the sequences.

Topics: Animals; Base Sequence; Bass; Encephalitis; Fish Diseases; Molecular Sequence Data; Nodaviridae; Phylogeny; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sea Bream; Sequence Analysis, DNA

The peacock grouper (Cephalopholis argus) was intentionally introduced to the Hawaiian coastal waters 50 years ago to enhance the local fisheries. Following introduction, this species spread rapidly and became extremely abundant. A comparison of the metazoan parasite community of C. argus was performed between its native range (Moorea Island, French Polynesia) and its introduced range (Oahu and Big Island, Hawaii). Polynesian groupers were infected with a highly diversified parasite community whereas Hawaiian groupers exhibited a depauperate ensemble of parasite species, C. argus having lost most of the parasites common in their native range. Interestingly, the grouper has not acquired new parasites present in Hawaiian waters. This study provides the first field evidence of significant parasite release in a wild but previously introduced fish in coral reefs and is discussed in relation to the Enemy-Release Hypothesis which has never been assessed in those ecosystems.

Topics: Animals; Bass; Crustacea; Ecosystem; Ectoparasitic Infestations; Fish Diseases; Hawaii; Host-Parasite Interactions; Polynesia; Trematoda; Trematode Infections

Intersex (specifically, testicular oocytes) has been observed in male smallmouth bass (SMB; Micropterus dolomieu) and other centrarchids in the South Branch of the Potomac River, U.S.A., and forks of the Shenandoah River, U.S.A., during the past five years. This condition often is associated with exposure to estrogenic endocrine-disrupting chemicals in some fish species, but such chemicals and their sources have yet to be identified in the Potomac. In an attempt to better understand the plausible causes of this condition, we investigated the reproductive health of bass sampled up- and downstream of wastewater treatment plant (WWTP) effluent point sources on the Potomac River in Maryland, U.S.A. Smallmouth bass were sampled from the Conococheague Creek and the Monocacy River, and largemouth bass (LMB; Micropterus salmoides) were collected near the Blue Plains WWTP on the mainstem of the Potomac River. Chemical analyses of compounds captured in passive samplers at these locations also were conducted. A high prevalence of intersex (82-100%) was identified in male SMB at all sites regardless of collection area. A lower prevalence of intersex (23%) was identified in male LMB collected at the Blue Plains site. When up- and downstream fish were compared, significant differences were noted only in fish from the Conococheague. Differences included condition factor, gonadosomatic index, plasma vitellogenin concentration, and estrogen to testosterone ratio. In general, chemicals associated with wastewater effluent, storm-water runoff, and agriculture were more prevalent at the downstream sampling sites. An exception was atrazine and its associated metabolites, which were present in greater concentrations at the upstream sites. It appears that proximity to effluent from WWTPs may influence the reproductive health of bass in the Potomac watershed, but inputs from other sources likely contribute to the widespread, high incidence of testicular oocytes.

Topics: Animals; Bass; Disorders of Sex Development; Endocrine Disruptors; Environmental Monitoring; Female; Fish Diseases; Gonads; Male; Maryland; Reproduction; Rivers; Virginia; Waste Disposal, Fluid; Water Pollutants, Chemical; West Virginia

The seasonal occurrence of organic contaminants, many of which are potential endocrine disruptors, entering the Potomac River, USA, watershed was investigated using a two-pronged approach during the fall of 2005 and spring of 2006. Passive samplers (semipermeable membrane device and polar organic chemical integrative sampler [POCIS]) were deployed in tandem at sites above and below wastewater treatment plant discharges within the watershed. Analysis of the samplers resulted in detection of 84 of 138 targeted chemicals. The agricultural pesticides atrazine and metolachlor had the greatest seasonal changes in water concentrations, with a 3.1- to 91-fold increase in the spring compared with the level in the previous fall. Coinciding with the elevated concentrations of atrazine in the spring were increasing concentrations of the atrazine degradation products desethylatrazine and desisopropylatrazine in the fall following spring and summer application of the parent compound. Other targeted chemicals (organochlorine pesticides, polycyclic aromatic hydrocarbons, and organic wastewater chemicals) did not indicate seasonal changes in occurrence or concentration; however, the overall concentrations and number of chemicals present were greater at the sites downstream of wastewater treatment plant discharges. Several fragrances and flame retardants were identified in these downstream sites, which are characteristic of wastewater effluent and human activities. The bioluminescent yeast estrogen screen in vitro assay of the POCIS extracts indicated the presence of chemicals that were capable of producing an estrogenic response at all sampling sites.

Topics: Animals; Bass; Disorders of Sex Development; Endocrine Disruptors; Environmental Monitoring; Female; Fish Diseases; Male; Maryland; Reproduction; Rivers; Virginia; Waste Disposal, Fluid; Water Pollutants, Chemical; West Virginia

Diseased wild redspotted grouper Epinephelus akaara were collected from Seto Inland Sea, Ehime Prefecture, in August 2002. Fish showed erratic swimming behavior and inflation of the swim bladder. The fish brains were positive for nodavirus in both RT-PCR and nested PCR. The sequence of the nested PCR product (177 nt) was closely related to that of a known betanodavirus, redspotted grouper nervous necrosis virus. When juvenile sevenband grouper E. septemfasciatus were challenged intravitreously with virus, abnormal swimming behavior and high mortality were observed. This is the first report on viral nervous necrosis in a wild population of redspotted grouper with clinical signs.

Topics: Animals; Bass; Brain; Capsid Proteins; Fish Diseases; Genes, Viral; Japan; Nodaviridae; Phylogeny; RNA Virus Infections; Virulence

The expression and regulation of sodium-independent glucose transporter (GLUT)-2, in relation to hypoxia has not yet been explored in fish or other vertebrates. In this study, the complete open-reading frame for sea bass GLUT2 was isolated and deposited in the GenBank. The predicted 12 transmembrane domains of the protein (508 amino acids) are presented. A phylogenetic tree was constructed on GLUT2 sequences of sea bass and those of other teleost, amphibian, avian, and mammalian species. We also analyzed acute and chronic hypoxia-induced changes in the expression of hepatic GLUT2 mRNA, using one-tube, two-temperature, real-time RT-PCR with which gene expression can be absolutely quantified by the standard curve method. The number of GLUT2 mRNA copies was significantly increased in response to both acute (1.9 mg/L, dissolved oxygen for 4 h) and chronic (4.3 mg/L, DO for 15 days) hypoxia conditions. The hypoxia-related changes in GLUT2 mRNA copy number support the view that GLUT2 is involved in the adaptation response to hypoxia in sea bass, a marine hypoxia-sensitive species. We realize that the GLUT2 mRNA levels in our study do not measure the physiological effects produced by the protein. Thus, we can only speculate that, under hypoxic conditions, GLUT2 probably functions to allow the glucose produced from liver glycogen to leave the hepatocytes.

Topics: Acute Disease; Amino Acid Sequence; Animals; Bass; Chronic Disease; Cloning, Molecular; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Glucose Transporter Type 2; Hypoxia; Molecular Sequence Data; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment

Farmed fish are usually exposed to routine procedures which have strong effects on stress responses. Rodlet cells may represent an useful biomarker for studies on the presence of stressors in aquaculture. This work focused on the localization of rodlet cells by light and electron microscopy in gills of sea bass (Dicentrarchus labrax) subjected to different conditions of overcrowding. In general, a significant increase in number of rodlet cells has been observed in all animals subjected to overcrowding stress. In gills of control group rare rodlet cells were detected at the level of both primary and secondary lamellae, whereas in stressed group clusters of rodlet cells have been found in the epithelium of primary and secondary lamellae indicating that these cells are influenced by stocking density.

Topics: Animals; Aquaculture; Bass; Biomarkers; Cell Physiological Phenomena; Cells; Crowding; Environmental Exposure; Fish Diseases; Gills; Hydrocortisone; Population Density; Seawater

Antimicrobial peptides (AMP) are an integral component of innate immunity. One of the most widespread AMP in fish are the piscidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. The widespread phylogenetic distribution of piscidins suggests that they might play an important host defense role in many fish. Quantifying their expression is important in understanding how and where they function. Using a novel piscidin (piscidin 4) that we recently isolated from commercially cultured hybrid striped bass (white bass, Morone chrysops Rafinesque, female symbolxstriped bass, Morone saxatilis Walbaum male symbol), we optimized the conditions for measuring this piscidin via sandwich ELISA. We used an antibody to the highly conserved amino terminus of all piscidins as the capture antibody and a peroxidase-labeled antibody specific for the carboxy terminus of piscidin 4 as the detecting antibody. Specificity of the detecting antibody was confirmed by lack of cross-reactivity with other piscidins in ELISA, as well as specificity for piscidin 4 in tissue extracts via Western blotting. The accuracy of the test, defined as piscidin 4 recovery, was 96-103%. Precision, measured by the coefficient of variation, was 13-19%, and parallelism, determined by linearity of the response, had an r(2)>0.99. The ELISA paralleled the results obtained via Western blotting. Piscidin 4 levels expressed in gill tissue of healthy hybrid striped bass were well within concentrations that are lethal to important fish pathogens. Mean gill piscidin 4 in healthy hybrid striped bass was significantly greater than in either nutritionally stressed fish or in diseased (ectoparasite-infested) fish, suggesting that piscidin 4 can be significantly downregulated with stress or disease. These data suggest that the piscidin 4 ELISA might be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Enzyme-Linked Immunosorbent Assay; Female; Fish Diseases; Immunoblotting; Male; Reproducibility of Results; Stress, Physiological

The European seabass (Dicentrarchus labrax), one of the most extensively cultured species in European aquaculture productions, is, along with the gilthead sea bream (Sparus aurata), a prospective model species for the Perciformes which includes several other commercially important species. Massive mortalities may be caused by bacterial or viral infections in intensive aquaculture production. Revealing transcripts involved in immune response and studying their relative expression enhances the understanding of the immune response mechanism and consequently also the creation of vaccines. The analysis of expressed sequence tags (EST) is an efficient and easy approach for gene discovery, comparative genomics and for examining gene expression in specific tissues in a qualitative and quantitative way.. Here we describe the construction, analysis and comparison of a total of ten cDNA libraries, six from different tissues infected with V. anguillarum (liver, spleen, head kidney, gill, peritoneal exudates and intestine) and four cDNA libraries from different tissues infected with Nodavirus (liver, spleen, head kidney and brain). In total 9605 sequences representing 3075 (32%) unique sequences (set of sequences obtained after clustering) were obtained and analysed. Among the sequences several immune-related proteins were identified for the first time in the order of Perciformes as well as in Teleostei.. The present study provides new information to the Gene Index of seabass. It gives a unigene set that will make a significant contribution to functional genomic studies and to studies of differential gene expression in relation to the immune system. In addition some of the potentially interesting genes identified by in silico analysis and confirmed by real-time PCR are putative biomarkers for bacterial and viral infections in fish.

Topics: Animals; Bass; Expressed Sequence Tags; Fish Diseases; Gene Expression Profiling; Gene Library; Microsatellite Repeats; Nodaviridae; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Sequence Analysis, DNA; Vibrio

The nematode Philometra rubra (Leidy, 1856) (Philometridae) is redescribed from subgravid females found in the abdominal cavity of the fish Morone saxatilis (Walbaum) from South Carolina, USA in November 2008. The species is characterized by the presence of 14 cephalic papillae arranged in two circles, a relatively long oesophagus with a distinct anterior inflation, and well-developed papilla-like caudal projections. Cephalic papillae of the external circle differ from those in other congeners in that the dorso-lateral and ventro-lateral papillae are large, dome-shaped, whereas the dorso-dorsal and ventro-ventral papillae are small.

Topics: Animals; Bass; Female; Fish Diseases; Microscopy, Electron, Scanning; Nematoda; Nematode Infections; Species Specificity; United States

Vaccinated, feed-trained largemouth bass fry (Micropterus salmoides floridanus) were cohabited with sham-vaccinated fish. Fish were exposed, under natural conditions, to Flavobacterium columnare, a ubiquitous bacterium associated with columnaris disease. During every time interval, the probability that a vaccinated fish would survive past time, t, was greater than for sham-vaccinated fish and survivor functions were significantly different (p-value <0.001). Overall, vaccinated fish had a 43% lower risk of death during the field trial. Overall incidence was 1.7 times greater for the sham-vaccinated (1.4%/d) as compared to the vaccinated fish (0.8%/d). Vaccination with AQUAVAC-COL (Intervet/Schering-Plough Animal Health) significantly reduced the risk of death from columnaris disease in feed-trained largemouth bass fry.

Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Survival Analysis

In response to fish kills at prominent fishing sites for largemouth bass Micropterus salmoides, such as Lake Fork and Sam Rayburn Reservoir, the Texas Parks and Wildlife Department began a systematic evaluation of state waters for the presence of largemouth bass virus (LMBV). The survey comprised 49 water bodies and 13 river basins, and a total of 2,876 adult bass were collected by electrofishing and angling during the summer and fall of 2000. The virus was initially detected by means of cell culture and its presence subsequently confirmed by polymerase chain reaction. Fourteen reservoirs in eight river basins in eastern and central Texas tested positive for LMBV. Lake Fork was also tested to determine the prevalence of infection following a 1999 LMBV fish kill. The overall prevalence was low in all of the water bodies tested (1.50 +/- 2.82% [mean +/- SD]) as well as those determined to contain LMBV (5.00 +/- 3.02%). Largemouth bass testing positive for LMBV had a significantly higher prevalence of swim bladder anomalies, but this condition was not a good indicator of LMBV infection. No significant relationships were found between LMBV-positive fish and other factors investigated, including the presence or absence of grossly visible injury, hook marks, external parasites, known water quality problems, gender, allozyme-phenotype, method of capture, length, weight, body condition (relative weight), or age. This survey provided a means of gathering scientific information about LMBV, including its distribution in Texas. From the information gained by this survey, prior fish kills, and previous sampling efforts, a total of 19 water bodies within 9 of the 13 major river basins in the state were found to contain the virus. These results were used to guide a statewide fish stocking strategy aimed at preventing the spread of LMBV in Texas and to contribute to a nationwide effort to understand this virus and its effects on largemouth bass fisheries.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Iridoviridae; Texas

Abstract An inactivated betanodavirus, red-spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 degrees C water temperature by a single intraperitoneal injection of formalin-inactivated RGNNV. Fish immunized at vaccine doses of 10(8.5), 10(8.0), 10(7.5), 10(7.0) and 10(6.5) TCID(50) per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post-immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (10(5.0) and 10(4.0) TCID(50)/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10(8.5), 10(8.0), 10(7.5) and 10(7.0) TCID(50) per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10(7.5) TCID(50) per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10(6.5) TCID(50) per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10(7.0) TCID(50) per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.

Topics: Animals; Antibodies, Viral; Bass; Fish Diseases; Nodaviridae; RNA Virus Infections; Time Factors; Vaccines, Inactivated; Viral Vaccines

Topics: Animals; Bass; Breeding; Female; Fish Diseases; Male; Muscle, Striated; Nematoda; Nematode Infections; Pancreas; Southeastern United States

A 22.4-ha impoundment experienced an outbreak of Largemouth bass (Micropterus salmoides) virus (LMBV) disease in the summer of 2006. All dead or dying largemouth bass observed throughout the entire event were recorded and removed. In this study, we estimated mortality and examined size distribution, condition, and biomass following the outbreak. Boat-mounted electrofishing was used to collect largemouth bass for a mark-recapture population estimate and other population metrics. Fish samples were examined for evidence of LMBV, other infectious diseases, and physical abnormalities. Cell cultures inoculated with samples from moribund fish developed cytopathic effects typical of LMBV, and polymerase chain reaction (PCR) confirmed the presence of LMBV. The total number (N+/-95% confidence interval) of stock-size largemouth bass remaining was estimated to be 2,301+/-528 fish (103 bass/ha). The total observed mortality, including dead and dying individuals, during the LMBV outbreak was 176 largemouth bass (7% of the initial population). The total biomass remaining was estimated at 1,592 kg of stock-size bass and a relative biomass of 71.5 kg of stock-size largemouth bass per hectare. Largemouth bass size structure was dominated by quality and preferred (300-510 mm) size classes, with very few memorable-size or larger (>510 mm) fish, and the relative weight of largemouth bass was unusually variable. These results demonstrate that largemouth bass abundance and biomass in the reservoir remained very high despite mortalities attributed to a LMBV outbreak.

Topics: Animals; Arkansas; Bass; Disease Outbreaks; DNA Virus Infections; Fish Diseases; Iridoviridae; Population Density; Water Microbiology

Singapore grouper iridovirus (SGIV), as a causative agent of serious systemic disease, causes significant economic losses in grouper aquaculture. In this study, a novel ICP18 homolog encoded by SGIV ORF086R was identified and characterized. Strikingly, ICP18 homologs can be found in all ranaviruses, but not in other sequenced large DNA viruses. SGIV ICP18 is an immediate-early gene and begins to be transcribed as early as 2 h post-infection (p.i.). Western blotting indicated that SGIV ICP18 is translated as early as 6 h p.i. and is a viral non-envelope protein. Subcellular localization analysis revealed that the SGIV ICP18 displays a finely punctate cytoplasmic pattern. Furthermore, overexpression of SGIV ICP18 can promote the growth of grouper embryonic cells (GP) and contribute to SGIV replication. These results should offer important insights into the pathogenesis of ranaviruses.

Topics: Amino Acid Sequence; Animals; Bass; Cell Proliferation; Cells, Cultured; Cytoplasm; DNA Virus Infections; Fish Diseases; Genes, Viral; Immediate-Early Proteins; Molecular Sequence Data; Open Reading Frames; Ranavirus; Sequence Alignment; Sequence Homology, Amino Acid; Virus Replication

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.

Topics: Animals; Bass; Disease Outbreaks; Evolution, Molecular; Fish Diseases; Fisheries; Genome, Viral; Nodaviridae; Phylogeny; Reassortant Viruses; Recombination, Genetic; RNA Virus Infections; Sea Bream; Sequence Analysis, DNA

Intersex occurrence in freshwater fishes was evaluated for nine river basins in the United States. Testicular oocytes (predominantly male testes containing female germ cells) were the most pervasive form of intersex observed, even though similar numbers of male (n=1477) and female (n=1633) fish were examined. Intersex was found in 3% of the fish collected. The intersex condition was observed in four of the 16 species examined (25%) and in fish from 34 of 111 sites (31%). Intersex was not found in multiple species from the same site but was most prevalent in largemouth bass (Micropterus salmoides; 18% of males) and smallmouth bass (M. dolomieu; 33% of males). The percentage of intersex fish per site was 8-91% for largemouth bass and 14-73% for smallmouth bass. The incidence of intersex was greatest in the southeastern United States, with intersex largemouth bass present at all sites in the Apalachicola, Savannah, and Pee Dee River Basins. Total mercury, trans-nonachlor, p,p'-DDE, p,p'-DDD, and total PCBs were the most commonly detected chemical contaminants at all sites, regardless of whether intersex was observed. Although the genotype of the intersex fish was not determined, the microscopic appearance of the gonads, the presence of mature sperm, and the concentrations of sex steroid hormones and vitellogenin indicate the intersex bass were males. Few reproductive endpoints differed significantly among male and intersex bass; plasma vitellogenin concentration in males was not a good indicator of intersex presence. Hierarchical linkages of the intersex condition to reproductive function will require a more quantitative measure of intersex (e.g. severity index) rather than presence or absence of the condition. The baseline incidence of intersex gonadal tissue in black basses and other freshwater fishes is unknown, but intersex prevalence may be related to collection season, age, and endocrine active compounds in the environment. Intersex was not found in largemouth bass older than five years and was most common in 1-3-year-old male largemouth bass. The cause(s) of intersex in these species is also unknown, and it remains to be determined whether the intersex we observed in largemouth and smallmouth bass developed during sex differentiation in early life stages, during exposure to environmental factors during adult life stages, or both.

Topics: Animals; Bass; Disorders of Sex Development; Female; Fish Diseases; Male; Oocytes; Prevalence; Rivers; Testis; United States; Water Pollutants, Chemical

A retrospective analysis of archived tissue blocks has revealed that mycobacteriosis was apparent in Chesapeake Bay striped bass as early as 1984. Of 37 cases available from the years 1975 to 1985, 2 fish were found positive based on histopathology and genus-specific PCR. Multi-gene sequencing places the bacteria from the 2 positive cases (1984 and 1985) within the Mycobacterium tuberculosis clade with closest resemblance to the recently described fish pathogen M. pseudoshottsii. Our data confirms that mycobacteriosis is not a new disease of Chesapeake Bay striped bass and underscores the value of archived tissues in epidemiological examinations.

Topics: Animals; Bass; Fish Diseases; Mycobacterium; Mycobacterium Infections, Nontuberculous; Oceans and Seas; Polymerase Chain Reaction

The life cycle of a new microsporidian of the genus Pleistophora is described. This parasite infects the epithelial cells of the gut and the peritoneal cavity of the Red Sea fish, Epinephelus chlorostignei. All stages develop within a special structure, the sporophorocyst, which is covered by a thick dense wall. This wall grows along with the growth of the parasites inside. Meronts are uni- to binucleate, which divide and constantly give rise to sporonts. During transition to sporonts, the cell border of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually, a uniform thick sporont wall is formed; then, the sporont cells detach themselves from the wall (future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores, and consists of two layers. Mature spores measure about 2.0 x 1.8 microm. They possess a polar filament with 20-28 coils, a posterior vacuole, and a polaroplast made up of an outer part of dense and closely spaced lamellae encircling an inner part of widely spaced lamellae. All morphological and ultrastructural features indicate that the described microsporidian parasite belongs to the genus Pleistophora.

Topics: Animals; Bass; Epithelial Cells; Fish Diseases; Gastrointestinal Tract; Host-Parasite Interactions; Microscopy; Microscopy, Electron, Transmission; Microsporidiosis; Peritoneal Cavity; Pleistophora

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1beta, TNF-alpha, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

Topics: Amino Acid Sequence; Animals; Bass; Brain; Fish Diseases; Galectin 1; Gene Expression Profiling; Interleukin-1beta; Kidney; Lectins, C-Type; Molecular Sequence Data; Nerve Tissue Proteins; Nodaviridae; Phylogeny; Recombinant Proteins; Respiratory Burst; RNA Virus Infections; Sequence Alignment; Tumor Necrosis Factor-alpha; Up-Regulation

The immune response of European sea bass, Dicentrarchus labrax, to a natural infection by the copepod parasite Lernanthropus kroyeri was evaluated for the first time in vivo. The results clearly demonstrated the triggering of the fish immune system by the parasite. Lysozyme activity and alternative complement pathway were involved in the early action against the parasitical infection, whilst classical complement and respiratory burst (RB) activity took over in the later stages of infection. It was hypothesized that the levels of alternative and classical complement and RB stimulation indexes may determine the resistance capacity of the fish to the parasite. It is not clear how parasites can survive despite the strong immunological arsenal deployed by the fish. The continual increase of prevalence and severity of parasite infection suggested that the parasite's mechanism of evasion of the immune system was extremely successful. The contrasting decrease in the negative effects of parasites on the fish health (such as gills anaemia) suggested that an equilibrium between the parasites and their hosts was reached in chronic infection. These dynamic interactions between parasites and fish hosts were probably the main determinant of host specificity.

Topics: Anemia; Animals; Bass; Blood Bactericidal Activity; Complement System Proteins; Copepoda; Female; Fish Diseases; Fisheries; Gills; Greece; Host-Parasite Interactions; Immunity, Innate; Male; Muramidase; Nitric Oxide; Respiratory Burst

The grow out of Asian seabass Lates calcarifer in marine net-cages is a popular aquaculture activity in Malaysia. Production of this species is greatly affected by the occurrence of vibriosis, which causes heavy mortality. Generally, young fish are more susceptible; they exhibit anorexia and skin darkening, followed by heavy mortality. The acutely affected older fish may also exhibit bloody lesions around the anus and the base of the fins. Twenty-one bacterial isolates obtained from internal organs (kidney, heart, spleen and liver) of the affected specimens were subjected to phenotypic characterization, testing for antibiotic susceptibility, and 16S ribosomal DNA sequencing. The sequencing result showed that all of the bacterial isolates belonged to Vibrio harveyi. The phenotypic characterization, however, identified 4 of the bacterial isolates as V. harveyi, 16 as V. parahaemolyticus, and 1 as V. alginolyticus. These findings suggest that biochemical features alone cannot be reliably used to identify bacterial pathogens, including V. harveyi, in aquaculture. Antibiotic susceptibility assays showed that some antibiotics, including oxytetracycline, nitrofurantoin, furazolidone, streptomycin, sulfamethoxazole, chloramphenicol, nalidixic acid, and oxolinic acid were effective against V. harveyi. Considering the side effects of these antibiotics, however, their use is not recommended in the aquaculture of Asian seabass.

Topics: Animals; Bass; Fish Diseases; RNA, Ribosomal, 16S; Vibrio; Vibrio Infections

Challenge studies with Mycobacterium marinum clearly demonstrate that a poor diet affects the progression and severity of mycobacteriosis in striped bass Morone saxatilis. Fish (n = 512 total, wt = 65 +/- 15 g) were inoculated intraperitoneally with 10(4) colony-forming units (CFU) g(-1) body weigth (BW) or a physiological saline solution (controls) and evaluated for 8 mo. Inoculated fish fed a low-ration diet (0.15% BW d(-1)) developed a severe, systemic infection characterized by a high bacterial load (>10(8) CFU g(-1) spleen) and poor granuloma formation, which commonly progressed to mortality by 6 wk. In contrast, inoculated fish fed an adequate ration diet (1% BW d(-1)) developed classic granulomatous inflammation of reduced severity and total body energy similar to that found in uninoculated controls (p > 0.05). After 4 wk, fish fed adequate rations maintained an equilibrium state throughout the study period, even though 10(6) CFU g(-1) spleen mycobacteria were consistently cultured. In a second study, reactivation of an acute inflammatory state was demonstrated by placing previously infected fish on reducing diets (0.073% BW d(-1)). In both studies, the energetic demand of this disease was only appreciable when associated with active, severe, inflammatory states. To our knowledge, this study is the first to demonstrate the interaction of diet and mycobacteriosis in fish.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Bass; Diet; Energy Metabolism; Erythrocyte Count; Fish Diseases; Heart; Kidney; Leukocyte Count; Liver; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Spleen; Time Factors

The aim of this study was to monitor erythrocyte nuclear abnormalities (NA) including micronuclei (MN) in cultured and wild sea bass Dicentrarchus labrax and wild mullet Mugil spp. Seasonal sampling was performed at seven locations along the eastern coast of the Adriatic Sea. The frequency of NA and MN was positively correlated to temperature (NA: P < 0.05, r = 0.11; MN P < 0.05, r = 0.10), and there was also a positive correlation between NA and MN frequency (P < 0.001, r = 0.43). The lowest NA and MN values for both fish species were recorded in spring, while the highest were recorded in autumn. Significantly higher frequency of NA was seen in D. labrax compared to Mugil spp., while MN frequency was low in both species and not significantly different. There was no significant difference in NA and MN frequency between cultured and wild D. labrax sampled in the same month, and there was no difference between wild Mugil spp. sampled near or far from fish farms. In view of sampling sites, the highest values were detected in fishes from the Limski Channel, the lowest from the Janjina location.

Topics: Animals; Aquatic Organisms; Bass; Cell Nucleus; Erythrocytes; Fish Diseases; Fisheries; Oceans and Seas; Seasons; Smegmamorpha; Temperature

Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a host's cellular protein response to viral infection on a translational basis.

Topics: Animals; Antigens; Bass; Blotting, Western; Cell Line; Cytopathogenic Effect, Viral; Fish Diseases; Gene Expression Profiling; Iridovirus

Fishborne zoonotic metacercariae have not been reported from brackish water and marine fish from Vietnam waters although these parasites are common in the country's freshwater fish. Both wild-caught and cultured grouper (Epinephelus coioides and Epinephelus bleekeri), and mullet (Mugil cephalus) from brackish and marine waters located in Khanh Hoa province in central coastal Vietnam were examined, and found positive for zoonotic trematode metacercariae. From grouper, Heterophyopsis continua and Procerovum varium were recovered. The prevalence of H. continua ranged from 2.0 to 6.0% and that for P. varium ranged from 11.6 to 15.8%. Mullet were infected with Pygidiopsis summa and H. continua both of these species are new records for Vietnam. The prevalence of P. summa in mullet was generally high, ranging from 17.6 to 75.5%, and was significantly higher than the prevalence of H. continua (2.5 to 32.4%). There were no significant differences in the prevalence of metacercariae between grouper from natural or cultured habitats, indicating that the highest risk of infection occurs in the wild-caught state prior to their placement in culture. Further, there was no difference in metacercarial prevalence between the 2 species of grouper. Infected wild-caught seed were only observed from January to October. Monthly variation in prevalence suggests seasonal variation in mullet infections occurs in this region with the highest transmission taking place from October to December. Basic investigations on the ecology and epidemiology of these intestinal flukes need to be carried out to determine their significance as a public health problem and the aspects of their biology that may be vulnerable to control interventions.

Topics: Animals; Bass; Fish Diseases; Fresh Water; Humans; Prevalence; Seawater; Smegmamorpha; Time Factors; Trematoda; Trematode Infections; Vietnam; Zoonoses

A DNA microarray containing all Singapore grouper iridovirus (SGIV) open reading frames (ORFs) was constructed to map the viral gene transcriptional profiles in virus-infected grouper spleen (GS) cells and in spleen tissues of virus-infected grouper. The results showed that viral genes started to be transcribed as early as 1 h postinfection (p.i.), and followed by a rapid increasing gene expression along with virus infection in cell cultures. The three temporal kinetic classes (15 immediate-early, 89 early and 53 late transcripts) were classified during an in vitro infection by their dependence on de novo protein synthesis and viral DNA replication inhibitors. In SGIV-infected grouper, Epinephulus coioides, most of the viral genes were expressed between 1 and 4 d p.i., and the number and expression levels started to decrease after 5 d p.i. These data were confirmed by real-time RT-PCR. This study provides an experimental basis for investigation of virus-host interactions and the development of control strategies against SGIV infection.

Topics: Animals; Base Sequence; Bass; Cells, Cultured; DNA Primers; DNA Virus Infections; DNA, Viral; Fish Diseases; Gene Expression Profiling; Genome, Viral; Iridovirus; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

We evaluated the alterations of organochlorinated compounds such as polychlorobiphenyls (PCB), dichloro-diphenyl-dichloroethylene (DDE) and dichloro-diphenyl-trichloroethane (DDT) on the thyroid in wild and cultured sea bass (Dicentrarchus labrax) at environmental concentrations. These compounds influence the endocrine system of many fish species and are qualified as endocrine disruptors. The thyroid seems to be a target organ. Two alteration endpoints: the thyroid histology and the muscular thyroid hormone concentrations, were used simultaneously. High concentrations in PCBs and DDT were detected in muscles, supporting the idea that the Mediterranean fauna could be more polluted than the Atlantic fauna. The high abundance of DDE indicates a progressive degradation of remnant DDT load and the absence of new inputs in this area. Aquaculture sea bass shows a significant higher amount of pollutants on fresh weight basis (especially PCBs) in their muscles compared to the wild sea bass. Those differences may be related mainly to the contaminations of diet. Thyroid parameters vary between wild and aquaculture sea bass, wild sea bass were characterized by higher follicle diameters, epithelial cell heights and muscular T(4) concentrations. A significant relationship between persistent organic pollutants (muscular PCBs and DDT concentration) and the different thyroid parameters (diameters of follicles, epithelial cell heights and muscular T(4) levels) could be observed, which support the hypothesis that these compounds have an adverse impact on thyroid morphometry and function.

Topics: Animals; Bass; Endocrine Disruptors; Fish Diseases; Mediterranean Region; Muscle, Skeletal; Oceans and Seas; Polychlorinated Biphenyls; Thyroid Gland; Water Pollutants, Chemical

Prodistomum lichtenfelsi n. sp. (Digenea: Lepocreadiidae) is described; it was obtained from the intestine of the longtail bass, Hemanthias leptus (Ginsburg), collected from the Bay of Campeche in the Gulf of Mexico. This is the first record of a parasite from this host. Prodistomum lichtenfelsi n. sp. is similar to P. hynnodi, P. menidiae, and P. waltairensis in that it possesses a prepharynx that is distinctly shorter (153 microm [110-210] long) than the combined length of the esophagus (163 microm [110-220] long) and the pseudoesophagus (137 microm [120-170] long), but it differs from them in having an excretory vesicle that extends into the forebody, a smooth ovary and testes, and vitellaria that extend to the posterior level of the esophagus. An updated key to the 12 nominal species within Prodistomum is given, and the diagnosis of the genus is emended to include species possessing a sinuous external seminal vesicle.

Topics: Animals; Bass; Fish Diseases; Mexico; Seawater; Trematoda; Trematode Infections

The striped bass (Morone saxatilis) is an economically and ecologically important finfish species along the Atlantic seaboard of the United States. Recent stock assessments in Chesapeake Bay (U.S.A.) indicate that non-fishing mortality in striped bass has increased since 1999, concomitant with very high (>50%) prevalence of visceral and dermal disease caused by Mycobacterium spp. Current fishery assessment models do not differentiate between disease and other components of non-fishing mortality (e.g., senescence, predation); therefore, disease impact on the striped bass population has not been established. Specific measurement of mortality associated with mycobacteriosis in wild striped bass is complicated because the disease is chronic and mortality is cryptic. Epidemiological models have been developed to estimate disease-associated mortality from cross-sectional prevalence data and have recently been generalized to represent disease processes more realistically. Here, we used this generalized approach to demonstrate disease-associated mortality in striped bass from Chesapeake Bay. To our knowledge this is the first demonstration of cryptic mortality associated with a chronic infectious disease in a wild finfish. This finding has direct implications for management and stock assessment of striped bass, as it demonstrates population-level negative impacts of a chronic disease. Additionally, this research provides a framework by which disease-associated mortality may be specifically addressed within fisheries models for resource management.

Topics: Age Distribution; Animals; Bass; Female; Fish Diseases; Male; Mycobacterium; Mycobacterium Infections, Nontuberculous; Oceans and Seas; United States

During 2004 and 2005 a survey was conducted to investigate the presence and geographic distribution of largemouth bass virus (LMBV) in New York State. This iridovirus is widely distributed across the eastern United States; however, it had not previously been reported in New York State. Two hundred and eighty-three wild largemouth bass Micropterus salmoides and 8 smallmouth bass M. dolomieu were collected from 37 locations across the state. No clinical signs of LMBV or mortalities attributable to the virus were observed in the fish collected. Using a quantitative polymerase chain reaction (QPCR) method, we detected LMBV in 28 fish from 13 locations. Viral cytopathic effect in cell culture was observed in 5 fish from 3 locations. The virus isolated from cell culture was confirmed to be LMBV by an independent PCR method. Statistical analysis of the largemouth bass samples collected during 2005 revealed a wide difference in prevalence between the QPCR results and the cell culture results. Analysis of possible predictors, including age, sex, and month collected, showed no significant associations with the QPCR results. This survey confirms the presence and wide distribution of a potentially pathogenic form of LMBV in multiple water systems across New York State.

Topics: Age Factors; Animals; Bass; DNA Virus Infections; DNA, Viral; Fish Diseases; Iridovirus; New York; Polymerase Chain Reaction; Seasons; Sex Factors; Water Microbiology

A detailed redescription of Lernanthropus kroyeri van Beneden, 1851 is provided based on observations made with the aid of scanning electron microscopy. Specimens were obtained from the host, the sea bass Dicentrarchus labrax (L., 1758) obtained from a commercial aquaculture enterprise in Izmir (western Turkey).

Topics: Animals; Aquaculture; Bass; Copepoda; Ectoparasitic Infestations; Female; Fish Diseases; Male; Microscopy, Electron, Scanning; Turkey

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR-RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.

Topics: Animals; Bass; Colony Count, Microbial; DNA, Bacterial; Fish Diseases; Mycobacterium; Mycobacterium Infections; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Prevalence; Species Specificity; Spleen

The biflagellated, single-celled parasite Ichthyobodo necator can cause significant losses among fish populations, particularly those cultured in tanks. Treatments of KMnO4 and CuSO4 were evaluated against a naturally occurring I. necator infestation on sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis) raised in tanks. Four-hour static treatments with 3 mg of KMnO4/L of water (2.5 mg/L above the determined KMnO4 demand) or 2 mg of CuSO4/L of water (total alkalinity = 207 mg/L; total hardness = 95 mg/L) were randomly applied to 4 tanks/treatment (23 fish/tank); the same treatments were reapplied 2 d later. Four tanks were used as positive controls. By 2 d posttreatment (after the second treatment), only 17.4% of the untreated control fish survived, and a sample of the remaining fish was heavily infested with I. necator. All remaining control fish were dead by 5 d posttreatment. The KMnO4 treatment significantly curtailed the initial mortality (survival = 92.4%) and slightly reduced the high parasite loads at 2 d posttreatment. However, fish mortalities increased dramatically over the next 3 d (survival at 5 d posttreatment = 37.5%), and parasite loads from sampled fish remained high. The CuSO4 treatment was effective in significantly lowering the parasite load (almost eliminating I. necator) and maintaining a high fish survival (87.5%) by 5 d posttreatment. The findings in this study clearly demonstrate that CuSO4 is a viable treatment for ichthyobodosis in tanks.

Topics: Animals; Antiprotozoal Agents; Aquaculture; Bass; Copper Sulfate; Female; Fish Diseases; Kinetoplastida; Male; Potassium Permanganate; Random Allocation

Nodavirus has become a serious pathogen for a wide range of cultured marine fish species. In the present work, the expression of genes related to immune and inflammatory responses of sea bream (Sparus aurata L.), considered as non susceptible species, was studied both in vitro and in vivo. No replication of the virus was observed in head kidney macrophages and blood leukocytes. Moreover, the enhancement of expression of several immune genes (tumor necrosis factor alpha (TNFalpha), interleukin-1-beta (IL-1beta), interferon-induced Mx protein) was not detected in both head kidney macrophages and blood leucocytes in response to an in vitro infection with nodavirus. However, in vivo, nodavirus was detected 1 day post-infection (p.i.) by a reverse transcription-polymerase chain reaction (RT-PCR) in blood, liver, head kidney and brain of experimentally infected sea bream, while its presence clearly decreased in blood after 3 days p.i. Also, a transitory increment of the expression of TNFalpha and IL-1beta was detected in the brain of intramuscular (i.m.) infected sea bream 3 days p.i. In head kidney, the over expression of TNFalpha was only observed 1 day p.i. The expression of Mx, an interferon induced gene, was increased in brain and head kidney of infected sea bream, reaching values of 1300-fold compared to controls in brain three days post-infection. For comparative purposes, we analyzed the expression of the same genes on a susceptible species, such as sea bass (Dicentrarchus labrax) and, although the same pattern of expression was observed both in brain and kidney, the magnitude was different mainly in the case of brain, the key organ of the infection, where higher expression of TNFalpha and lower expression of Mx compared with control was observed.

Topics: Animals; Bass; Brain; Cytokines; DNA, Viral; Fish Diseases; Fish Proteins; Gene Expression Regulation; Inflammation Mediators; Kidney; Leukocytes; Macrophages; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; Sea Bream; Virus Replication

Largemouth bass (Micropterus salmoides) and common carp (Cyprinus carpio) were collected from 13 sites located in the Mobile (MRB), Apalachicola-Flint-Chattahoochee (ARB), Savannah (SRB), and Pee Dee (PRB) River Basins to document spatial trends in accumulative chemical contaminants, health indicators, and reproductive biomarkers. Organochlorine residues, 2,3,7,8-tetrachlorodibenzo-p-dioxin-like activity (TCDD-EQ), and elemental contaminants were measured in composite samples of whole fish, grouped by species and gender, from each site. Mercury (Hg) and polychlorinated biphenyls (PCBs) were the primary contaminants of concern. Concentrations of Hg in bass samples from all basins exceeded toxicity thresholds for piscivorous mammals (>0.1 microg/g ww), juvenile and adult fish (>0.2 microg/g ww), and piscivorous birds (>0.3 microg/g ww). Total PCB concentrations in samples from the MRB, ARB, and PRB were >480 ng/g ww and may be a risk to piscivorous wildlife. Selenium concentrations also exceeded toxicity thresholds (>0.75 microg/g ww) in MRB and ARB fish. Concentrations of other formerly used (total chlordanes, dieldrin, endrin, aldrin, mirex, and hexachlorobenzene) and currently used (pentachlorobenzene, pentachloroanisole, dacthal, endosulfan, gamma-hexachlorocyclohexane, and methoxychlor) organochlorine residues were generally low or did not exceed toxicity thresholds for fish and piscivorous wildlife. TCDD-EQs exceeded wildlife dietary guidelines (>5 pg/g ww) in MRB and PRB fish. Hepatic ethoxyresorufin O-deethylase (EROD) activity was generally greatest in MRB bass and carp. Altered fish health indicators and reproductive biomarker were noted in individual fish, but mean responses were similar among basins. The field necropsy and histopathological examination determined that MRB fish were generally in poorer health than those from the other basins, primarily due to parasitic infestations. Tumors were found in few fish (n=5; 0.01%); ovarian tumors of smooth muscle origin were found in two ARB carp from the same site. Intersex gonads were identified in 47 male bass (42%) representing 12 sites and may indicate exposure to potential endocrine disrupting compounds. Comparatively high vitellogenin concentrations (>0.35 mg/mL) in male fish from the MRB, SRB, and PRB indicate exposure to estrogenic or anti-androgenic chemicals.

Topics: Animals; Bass; Carps; Cytochrome P-450 CYP1A1; Dioxins; Estradiol; Female; Fish Diseases; Gonads; Hydrocarbons, Chlorinated; Lipids; Male; Metals, Heavy; Pesticides; Rivers; Selenium; Southeastern United States; Testosterone; Vitellogenins; Water Pollutants, Chemical

Photobacterium damselae subsp. piscicida is a bacterial fish pathogen that causes a disease known as pasteurellosis. Two transferable multiple-drug resistance (R) plasmids, pP99-018 (carrying resistance to kanamycin, chloramphenicol, tetracycline, and sulfonamide) and pP91278 (carrying resistance to tetracycline, trimethoprim, and sulfonamide), isolated from P. damselae subsp. piscicida strains from Japan (P99-018) and the United States (P91278), respectively, were completely sequenced and analyzed, along with the multiple-drug resistance regions of three other R plasmids also from P. damselae subsp. piscicida strains from Japan. The sequence structures of pP99-018 (150,057 bp) and pP91278 (131,520 bp) were highly conserved, with differences due to variation in the drug resistance and conjugative transfer regions. These plasmids, shown to be closely related to the IncJ element R391 (a conjugative, self-transmitting, integrating element, or constin), were divided into the conjugative transfer, replication, partition, and multiple-drug resistance regions. Each of the five multiple-drug resistance regions sequenced exhibited unique drug resistance marker composition and arrangement.

Topics: Animals; Bass; Conjugation, Genetic; Drug Resistance, Multiple, Bacterial; Fish Diseases; Gram-Negative Bacterial Infections; Japan; Molecular Sequence Data; Perciformes; Photobacterium; R Factors; Sequence Analysis, DNA; United States

The sea bass, Dicentrarchus labrax, is one of the most extensively farmed marine fishes in the Mediterranean. Under the high-density condition common in aquaculture, the monogenean gill parasite Diplectanum aequans can cause significant economic losses. This study used real-time quantitative PCR to investigate the dynamic expression of immune response genes in sea bass infected with Diplectanum aequans. The target genes, interleukin-1 (IL-1beta, transforming growth factor (TGF-beta and T-cell receptor (TCR-beta), were studied in the gills and spleen of the sea bass from the first day of infection until thirty days post- infection. Our results showed that there was an increase in IL-1beta gene expression in the spleen and gills and in TGF-beta gene expression in the gills of infected fish. These results show that parasitic infection induced a local inflammatory reaction and that reaction was restricted to the site of infection. Finally, the absence of relationship between TCR-beta expression and the parasitic infection suggests that the adaptive immune system is not involved in the response against this parasite.

Topics: Animals; Bass; Body Size; Fish Diseases; Gene Expression Regulation; Genes, T-Cell Receptor beta; Gills; Interleukin-1beta; Spleen; Transforming Growth Factor beta1; Trematoda; Trematode Infections

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.

Topics: Animals; Bass; Cell Line; Colony Count, Microbial; Fish Diseases; Hemolysis; Injections, Intraperitoneal; Phagocytosis; Random Allocation; Species Specificity; Streptococcal Infections; Streptococcus; Virulence Factors

Abstract The antiparasitic effects of piscidin 2, an antimicrobial polypeptide (AMPP) first isolated from mast cells of hybrid striped bass, were tested against three protistan ectoparasites of marine fish (the ciliates Cryptocaryon irritans and Trichodina sp., and the dinoflagellate Amyloodinium ocellatum) and one ciliate ectoparasite of freshwater fish (Ichthyophthirius multifiliis). I. multifiliis was the most susceptible parasite, with all theronts killed at 6.3 microg mL(-1) piscidin 2. The most resistant parasite was Trichodina, where a few cells were killed at 12.5 microg mL(-1), but several were still alive even at 100 microg mL(-1). C. irritans was of intermediate sensitivity, with some theronts killed at 12.5 microg mL(-1) and all killed at 25 microg mL(-1). High parasite density apparently exhausted the piscidin 2 before it could attain its maximal effect, but surviving parasites were often visibly damaged. The lower efficacy of piscidin 2 against marine parasites compared with the freshwater ciliate might be related to the inhibitory effects of high sea water cation levels. The tissue concentration of piscidins estimated in healthy hybrid striped bass gill (40 microg mL(-1)) suggests that piscidin 2 is lethal to the parasites tested at physiological concentrations and is thus an important component of innate defence in fish expressing this type of AMPP.

Topics: Animals; Antimicrobial Cationic Peptides; Bass; Ciliophora; Ectoparasitic Infestations; Fish Diseases; Fish Proteins; Gills; Parasitic Sensitivity Tests; Survival Analysis

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

Topics: Animals; Bass; Crosses, Genetic; DNA, Ribosomal Spacer; Fish Diseases; Heat-Shock Proteins; Hybridization, Genetic; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Spleen; Virulence; Zebrafish

Cultured and wild sea bass (Dicentrarchus labrax) from the Arade Estuary were sampled in summer and winter and the degree of exposure to metals and polycyclic aromatic hydrocarbons (PAHs) assessed, together with some biochemical responses against those and other pollutants. The highest levels of copper (up to 997 microg g-1 dry weight) and cadmium (up to 4.22 microg g-1 dry weight) were detected in the liver and kidney of cultured specimens, whereas the highest exposure to PAHs was observed in wild fish. Significant alterations in some biochemical markers were detected and associated to pollutant exposure. Thus, metallothionein concentrations were higher in the tissues of cultured fish and positively correlated with metal residues. The activity 7-ethoxyresorufin O-deethylase ranged from 28 pmol/min/mg protein in cultured fish to 83 pmol/min/mg protein in wild fish collected near a marina area. Cultured fish and wild fish from the marina area had depressed acetylcholinesterase in muscle tissue and a parasitic infection in the gonads. The obtained results support the usefulness of the combined use of chemical and biochemical markers to assess the impact of anthropogenic pollutants in both wild and cultured fish.

Topics: Acetylcholinesterase; Animals; Animals, Wild; Bass; Cytochrome P-450 CYP1A1; Fish Diseases; Fusobacterium Infections; Male; Metallothionein; Metals, Heavy; Organ Specificity; Polycyclic Aromatic Hydrocarbons; Portugal; Seasons; Testis; Tissue Distribution; Water Pollutants, Chemical

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

Topics: Amino Acid Sequence; Animals; Bacteremia; Base Sequence; Bass; Caspase 9; Cloning, Molecular; DNA, Complementary; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Molecular Sequence Data; Photobacterium; Phylogeny; Sequence Homology, Amino Acid

The mortality of juvenile European sea bass, Dicentrarchus labrax (L.), in the spring of the last 5 years in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans (Wagener 1857) Diesing 1858. The histopathological examination of 38 sets of gills from hosts measuring 16.46 +/- 0.26 cm in total length (mean+/-S.E.) and weighing 45.98 +/- 2.37 g (mean+/-S.E.) were conducted using light and transmission electron microscopy. Twenty-eight (73.6%) D. labrax specimens were infected (34.61 +/- 4.42, mean intensity+/-S.E.; 5-100, range) with the majority of D. aequans attaching to the median and apical portions of the primary gill filaments. The sites of attachment were marked by the common presence of haemorrhages and a white mucoid exudate. In histological sections, the opisthaptors of the parasites were observed to penetrate deeply, lying in close proximity to the basal membrane of primary lamella where they induced a hyperplastic response. Disruption and fusion of the secondary lamellae were common in all infected specimens with several individuals also exhibiting a marked erosion and inflammation of the epithelium of the primary and secondary lamellae. In infected fish, cellular changes in the epithelium underlying the bodies of worms were noted typified by an elevation in the number of mucous and rodlet cells and a reduction in the number of chloride cells.

Topics: Animals; Bass; Fish Diseases; Gills; Helminthiasis, Animal; Platyhelminths

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Topics: Animals; Bacterial Capsules; Bass; Carbohydrates; Cell Line; Epithelial Cells; Fish Diseases; Gene Expression Regulation, Bacterial; Macrophages; Molecular Sequence Data; Phagocytosis; Streptococcal Infections; Streptococcus; Virulence

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.

Topics: Agglutination Tests; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Bass; Complement System Proteins; DNA, Bacterial; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Kaplan-Meier Estimate; Vaccines, DNA; Vibrio; Vibrio Infections

Topics: Animals; Bass; Disease Transmission, Infectious; Eukaryota; Fish Diseases; Host-Parasite Interactions; Intestines; Protozoan Infections, Animal; Sea Bream

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

Topics: Animals; Bass; Cloning, Molecular; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Interleukin-1beta; Models, Molecular; Molecular Sequence Data; Photobacterium; Phylogeny; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Tumor Necrosis Factor-alpha

Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)-stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22-51 and g-ple 22-42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Base Sequence; Bass; DNA Primers; Fish Diseases; Fish Proteins; Gene Expression; Gills; Intestinal Mucosa; Models, Molecular; Molecular Sequence Data; Recombinant Proteins; RNA, Messenger

To rapidly determine the causative agent of mass death in Lateolabrax japonicus in Zhelin Bay of Guangdong Province in China in April 2004.. Thirty-six strains, numbered sequentially from RP01 to RP36, were isolated from six diseased fish. All of the strains were identified as being of the same vibrio species according to the results of universal primer PCR combined with DGGE (UPPCR-DGGE). RP30 was one of these strains that was randomly selected and analysed by using a morphological, physiological and biochemical plate, Biolog GN2 Microplate System and API 20E system. Furthermore, RP30' 16S rDNA was sequenced and aligned in Genbank. Its virulence to Lateolabrax japonicus (Cuvier in Cuvier and Valenciennes) was also tested. RP30 is most closely related to four Vibrio ponticus strains (99.3% similarity). LD50s were 2.5 (x103 CFU per fish for intraperitoneal inoculation (IP) and 3.2 (x103 CFU per fish for intramuscular inoculation (IM), respectively.. The investigated pathogenic agent of Lateolabrax japonicus (Cuvier in Cuvier and Valenciennes) was V. ponticus.. UPPCR-DGGE is very helpful in epidemiologic investigation. Interestingly, this is the first report that V. ponticus infects cultured marine fish. DGGE was likewise first introduced to epidemiologic investigation of fish disease.

Topics: Animals; Aquaculture; Bacterial Typing Techniques; Bass; Electrophoresis, Polyacrylamide Gel; Fish Diseases; Molecular Sequence Data; Polymerase Chain Reaction; Sequence Analysis, DNA; Vibrio; Vibrio Infections; Virulence

The antigenicity of Photobacterium damselae (Ph. d.) subsp. piscicida, cultured in four different growth media [tryptone soya broth (TSB), glucose-rich medium (GRM), iron-depleted TSB (TSB + IR(-)), and iron-depleted GRM (GRM + IR(-))] was compared by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using sera obtained from sea bass (Dicentrarchus labrax) raised against live or heat-killed Ph. d. subsp. piscicida. The antigenic expression of Ph. d. subsp. piscicida was found to differ depending on the culture medium used. A significantly higher antibody response was obtained with iron-depleted bacteria by ELISA compared with non-iron depleted bacteria obtained from the sera of sea bass raised against live Ph. d. subsp. piscicida. The sera from sea bass raised against live bacteria showed a band at 22 kDa in bacteria cultured in TSB + IR(-) or GRM+ IR(-) when bacteria that had been freshly isolated from fish were used for the screening, while bands at 24 and 47 kDa were observed with bacteria cultured in TSB or GRM. When bacteria were passaged several times on tryptic soya agar prior to culturing in the four different media, only bands at 24 and 47 kDa were recognized, regardless of the medium used to culture the bacteria. It would appear that the molecular weight of Ph. d. subsp. piscicida antigens change in the presence of iron restriction, and sera from sea bass infected with live bacteria are able to detect epitopes on the antigens after this shift in molecular weight.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bass; Blotting, Western; Cell Count; Culture Media; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Molecular Weight; Pasteurella Infections; Photobacterium

Streptococcus iniae is a leading pathogen of intensive aquaculture operations worldwide, although understanding of virulence mechanisms of this pathogen in fish is lacking. S. iniae possesses a homolog of streptolysin S (SLS), a secreted, pore-forming cytotoxin that is a proven virulence factor in the human pathogen S. pyogenes. Here we used allelic exchange mutagenesis of the structural gene for the S. iniae SLS precursor (sagA) to examine the role of SLS in S. iniae pathogenicity using in vitro and in vivo models. The isogenic Delta sagA mutant was less cytotoxic to fish blood cells and cultured epithelial cells, but comparable to wild-type (WT) S. iniae in adherence/invasion of epithelial cell monolayers and resisting phagocytic killing by fish whole blood or macrophages. In a hybrid striped bass infection model, loss of SLS production led to marked virulence attenuation, as injection of the Delta sagA mutant at 1000x the WT lethal dose (LD80) produced only 10% mortality. The neutralization of SLS could represent a novel strategy for control of S. iniae infection in aquaculture.

Topics: Animals; Bacterial Proteins; Bass; Brain; Carps; Cell Line; Colony Count, Microbial; Fish Diseases; Gene Expression Regulation, Bacterial; Genetic Engineering; Hemolysis; Kaplan-Meier Estimate; Mutation; Streptococcal Infections; Streptococcus; Streptolysins; Time Factors; Virulence; Virulence Factors

A survey of the gill parasites of Epinephelus costae (Teleostei: Serranidae) was conducted between 2001 and 2005 in the Gulf of Gabès (Tunisia). Five new species of Diplectanidae (Monogenea) were collected, all belonging to Pseudorhabdosynochus Yamaguti, 1958: P. bouaini sp. n., P. dolicocolpos sp. n., P. enitsuji sp. n., P. sinediscus sp. n., and P. sosia sp. n. These five species differ from each other and from all described species of Pseudorhabdosynochus by the morphology and size of their sclerotized vagina. These diplectanids (except P. sinediscus) were also collected from the same host off Dakar in 1981 and 1989. The present paper includes the descriptions and taxonomic considerations of each of these species in addition to an amended diagnosis of Pseudorhabdosynochus. A key to the five new species parasitizing E. costae is provided. These five species are the first diplectanids described from E. costae.

Topics: Animals; Bass; Fish Diseases; Gills; Trematoda; Trematode Infections; Tunisia

Monogeneans from three species of Cephalopholis, namely C. argus, C. sonnerati and C. boenak, are described from fish caught off New Caledonia, South Pacific, with comparisons with material from off Queensland, Australia. Pseudorhabdosynochus argus n. sp. from C. argus is present off New Caledonia and Australia; it is characterised by its male quadriloculate organ with very elongate cone, and its sclerotised vagina with anterior trumpet, coiled primary canal and distal part with two chambers and an accessory part. C. boenak has no monogeneans off New Caledonia, but off Australia it harbours Pseudorhabdosynochus sp., a new species which is morphologically related to P. argus. P. minutus n. sp. from C. sonnerati is characterised by its minute body and a sclerotised vagina with two spherical chambers. Diplectanum nanus n. sp. from C. sonnerati is characterised by its very small funnel-shaped male copulatory organ and minute body. A new species, Haliotrema sp. from C. sonnerati is characterised by a very elongate tubular penis; it is distinct from H. cromileptis Young, 1968 (redescribed herein from specimens collected from Cromileptes altivelis off New Caledonia). The species described here include the first members of Pseudorhabdosynochus and the first diplectanids described from species of Cephalopholis. There is no evidence for a clade of Pseudorhabdosynochus species specific to members of Cephalopholis, since the species described here share similarities with other species from Epinephelus. However, it is suggested that the gill structure of Cephalopholis spp. imposes selection toward small body sizes for monogeneans.

Topics: Animals; Bass; Female; Fish Diseases; Gills; Male; New Caledonia; Queensland; Species Specificity; Trematoda; Trematode Infections

Eighty striped bass Morone saxatilis were obtained from Delaware Bay using commercial gill nets set adjacent to Woodland Beach (n = 70) and Bowers Beach (n = 10) in December 2003. Fish were examined for gross lesions. Total lengths (TLs) and eviscerated weights were determined to calculate condition factors (K). Portions of spleens were aseptically harvested for bacterial culture, and portions of spleens, kidneys (anterior and posterior), livers, and gonads were obtained for histological examination. The size distribution of the striped bass was relatively homogeneous; the mean TL was about 600 mm for all samples. Mean K exceeded 0.95 in all samples and was not significantly different (P > 0.05) among samples. Significant differences in mycobacterial infection prevalence (P < or = 0.05) were observed among samples; samples obtained at Woodland Beach (WB) on December 10 (53.8%, n = 13) and December 17 (7.1%, n = 42) exhibited the most striking differences in prevalence. Mycobacterial infection intensity ranged from 1 X 10(2) to 1 X 10(7) colony-forming units per gram of spleen. Acanthocephalan infection prevalence and intensity, non-acid-fast bacterial infection prevalence, and fish sex ratio were also significantly different among the samples (P < or = 0.05). Similar to the mycobacterial infections, differences in sex ratio, acanthocephalan infection, and non-acid-fast bacterial infection were observed between the WB samples taken on December 10 and 17. However, no significant associations (P > 0.05) were observed between sex ratio or these infections and mycobacterial infection. The differences in bacterial and parasite infection prevalence and intensity and fish sex ratio in some samples indicate that these fish had a different history and that the epizootiology of mycobacterial infection in striped bass from Delaware Bay may be relatively complex.

Topics: Animals; Bass; Colony Count, Microbial; Delaware; Female; Fish Diseases; Gonads; Kidney; Liver; Male; Mycobacterium; Mycobacterium Infections; Prevalence; Sex Factors; Spleen

Acolpenteron ureteroecetes infections in the kidneys of largemouth bass Micropterus salmoides have been reported, but the time course of infection and progression of pathology in experimentally infected fish remain unknown. We exposed 299 naive juvenile largemouth bass at 19.8 degrees C to A. ureteroecetes-infected largemouth bass via recirculating water without direct contact. For 7 months, prevalence and density were determined monthly in squashes of posterior kidney (20 exposed fish, 20 nonexposed fish), and histopathology was assessed in 5 fish from each group. Prevalence increased steadily from months 1 (5%) to 4 (85%), thereafter remaining relatively stable. Mean density of infection doubled monthly (month 1, 0.1 individuals/2 cm2 squash; month 7, 15.1 individuals/2 cm2 squash). Eggs were first observed at month 3, and mean density increased markedly from month 4 to month 5 (2.9-15.3 eggs/2 cm2 squash). Histopathology showed damage in renal collecting ducts that got progressively worse between 5 and 7 months. The infected ducts were dilated, had a hyperplastic epithelium, and were surrounded by chronic inflammation, including eosinophilic granular cells and varying degrees of fibrosis. Eggs within granulomas were present in the interstitium; this response is newly reported. The infection system developed in this study provides a reproducible and consistent source of infected individuals that will facilitate further study of the parasite and potential treatments.

Topics: Animals; Bass; Fish Diseases; Host-Parasite Interactions; Kidney; Population Density; Prevalence; Trematoda; Trematode Infections

An experimental feeding trial was performed to evaluate the efficacy of florfenicol (FFC) in controlling Streptococcus iniae infection in sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis). Five dosage levels of FFC in medicated feed were administered daily: 0, 5, 10, 15, and 30 mg of active ingredient/kg of fish body weight. Treatment was started within 22-24 h postchallenge by waterborne exposure to virulent S. iniae. The FFC medication was continued for 10 consecutive days, followed by a 25-d posttreatment observation. At the conclusion of the experiment, FFC treatment significantly increased the survival of S. iniae-challenged sunshine bass from 4.2% in the nonmedicated (positive control) group to 69.2% in the 5-mg/kg dosage group, 86.7% in the 10-mg/kg group, and 94.2% in the 15- and 30-mg/kg groups. Survival was significantly higher in the 15- and 30-mg/kg treatment groups than in the 5-mg/kg treatment group; differences among the 10-mg/kg and higher dosage groups were not significant. Survival curve analysis using a log-rank test indicated no significant difference between curves for the 10- and 15-mg/kg groups but a significant difference between curves for the 5- and 10-mg/kg groups. At the end of the experiment, no carriers were detected in any challenged group receiving an FFC-medicated diet, but the bacterium was recovered from the nonmedicated challenged survivors of the infection. The results of the experiment suggest that the optimum therapeutic daily dose of FFC is between 10 and 15 mg/kg body weight for 10 d.

Topics: Animals; Anti-Bacterial Agents; Bass; Dose-Response Relationship, Drug; Fish Diseases; Kaplan-Meier Estimate; Streptococcal Infections; Streptococcus; Thiamphenicol; Time Factors; Treatment Outcome

The use of quantitative polymerase chain reaction (QPCR) to test for largemouth bass virus (LMBV) was evaluated during a challenge experiment in which largemouth bass Micropterus salmoides were immersed in the type strain of LMBV. The real-time PCR and cell culture methods were both used to measure LMBV present in the inoculum. Additional samples tested by QPCR included gill, gonad, kidney, liver, mucus, spleen, and swim bladder. A plasmid clone containing a 248-base pair (bp) fragment of the major capsid protein gene (MCP*) was serially diluted and used as a standard to quantify the number of LMBV DNA copies present in the samples tested. A 62-bp fragment of DNA located in MCP* was amplified in the real-time PCR assay. This work has demonstrated the value of the QPCR assay in LMBV surveys.

Topics: Animals; Bass; Capsid Proteins; Cell Line; DNA Primers; DNA Virus Infections; Fish Diseases; Polymerase Chain Reaction; Ranavirus; Reference Values; Reproducibility of Results; Sensitivity and Specificity

Intersex, or the presence of characteristics of both sexes, in fishes that are normally gonochoristic has been used as an indicator of exposure to estrogenic compounds. In 2003, during health assessments conducted in response to kills and a high prevalence of skin lesions observed in smallmouth bass Micropterus dolomieu in the South Branch of the Potomac River, the presence of immature oocytes within testes was noted. To evaluate this condition, a severity index (0-4) was developed based on the distribution of oocytes within the testes. Using gonad samples collected from 2003 to 2005, the number of histologic sections needed to accurately detect the condition in mature smallmouth bass was statistically evaluated. The reliability of detection depended on the severity index and the number of sections examined. Examining five transverse sections taken along the length of the gonad resulted in a greater than 90% probability of detecting testicular oocytes when the severity index exceeded 0.5. Using the severity index we compared smallmouth bass collected at selected sites within the South Branch during three seasons in 2004. Seasonal differences in severity and prevalence were observed. The highest prevalence and severity were consistently noted during the prespawn-spawning season, when compared with the postspawn season. In 2005, smallmouth bass were collected at selected out-of-basin sites in West Virginia where fish kills and external skin lesions have not been reported, as well as at sites in the Shenandoah River, Virginia (part of the Potomac drainage), where kills and lesions occurred in 2004-2005. The prevalence of testicular oocytes is discussed in terms of human population and agricultural intensity.

Topics: Agriculture; Animals; Bass; Disorders of Sex Development; Female; Fish Diseases; Humans; Male; Oocytes; Population Density; Prevalence; Risk Factors; Rivers; Severity of Illness Index; Testis; Virginia; West Virginia

In aquaculture management it is important to establish objective criteria to assess health and welfare of the fish. Here we show that European sea bass (Dicentrarchus labrax) confronted with husbandry-associated stress (tank cleaning, i.e. scrubbing, and water temperature variation) during early life stages show poorer survival and disease resistance as juveniles. We evaluated several parameters for stress (plasma cortisol, glucose and lactate, hydromineral status), growth performance, the immune response (plasma IgM levels) and the effects of a nodavirus challenge. Principal component analysis allowed the establishment of a stress panel including plasma cortisol, osmolality, IgM levels and weight. Sea bass juveniles reared during early life in high and constant temperature perform best in terms of stress-related parameters assessed by principle component analysis. Variable water temperature triggers dramatic changes in plasma cortisol, osmolality, IgM levels, body weight and susceptibility to nodavirus that suggest a strong and prolonged activation of the HPI axis. Scrubbing induces some disturbances typical for mild short-term, acute stress, viz. increased plasma osmolality and decreased IgM levels, but does not affect plasma cortisol, growth or susceptibility to nodavirus of sea bass. Our data fit well with the concept of allostasis. We discuss the relevance of our work for sea bass aquaculture.

Topics: Animal Husbandry; Animals; Aquaculture; Bass; Blood Glucose; Fish Diseases; Health Status; Hydrocortisone; Immunity, Innate; Immunoglobulin M; Lactic Acid; Nodaviridae; Principal Component Analysis; RNA Virus Infections; Stress, Physiological

In each challenge 30 sea bass juveniles (mean weight 3.3 +/- 0.2 g SD) were used. During the whole experiment (water T: 18 +/- 1 degrees C) the fish were held in four 50l seawater independent recirculation systems (one fish group per 50l system). The protection to the pathogen Vibrio anguillarum was tested on booster vaccinated sea bass (Dicentrarchus labrax L.) juveniles. The vaccination was performed by immersion for 60 s in a commercial anti-V. anguillarum vaccine suspension. Booster vaccination took place 60 days after the initial immunization. Thirty days after the booster vaccination all the fish received intraperitoneally (IP) 3.0 x 10(6) cfu/fish (colony forming units) virulent V. anguillarum bacteria. The booster vaccination showed a strong protection effect on the challenged sea bass. In the next 20 days after the challenge the mortality was 0% among the booster vaccinated sea bass, 10% among the once vaccinated fish and 50% in the control group (unvaccinated fish). No mortality was observed among the unvaccinated sea bass injected IP with sterile normal saline by the challenge.

Topics: Animals; Animals, Newborn; Aquaculture; Bacterial Vaccines; Bass; Colony Count, Microbial; Fish Diseases; Gram-Negative Bacterial Infections; Immunization, Secondary; Treatment Outcome; Vaccination; Vibrio

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.

Topics: Animals; Base Sequence; Bass; Cell Line; Cytopathogenic Effect, Viral; Fish Diseases; Molecular Sequence Data; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; Temperature; Time Factors; Virus Replication

The role of hepcidin in iron metabolism regulation and bacterial infection has been the focus of recent attention. However, in spite of the growing number of hepcidin genes known from different organisms, little is known about its putative dual function in fish. The aim of this study was to characterize the sea bass hepcidin gene and to study its role in iron metabolism and infection. The novel sea bass hepcidin gene was found to be organized into two introns and three exons with several copies present in the genome. The transcript showed a constitutive low basal expression being mainly expressed in liver and encoding a putative 85 residues long peptide. Fish were submitted either to iron status modulation or bacterial infection and the hepcidin transcript levels were analysed along with a number of other parameters. Liver hepcidin expression was found to increase in both the iron-overloaded and infected fish, while in the iron-deficient fish no alteration in expression levels was detected. These results point to the evolutionary conservation of hepcidin's dual functions.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Base Sequence; Bass; Erythrocyte Count; Fish Diseases; Gram-Negative Bacterial Infections; Hematocrit; Hepcidins; Iron Overload; Liver; Molecular Sequence Data; Photobacterium; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Transferrin

A hepcidin gene was amplified from the liver of Lateolabrax japonicus challenged with a mixed bacterial suspension. Using RT-PCR and RACE, a full length cDNA sequence of the hepcidin like antimicrobial peptide was determined. The complete hepcidin cDNA Hepc2 is 581 bases and contains an ORF of 258 bases with a coding capacity of 86 amino acids. The deduced amino acid sequence, which shares eight cysteines at the identical conserved positions, and gene organisation are conserved between Japan sea bass and other fish species. The predicted molecular weight of the peptide is 9.4 kDa. The 3'-untranslated region is composed of 225 bp with a polyadenylation signal AATAAA sequence appearing at 189 nt and the poly(A) tail at 212 nt downstream of stop codon TGA. The predicted signal peptide cleavage site of its deduced peptide is between codons 24 and 25. Japan sea bass hepcidin-like genomic DNA hepc2 sequence including upstream and downstream regions was composed of two introns and three exons. The cloned 173-bp upstream sequence of Japan sea bass hepcidin-like gene contains putative regulatory elements and several binding motifs for transcription factors. High homologies with hepcidin cDNAs and peptides of white bass (Morone chrysops), human and other fish were shown. Hepc2 of Lateolabrax japonicus is a new member of the hepcidin gene family.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Base Sequence; Bass; Cloning, Molecular; DNA Primers; DNA, Complementary; Fish Diseases; Hepcidins; Humans; Liver; Molecular Sequence Data; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid

Host fish acquire resistance to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). Glochidia metamorphose into juvenile mussels while encysted on host fish. We investigated the duration of acquired resistance of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received three successive priming infections with glochidia to induce an immune response. Primed fish were held at 22-23 degrees C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but not in naive control fish. Acquired resistance of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern.

Topics: Age Factors; Animals; Antibodies; Antigens; Bass; Bivalvia; Fish Diseases; Host-Parasite Interactions; Immunity, Innate; Larva; Metamorphosis, Biological; Time Factors

The grouper is a high-value fish in the seafood market. Grouper nervous necrosis virus (GNNV) causes mass mortality, near 100% in larvae and juveniles, which has great economic impact on the aquaculture of the marine fish. Since vaccination is one of the best methods against viral diseases, grouper Epinephelus lanceolatus was injected with virus-like particles (VLPs) of GNNV at different dosages and injection frequencies. The anti-sera of vaccinated fish were analyzed with antigen-capture ELISA to quantify immunization titer. The antibody titers in the vaccinated fish increased remarkably within 4 weeks, during which time the antibody was definitely capable to neutralize the native virus. With one shot of 10-250 microg VLPs, the stimulated antibody titer reached a steady saturation level in 1 month, among which the titers by one shot of 100 and 250 microg VLPs were 13% higher than by 10 microg. Two shots of 10 and 100 microg VLPs increased to maximum titer, which was 29% higher than one shot, whereas two shots of 250 microg VLPs and four shots of 100 microg VLPs dramatically downgraded the titers by -23% and -44%, respectively. These results imply that the overdose effects occurred in total dosages higher than 200 microg VLPs. The experiments of VLP vaccine with adjuvant revealed that the adjuvant is not required for increasing the efficacy of the VLP vaccine. Immunization with the VLPs can also stimulate fish to produce high antibody titer for more than 5 months, which can be correlated to long-term protection. When VLPs are used as vaccine agent, a dosage at 1 microg/g of fish body weight is enough to stimulate a full-scale immune response.

Topics: Animals; Antibody Formation; Bass; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Freund's Adjuvant; Nodaviridae; Vaccination; Viral Vaccines

Vibrio alginolyticus was isolated from the internal organs of diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) cultured in two fish farms located on the Tunisian Mediterranean coast, from 2003 to 2005. After phenotypic characterisation, a selection of 34 isolates from gilthead sea bream and sea bass were molecularly typed by repetitive intergenic consensus PCR (ERIC-PCR) showing a high polymorphism among the isolated strains (19 genotypes). Most of the isolates were resistant to atleast two antimicrobial agents. All the tested strains were resistant to ampicillin. However, 91.17% were resistant to nitrofurantoin and 35.29% to tetracycline. Several strains isolated from diseased gilthead sea bream and sea bass were tested for virulence in both fish species by intraperitoneal injection. The selected isolates (n=7) were pathogenic for gilthead sea bream and sea bass. LD(50) values ranged from 5.01 x 10(4) to 6.20 x 10(7)CFU/fish. This is the first report on characterisation and virulence of V. alginolyticus for sea bass and sea bream in Tunisia.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bacterial Typing Techniques; Bass; Disease Outbreaks; Drug Resistance, Bacterial; Fish Diseases; Lethal Dose 50; Microbial Sensitivity Tests; Polymerase Chain Reaction; Sea Bream; Tunisia; Vibrio alginolyticus; Vibrio Infections; Virulence

Leptorhynchoides thecatus (Linton, 1891), an acanthocephalan parasite of freshwater fishes, varies in host use, development, and habitat use throughout North America. Spatial structure of these characteristics was examined from data extracted from the literature. Geographic patterns were inferred from point comparisons using correllograms and then tested with Moran's I statistic for global and local significance, and visually from regional means within major river drainages. Species of Micropterus Lacepède, 1802 (black basses) were common hosts in most regions, except the Lower Mississippi and South Atlantic regions where species of Lepomis Rafinesque, 1819 (sunfishes) were common hosts. Development, described as the proportions of adults relative to cystacanths (extraintestinal juveniles), decreased with latitude. Habitat use of L. thecatus showed marked geographic patterns. Leptorhynchoides thecatus occurred in the intestine of sunfishes in the South Atlantic and Lower Mississippi regions, in the ceca in fish of all species included in the study in the Missouri and Texas-Gulf regions, and both in ceca and intestines in fish of all species in northern regions. Leptorhynchoides thecatus showed geographic patterning within the variable traits across the range of the species. These patterns may be the result of ecological factors or of genetic differences that might indicate L. thecatus comprises multiple cryptic species.

Topics: Acanthocephala; Animals; Bass; Cecum; Environment; Fish Diseases; Gadiformes; Geography; Helminthiasis, Animal; Host-Parasite Interactions; Ictaluridae; Intestines; Perciformes; Prevalence; United States

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.

Topics: Agglutination Tests; Animals; Antibodies, Bacterial; Bacterial Vaccines; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Immunization, Secondary; Survival Analysis; Time Factors; Vibrionaceae

Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Virus particles contain a single type of coat protein that spontaneously assembles into virus-like particles (VLPs) when expressed in a baculovirus expression system. In the present study, the immunogenicity of betanodavirus VLPs and the protection they confer against VNN in the European sea bass Dicentrarchus labrax were investigated. Enzyme-linked immunosorbent assay and seroneutralization tests performed on plasma from fish vaccinated intramuscularly with doses as low as 0.1 microg of VLPs indicated that the VLPs elicited the synthesis of specific antibetanodavirus antibodies with neutralizing activity. Moreover, fish vaccinated with VLPs were protected from challenge with live virus. Both the immune response and the protective effect against viral challenge were dose dependent. Reverse transcription-PCR data indicated that higher doses of vaccine also reduced the number of fish containing detectable quantities of betanodavirus RNA on day 30 after challenge. Taken together these data strongly support the hypothesis that VLPs obtained in the baculovirus expression system may represent an effective vaccine against VNN.

Topics: Animals; Antibodies, Viral; Bass; Central Nervous System Viral Diseases; Disease Models, Animal; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Neutralization Tests; Nodaviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Survival Analysis; Virosomes

Necropsy was performed on 2 smallmouth bass (Micropterus dolomieu) that died 8 days after experimental gill inoculation with Actinonaias pectorosa glochidia. The salient feature at necropsy was ragged gills containing multiple petechial hemorrhages and numerous white, iridescent dust-like particles easily confirmed as glochidia in squash preparations of the gill filaments. Microscopically, the gills contained multiple rounded glochidia encysted at the tips or along the length of the filaments. Parasitized filaments were thickened, blunted, and often fused. Lamellae were extensively fused and obliterated, with proliferation of the epithelial cells giving the filament a smooth outline. Sometimes glochidia were associated with necrosis and/or hemorrhage. These fish most likely died due to asphyxia associated with the severe branchial lesions caused by the glochidia.

Topics: Animals; Bass; Bivalvia; Fish Diseases; Gills; Parasitic Diseases, Animal

A new species, Prosorhynchus maternus sp. n., is described from the serranid fish Epinephelus malabaricus (Bloch et Schneider) in the waters off New Caledonia. It belongs to a group of Prosorhynchus species from serranids in which the uterus is restricted to the postovarian region. Its distinguishing features include the vitellarium relatively distant from the rhynchus, the cirrus-sac relatively distant from the posterior testis, the distinctly pre-equatorial mouth and several other somatic ratios. New records of Prosorhynchus longisaccatus Durio et Manter, 1968 from Epinephelus cyanopodus (Richardson) and Prosorhynchoides lamprelli Bott et Cribb, 2005 from Caranx papuensis Alleyne et Macleay off New Caledonia are also included.

Topics: Animals; Bass; Fish Diseases; New Caledonia; Trematoda; Trematode Infections

The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue.

Topics: Animals; Bass; Communicable Diseases, Emerging; DNA Primers; DNA, Bacterial; Fish Diseases; Fisheries; Francisella; Fresh Water; Gram-Negative Bacterial Infections; Kidney; Macrophages; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S; Spleen

The post-challenge immune and histopathologic responses of hybrid striped bass vaccinated with a DNA vaccine encoding the Mycobacterium marinum Ag85A gene and subsequently challenged with M. marinum were investigated. Juvenile hybrid striped bass Morone saxatilis x M. chrysops were injected intramuscularly with 25 or 50 microg DNA plasmid and developed significant specific protective responses to live bacterial challenge 120 d post-vaccination. Both vaccine groups demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups 14 d after challenge with approximately 8 x 10(5) cfu M. marinum g(-1) fish body wt. The vaccine groups also developed more rapidly and significantly increased antibody and lymphoproliferative responses post-challenge compared to control groups, and these post-challenge immune responses appear to be vital against M. marinum infection in vaccinated hybrid striped bass. No significant differences in immune responses were recognized between the 25 and 50 microg vaccination groups, and these groups eventually experienced mortalities, splenic bacterial counts, and granuloma formation 28 d post-challenge comparable to those of the control groups at 14 d post-challenge. Therefore, vaccination of hybrid striped bass with a DNA vaccine encoding the M. marinum Ag85A gene provided significant but limited duration of protection against an acute high-dose M. marinum challenge.

Topics: Acyltransferases; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Vaccines; Bass; Fish Diseases; Granuloma; Lymphocytes; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Time Factors; Vaccines, DNA

We studied the natural progression of viral nerve necrosis (VNN) in larvae of Asian seabass Lates calcarifer Bloch from 0 to 40 days post-hatch (dph). The hatchlings were reared in the vicinity of a confirmed nodavirus-affected older batch. Using light and electron microscopy (EM), we made a sequential analysis of histopathological manifestations in nerve tissue and other organs. There were no changes from the day of hatching until 4 dph. Larvae at 4 dph had viral particles in the intramuscular spaces underlying the skin, but the nerve cells of the brain were normal. The first signs of necrosis of the brain cells were observed at 6 dph. EM observations revealed characteristic membrane-bound viral particles measuring 30 nm in the cytoplasm of nerve cells of the brain, spinal cord and retina. Histological samples of fry examined when group mortalities reached 20 to 35% revealed highly vacuolated brains, empty nerve cell cytoplasm and viral particles in the intercellular spaces. Viral particles occurred extensively in the intramuscular spaces and the epidermal layers. These observations were corroborated by positive immunostaining of the virus-rich intramuscular spaces. EM studies also revealed progressive necrotic changes in the cells harboring the virus. Results emphasize the need to maintain hygiene in the hatchery environment and to develop strategies for prevention of disease spread among cohabiting seabass and other susceptible fish larvae. Intramuscular localization of the nodavirus in both preclinical healthy-looking and post-clinical moribund larvae suggests that virus neutralization strategies during larval development could be effective in controlling VNN-associated mortalities.

Topics: Animals; Bass; Brain; Fish Diseases; Larva; Microscopy, Electron, Transmission; Muscles; Necrosis; Neurons; Nodaviridae; RNA Virus Infections; Skin; Spinal Cord

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Topics: Animals; Antibodies, Bacterial; Bass; DNA, Bacterial; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunohistochemistry; Kidney; Liver; Piscirickettsia; Piscirickettsiaceae Infections; Polymerase Chain Reaction; Time Factors

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

Topics: Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Base Sequence; Bass; Chromatography, High Pressure Liquid; Disulfides; DNA Primers; Fish Diseases; Hepcidins; Liver; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Mycobacteriosis, caused by numerous Mycobacterium spp., can be a devastating disease of both wild and cultured fishes. As no efficacious treatment exists, a vaccine against fish mycobacteriosis is essential for prevention and control of this disease. Thus, a DNA vaccine was constructed using the Mycobacterium marinum Ag85A gene that encodes one of the major secreted fibronectin-binding proteins of Mycobacterium spp., which was isolated and then subcloned into a commercially available eukaryotic expression vector. Juvenile hybrid striped bass (Morone saxatilis x M. chrysops), a species known to be particularly susceptible to this disease, were immunized by i.m. and i.p. injection with the resulting construct and as a result produced specific immune responses towards the Ag85A. Increasing concentrations of humoral antibodies to the Ag85A antigen were generated in all DNA vaccine groups, while macrophage phagocytosis and respiratory burst functions failed to exhibit upregulation after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to a live M. marinum challenge 90 days post-inoculation, as demonstrated by increased survival of vaccinated fish over control fish and by reduced splenic bacterial counts in vaccinated fish. Furthermore, humoral immune responses and protective effects were significantly increased at higher vaccine doses using the i.m. injection route.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Vaccines; Bass; Cell Proliferation; DNA, Bacterial; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Flow Cytometry; Immunization; Lymphocytes; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Phagocytosis; Polymerase Chain Reaction; Recombinant Proteins; Survival Analysis; Vaccines, DNA

A long-term epidemiological study of Cryptosporidium molnari in aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend, C. molnari was more prevalent in GSB than in ESB. Data on the distribution pattern of C. molnari in total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.

Topics: Animals; Aquaculture; Bass; Cryptosporidiosis; Cryptosporidium; Fish Diseases; France; Sea Bream; Spain

Sea bass (Dicentrarchus labrax L.) were injected intraperitoneally with monovalent (Photobacterium damselae subsp. piscicida or Vibrio anguillarum) and divalent (Ph. damselae subsp. piscicida and V. anguillarum) vaccine formulations, with or without adjuvants (mineral oil, liposome or alginate), to evaluate the short and long-term pathological effects. Eight animals from each group were sampled one, two, four and 11 months after intraperitoneal injection. The acute peritoneal response and the progression to a chronic status were evaluated by analysing peritoneal leucocytes collected during the first days post-injection. To evaluate the chronic response, the late peritoneal leucocyte response was analysed and the peritoneal cavity was examined and the intra-abdominal lesion level scored based on a pre-defined scale. Correlation between leucocyte exudative response, tissue inflammatory response and the development of granuloma were sought. The acute leucocyte response was characterized by an early (24-48 h) mobilization of neutrophils and macrophages, with phagocyte numbers dependent on the formulation, but no significant variations were observed in lymphocytes/small cells and EGCs. Later on, a steady increase occurred in lymphocytes/small cells and EGCs and a high concentration in neutrophils and macrophages was maintained up to 30-60 days in groups i.p. injected with oil adjuvanted formulations with antigen. All the lesions observed were moderate, indicating that in sea bass, the pathological effects due to intraperitoneally injected vaccines are less severe than in other fish species. The divalent oil adjuvanted vaccine induced the most severe side effects, with macroscopic granulomas consistently present up to 11 months.

Topics: Adjuvants, Immunologic; Animals; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Granuloma; Immunohistochemistry; Leukocytes; Microscopy, Electron, Transmission; Peritoneal Cavity; Photobacterium; Vaccination; Vibrio

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass (Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 microg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.

Topics: Aeromonas; Analysis of Variance; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bass; DNA Primers; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; HeLa Cells; Hemorrhagic Septicemia; Humans; Immunoblotting; Nucleic Acid Amplification Techniques; Pacific Ocean; Plasmids; Vaccination; Vaccines, DNA

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis type-strain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.

Topics: Animals; Aquaculture; Base Sequence; Bass; Brain; Cluster Analysis; DNA Primers; DNA, Ribosomal; Fish Diseases; Fluorescent Antibody Technique, Indirect; Greece; Immunohistochemistry; Molecular Sequence Data; Phylogeny; Piscirickettsiaceae; Piscirickettsiaceae Infections; RNA, Transfer; Sequence Alignment; Sequence Analysis, DNA; Species Specificity

Mammalian bone is an active tissue in which osteoblasts and osteoclasts balance bone mass. This process of adaptive modelling and remodelling is probably regulated by strain-sensing osteocytes. Bone of advanced teleosts is acellular yet, despite the lack of osteocytes, it is capable of an adaptive response to physical stimuli. Strenuous exercise is known to induce lordosis. Lordosis is a ventrad curvature of the vertebral column, and the affected vertebrae show an increase in bone formation. The effects of lordosis on the strain distribution in sea bass (Dicentrarchus labrax L.) vertebrae are assessed using finite element modelling. The response of the local tissue is analyzed spatially and ontogenetically in terms of bone volume. Lordotic vertebrae show a significantly increased strain energy due to the increased load compared with normal vertebrae when loaded in compression. High strain regions are found in the vertebral centrum and parasagittal ridges. The increase in strain energy is attenuated by a change in architecture due to the increased bone formation. The increased bone formation is seen mainly at the articular surfaces of the vertebrae, although some extra bone is formed in the vertebral centrum. Regions in which the highest strains are found do not spatially correlate with regions in which the most extensive bone apposition occurs in lordotic vertebrae of sea bass. Mammalian-like strain-regulated bone modelling is probably not the guiding mechanism in adaptive bone modelling of acellular sea bass vertebrae. Chondroidal ossification is found at the articular surfaces where it mediates a rapid adaptive response, potentially attenuating high stresses on the dorsal zygapophyses.

Topics: Adaptation, Physiological; Animals; Bass; Biomechanical Phenomena; Finite Element Analysis; Fish Diseases; Lordosis; Osteogenesis; Phylogeny; Spine

Parasitic disease in fishes is one of the most important factors limiting aquaculture production and its economic viability. Cymothoa indica, a cymothoid isopod, is reported here for the first time parasitizing cultured larvae of the Asian seabass Lates calcarifer in India. Fourteen-day-old L. calcarifer larvae of mean weight 8.73 +/- 0.03 mg were fed with wild zooplankton in the laboratory. On Day 14 of rearing, larvae were found parasitized by cymothoids. Infected larvae reached a mean (+/- SE) weight of 98.86 +/- 0.30 mg, while uninfected specimens weighed 117 +/- 0.43 mg at the end of the experiment (Day 21). C. indica occurred in the branchial and anterodorsal regions of infected fish, where resultant skin lesions were red, hemorrhagic, without scales and with abundant secreted mucus. The cumulative mortality over the 3 wk period was 16.54 %. These parasites are transferred to the host via the zooplankton used as food; this could easily be overcome, either by filtering wild zooplankton to remove the infectious swimming larvae of C. indica or by using cultured copepods.

Topics: Animals; Aquaculture; Bass; Disease Outbreaks; Ectoparasitic Infestations; Fish Diseases; India; Isopoda; Larva

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Fish Proteins; Gene Deletion; Genes, Bacterial; Molecular Sequence Data; Mutation; Phosphoglucomutase; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Virulence; Virulence Factors

An epidemic of trichodinosis associated with severe epidermal hyperplasia occurred in adult largemouth bass (Micropterus salmoides) from the Chowan River drainage, North Carolina (USA) in late winter to early spring 2002. Initial reports by anglers of fish with a "jelly-like slime coat" on the skin prompted an electrofishing survey in which about 10% of sampled largemouth bass had a very thick, bluish-white "mucoid layer" on the body and fins. Moderate to heavy infestations of the ciliate Trichodina were detected in wet mounts of skin from five of five fish having the mucoid layer; these fish also had significant gill infestations. An additional two fish with only mild reddening and four asymptomatic fish (no skin lesions) had mild skin infestations but no gill infestations. Two asymptomatic fish had no skin parasites. Four fish with the mucoid layer were necropsied and had extremely severe epidermal hyperplasia on the body and fins. The hyperplasic epidermis had relatively few mucus cells and typically was about 5-10 times thicker than healthy epidermis. The upper four fifths of the epidermis consisted of finely vacuolated, highly flattened, somewhat disorganized epithelial cells. No other significant clinical or histopathologic abnormalities were detected. No systemic infection by pathogenic bacteria was noted. The environmental cause of the epidemic is uncertain but the lesions suggest that some chronic stressor was involved.

Topics: Animals; Bass; Ectoparasitic Infestations; Fish Diseases; Gills; North Carolina; Oligohymenophorea; Seasons

Topics: Animals; Bass; Capsid Proteins; Eye Diseases; Fish Diseases; Genotype; Mediterranean Sea; Nodaviridae; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sequence Analysis, DNA

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Topics: Animals; Apoptosis; Bacterial Toxins; Base Sequence; Bass; Cell Line; Citrobacter rodentium; DNA, Bacterial; Escherichia coli O157; Fish Diseases; Gram-Negative Bacterial Infections; Immunization, Passive; Macrophages; Molecular Sequence Data; Neutrophils; Photobacterium; Plasmids; Recombinant Proteins; Virulence Factors

Topics: Animals; Arkansas; Bass; Chlamydia; Chlamydia Infections; Disease Outbreaks; Dose-Response Relationship, Drug; Epithelial Cells; Fish Diseases; Gills; Kidney; Oxytetracycline

One new species of diplectanid monogenean, Pseudorhabdosynochus summanoides n. sp., is reported and described from the marine fish Epinephelus coioides off Nan'ao Shenzhen, China. P. summanoides differs from its closest relative, P. summanae (Young, 1968), by the shape of its vaginal hard-parts, which have a tightly twisted distal region and an accessory patch on the proximal curve. During the course of this work, type-material of several species of Pseudorhabdosynochus was examined and aspects of the vagina and haptor are redescribed and/or figured. These species include P. americanus (Price, 1937), P. hargisi (Oliver & Paperna, 1984), P. amplidiscatus (Bravo-Hollis, 1954), P. epinepheli (Yamaguti, 1938), P. riouxi (Oliver 1986), P. melanesiensis (Laird, 1958), P. cupatus (Young, 1969), P. bocquetae (Oliver & Paperna, 1984), P. kritskyi Dyer et al., 1995, P. capurroi Vidal-Martinez, 1998, P. querni (Yamaguti, 1968) and P. summanae (Young, 1969). Several closely related species are considered in terms of their conspecificity: P. hargisi is proposed as a junior synonym of P. americanus; P. capurroi is suggested as a likely synonym of P. kritskyi; and it is suggested that P. cupatus and P. bocquetae may eventually be demonstrated to be consepcific with P. melanesiensis.

Topics: Animals; Bass; China; Female; Fish Diseases; Gills; Male; Trematoda; Trematode Infections

During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish > or = 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained < or = 10(3) mycobacteria g(-1).

Topics: Age Factors; Analysis of Variance; Animals; Bass; Colony Count, Microbial; Evaluation Studies as Topic; Fish Diseases; Mid-Atlantic Region; Mycobacterium; Mycobacterium Infections; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Spleen

We tested whether host fish that acquired resistance to glochidia of one mussel species were cross-resistant to glochidia of other species. Largemouth bass (Micropterus salmoides) were primed with 4-5 successive infections of glochidia of Lampsilis reeveiana. The percentage of attached glochidia that survived and transformed to the juvenile stage (transformation success) was compared between primed fish and naïve controls. Transformation success of L. reeveiana, Lampsilis abrupta, Villosa iris, and Utterbackia imbecillis was significantly lower on primed fish (37.8%, 43.5%, 67.0%, and 13.2%, respectively) than on control fish (89.0%, 89.7%, 90.0%, and 22.2% respectively). Immunoblotting was used to analyze the binding of serum antibodies from primed fish with glochidia proteins. Antibodies bound to glochidia proteins of similar molecular weight from L. reeveiana and L. abrupta. Bound proteins of V. iris differed in molecular weight from those of the Lampsilis species. There was no binding to specific glochidia proteins of U. imbecillis or Strophitus undulatus. Our results indicate that host-acquired resistance can extend across mussel genera and subfamilies and might involve both specific and nonspecific mechanisms. Understanding the specificity of acquired resistance of hosts to glochidia could enhance understanding of the evolutionary and ecological relationships between mussels and their host fishes.

Topics: Animals; Antibodies; Bass; Cross Reactions; Ectoparasitic Infestations; Electrophoresis, Polyacrylamide Gel; Female; Fish Diseases; Immunity, Active; Immunoblotting; Larva; Mice; Mice, Inbred BALB C; Species Specificity; Unionidae

Gilt-head sea bream, Sparus aurata L., the Mediterranean's most important mariculture species, has been cultured for the last 30 yr in Eilat (Israeli Red Sea). Kudoa sp. was the first myxosporean parasite reported from this species. In recent years, an increase in prevalence in both land-based and sea-cage facilities in Eilat has been observed. Infections with the same Kudoa species appeared in cultured European sea bass Dicentrarchus labrax (L.) and grey mullet Mugil cephalus in the same farms, as well as in 10 species of wild Red Sea reef fish, indicating that Kudoa sp. is not fastidious with regard to its host. All affected species displayed 1- to 2-mm (up to 5 mm) whitish, spherical, or oval polysporous plasmodia. The parasite established multiple site infections, most commonly in the muscles and intracranial adipose tissue of the brain and eye periphery. Other sites were subcutaneous adipose tissue, nerve axons, mouth, eye, mesenteries, peritoneum, swim bladder, intestinal musculature, heart, pericardium, kidney, and ovary. On the basis of spore morphology, the parasite was identified as Kudoa iwatai Egusa and Shiomitsu, 1983. Ultrastructural features were comparable to those of previously studied Kudoa species. The 18S rDNA from 7 Red Sea isolates was sequenced and compared with the sequence of the same gene from K. iwatai isolated from cultured red sea bream, Pagrus major, in Japan. The phylogenetic position of K. iwatai within the genus was determined using sequence analysis of all related taxa available in GenBank. The 3 isolates of K. iwatai clustered together on a newly formed, highly supported clade. The Red Sea strain of K. iwatai is apparently native to the region. In the absence of records of this Kudoa sp. from the extensive Mediterranean sea bream and sea bass production industries, introduction with its Mediterranean hosts seems unlikely. Therefore, we conclude that K. iwatai is an Indo-Pacific species that, in the Red Sea, has extended its host range to include the allochthonous gilt-head sea bream, European sea bass, and grey mullet.

Topics: Animals; Base Sequence; Bass; DNA, Protozoan; DNA, Ribosomal; Eukaryota; Fish Diseases; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; Protozoan Infections, Animal; RNA, Ribosomal, 18S; Sea Bream; Smegmamorpha; Spores, Protozoan

One new species of diplectanid, Pseudorhabdosynochus shenzhenensis n. sp., is reported and described from the marine fish Epinephelus coioides off Nan'ao, Shenzhen, China. It can be differentiated from previously described species of the same genus by features of the haptoral and vaginal hard-parts. A second species, P. serrani Yamaguti, 1953, originally described from Serranus sp. from the western Pacific Ocean off the Celebes (now called Sulawesi), is redescribed based on new material from E. coioides.

Topics: Animals; Bass; China; Fish Diseases; Platyhelminths; Prevalence; Trematode Infections

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Cation Transport Proteins; DNA, Complementary; Fish Diseases; Gene Expression Regulation; Macrophages; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; Mycobacterium; Mycobacterium Infections; Phylogeny; Sequence Homology, Amino Acid

The ability of two commercial rapid identification systems, API 20E and BBL Crystal E/NF, to reliably identify bacterial isolates from the internal organs of reared sea bass were compared. The tests gave different results: API 20E identified bacteria as Pseudomonas spp. with 37% accuracy, while BBL Crystal E/NF identified them as Flavobacterium odoratum with 99% accuracy. Although F. odoratum is not a marine fish pathogen, conventional tests conducted with the same isolates were more indicative of them being Flavobacterium spp. than Pseudomonas spp., suggesting that BBL Crystal E/NF was more reliable in this identification. Both systems were found to be applicable for diagnostics of marine fish pathogens, but should be used with caution because of possible misinterpretation.

Topics: Animals; Aquaculture; Bacterial Typing Techniques; Bass; Diagnosis, Differential; DNA, Bacterial; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Pseudomonas; Pseudomonas Infections; Sensitivity and Specificity

Topics: Animals; Bass; Euglena; Fish Diseases; Fresh Water; Ictaluridae; Neurotoxins

Topics: Aeromonas; Animals; Aquaculture; Bacterial Infections; Bass; Fish Diseases; Histological Techniques; Hybridization, Genetic; Liver; Mycobacterium; Vibrio

Three feeding trials were conducted to evaluate potential immunomodulatory effects of nucleotides in the diet of hybrid striped bass. A basal diet was formulated from menhaden fish meal to contain 40% crude protein and 10% lipid. An oligonucleotide product (Ascogen P) from brewer's yeast was added to the basal formulation at the manufacturer's recommended rate of 0.5% to produce the experimental diet. Each diet was fed to four replicate groups of juvenile hybrid striped bass for seven or eight weeks in two separate trials. After Trials 1 and 2, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. No significant difference in growth performance was observed between fish fed the basal and experimental diets. Body composition of whole fish, hematocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by dietary nucleotides. Neutrophil oxidative radical production of fish fed the nucleotide-supplemented diet was significantly (P=0.011) higher than in fish fed the basal diet. Significantly (P<0.05) enhanced survival after exposure to S. iniae also was generally observed in fish fed the nucleotide-supplemented diet. In addition, fish fed the nucleotide-supplemented diet tended to have a higher antibody response based on microtitration agglutination; however, the difference was not statistically significant because of high variation between individual fish. Long-term (16 weeks) administration of oligonucleotides in Trial 3 failed to show enhancement of immune responses between treatments. It is concluded that dietary oligonucleotides positively influenced immune responses and resistance of juvenile hybrid striped bass to S. iniae infection.

Topics: Agglutination Tests; Animals; Bass; Body Composition; Diet; Fish Diseases; Hematocrit; Immunity, Innate; Muramidase; Oligonucleotides; RNA; Saccharomyces cerevisiae; Streptococcal Infections; Streptococcus

Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38 per thousand. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 degrees C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection.

Topics: Amino Acid Sequence; Animals; Antibodies, Bacterial; Bacillus; Bass; Blotting, Western; Culture Media; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Hybridization, Genetic; Immunity, Active; Israel; Membrane Proteins; Molecular Sequence Data; Pasteurella Infections; Photobacterium; Sequence Analysis, DNA; Sodium Chloride; Vaccination

A number of factors can influence immune function affecting the magnitude and duration of specific immune responses. One factor which has been noted to affect immune responses is age of animal. In mammals, juveniles have a lower immune response when compared to adults. In fish, fry have a lower immune response than adults; however, few studies have investigated the immune response in juveniles, the age when most fish are reared in aquaculture settings. The humoral immune responses of hybrid striped bass (Morone saxatilis x Morone chrysops) at five different ages were compared to determine any maturational changes. Fifty fish were bath immunized with a commercial Vibrio vaccine at 4, 6, 9, 15, and 19 months of age. The antibody response in these fish was monitored by an enzyme-linked immunosorbent assay (ELISA) for 106 days post-vaccination. The magnitude, duration, and time to peak level were compared to controls and between the different ages using analysis of variance (ANOVA). Younger fish exhibited significantly lower antibody levels indicating that juvenile fish may not be able to mount as effective an immune response as young adult fish.

Topics: Age Factors; Animals; Antibodies, Viral; Aquaculture; Bass; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunization; Vibrio; Viral Vaccines

A total of 65 largemouth bass, Micropterus salmoides, and 27 smallmouth bass, M. dolomieu, collected in April-September 2000 and April-July 2001 from Gull Lake, Michigan, were examined for acanthocephalans. Leptorhynchoides thecatus and Neoechinorhynchus cylindratus infected all the bass examined. Leptorhynchoides thecatus had the highest mean intensity (258.2 +/- 185.4 in 2000 and 145.0 +/- 61.0 in 2001) of the species infecting smallmouth bass. Although N. cylindratus had higher mean intensities (42.1 +/- 37.9 in 2000 and 68.9 +/- 70.5 in 2001) than did L. thecatus in largemouth bass, the values were not significantly different between bass species. The prevalence, mean intensity, and mean abundance of Pomphorhynchus bulbocolli in the bass species were below the values for the other acanthocephalan species. Leptorhynchoides thecatus and N. cylindratus are the most abundant intestinal helminths in bass from Gull Lake.

Topics: Acanthocephala; Animals; Bass; Female; Fish Diseases; Fresh Water; Helminthiasis, Animal; Male; Michigan; Prevalence

The distribution of Sphaerospora dicentrarchi Sitjà-Bobadilla et Alvarez-Pellitero, 1992 and S. testicularis Sitjà-Bobadilla et Alvarez-Pellitero, 1990, myxozoan parasites of European seabass Dicentrarchus labrax (L.), was investigated in different farming systems in Italy. In total, 1406 fish were examined. High S. dicentrarchi prevalence was observed in all the farming systems involved in this survey (extensive farms: 51.5%; intensive farms: inland 59.6%, inshore floating cages 76.2%, offshore floating cages 41.6%) except for submersible cages (7.4%). S. testicularis was detected only in nine male fish from two intensive farms. The epidemiology and pathological effects of the parasites are discussed.

Topics: Animals; Aquaculture; Bass; Eukaryota; Fish Diseases; Italy; Prevalence; Protozoan Infections, Animal; Species Specificity; Spores, Protozoan

Topics: Animal Feed; Animals; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Iron, Dietary; Liver; Photobacterium; Spleen; Time Factors

Mycobacteriosis in striped bass Morone saxatilis of Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at 23 degrees C. Mycobacteria were recovered from 76% (n = 149/196) of fish examined. Mycobacterial densities exceeded 10(4) colony forming units x g tissue(-1) in 38% of samples (n = 63/168) that were examined using a quantitative approach. The most frequently recovered mycobacterium, present in 57% (n = 109/192) of characterized samples, was the recently named new species Mycobacterium shottsii. Polyinfections of M. shottsii and other mycobacteria were observed in 25% of samples (n = 47/192) with densities of M. shottsii usually 1 or more orders of magnitude higher than co-isolate(s). Other mycobacteria recovered included isolates that, based on phenotypic traits, resembled M. interjectum, M. marinum, M. scrofulaceum, M. szulgai and M. triplex. M. marinum, commonly associated with fish mycobacteriosis and human disease, was recovered infrequently (3%, n = 6/192). The presence of multiple mycobacterial types occurring at high densities suggests that a variety of mycobacteria could be causative agents of mycobacteriosis in striped bass from the Chesapeake Bay. Striped bass is the major recreational fish species in the Chesapeake Bay, and the significance of the current epizootic to human health and the potential adverse effects on fish stocks are not known.

Topics: Animals; Bass; Colony Count, Microbial; Fish Diseases; Histological Techniques; Mycobacterium; Mycobacterium Infections; Phenotype; Spleen; Virginia

In a previous study, we discovered that acute confinement stress causes rapid ulceration of the fins of hybrid striped bass Morone saxatilis female x M. chrysops male (Noga et al. 1998. Vet Pathol 35:102-107). In this paper, we report the development of a reproducible model for studying this phenomenon in juvenile hybrid striped bass. We also determined how quickly ulceration could develop in acutely stressed fish and documented the sequential light microscopic and ultrastructural changes associated with this response. When hybrid striped bass were subjected to a standardized confinement protocol, the pathological response was extremely rapid (fin ulceration began to develop within 15 min of confinement). Grossly, the distal edges of the fins became blanched, and melanophores aggregated near the basement membrane and dermis after 15 min of confinement. Microscopically, the earliest detectable change in the fins, which occurred within 15 min of confinement, was swelling and loss of microridges of the outermost epidermal cells; this was followed by epidermal erosion. After 30 min of stress, epidermal ulceration developed at the distal edges of the fins. At this time, both necrotic and apoptotic epidermal cells were present. The middle and basal epidermal layers were severely spongiotic and the dermis and hypodermis were edematous. Over longer periods (up to 2 h), lesions were similar but increasingly more severe, progressing from the distal edge of the fin towards the base. The response to acute stress showed a significant correlation between confinement period and severity of the pathological changes (epidermal degeneration, epidermal ulceration and leukocyte infiltration). Also, we demonstrated that epidermal damage was not restricted to the fins but also affected the body skin and eyes. The ventral area of the body and the corneal epithelium of stressed fish were ulcerated; however, skin on the head and operculum was not affected, suggesting a site-specific mode of damage. In stressed fish, epidermal ulceration was found in 67 to 97% of all fins, 88% of skin on the ventrum, and 67% of corneas, while control fish had only very mild epidermal ulceration in the few fish in which it was present (on 5 to 10% of the fins, but not on the ventral skin or corneas). Due to the widespread damage to epidermal tissues of the body surface, we have named this the acute ulceration response (AUR). Our study indicates that acute confinement can rapidly cause significant d

Topics: Analysis of Variance; Animals; Bass; Crowding; Epidermis; Fish Diseases; Histological Techniques; Microscopy, Electron, Transmission; Skin Ulcer; Stress, Physiological; Time Factors

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close association of lysosomes with mycobacterial phagosomes, as well as the presence of electron-opaque material within phagosomes, suggested phagolysosomal fusion. Development of granulomas involved epithelioid transformation of macrophages, followed by appearance of central necrosis. Desmosomes were present between mature epithelioid cells. The necrotic core region of M. marinum granulomas was separated from overlying epithelioid cells by several layers of flattened, electron-opaque spindle-shaped cells. These cells appeared to be formed by compression of epithelioid cells and, aside from a flattened nucleus, did not possess recognizable organelles. Following the development of well-defined, paucibacillary granulomas, secondary disease was observed. Recrudescence was marked by bacterial replication followed by disruption of granuloma architecture, including loss of epithelioid and spindle cell layers. In advanced recrudescent lesions, normal tissue was replaced by macrophages, fibroblasts, and other inflammatory leukocytes. Large numbers of mycobacteria were observed, both intracellular and suspended in cellular debris.

Topics: Animals; Bass; Fish Diseases; Granuloma; Microscopy, Electron, Transmission; Mycobacterium marinum; Phagosomes; Recurrence

Thiamine deficiency has been linked to early mortality syndrome in salmonids in the Great Lakes. This study was conducted to compare thiamine concentrations in American alligators (Alligator mississippiensis) and Florida largemouth bass (Micropterus salmoides floridanus) eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Lakes Apopka and Griffin, and Emeralda Marsh, Florida, USA) to those from sites that have historically exhibited low embryo mortality and low OCPs (Lakes Woodruff and Orange, Florida). During June-July 2000, 20 alligator clutches were collected from these sites, artificially incubated, and monitored for embryo mortality. Thiamine and OCPs were measured in one egg/clutch. During February 2002, 10 adult female bass were collected from Emeralda Marsh and Lake Woodruff and mature ovaries analyzed for thiamine and OCP concentrations. Although ovaries from the Emeralda Marsh bass contained almost 1,000-fold more OCPs compared with the reference site, Lake Woodruff, there were no differences in thiamine concentrations between sites (11,710 vs. 11,857 pmol/g). In contrast, alligator eggs from the reference site had five times the amount of thiamine compared with the contaminated sites (3,123 vs. 617 pmol/g). Similarly, clutches with >55% hatch rates had significantly higher concentrations of thiamine compared with clutches with <54% hatch rates (1,119 vs. 201 pmol/g). These results suggest that thiamine deficiency might be playing an important role in alligator embryo survival but not in reproductive failure and recruitment of largemouth bass. The cause(s) of this thiamine deficiency are unknown but might be related to differences in the nutritional value of prey items across the sites studied and/or to the presence of high concentration of contaminants in eggs.

Topics: Alligators and Crocodiles; Animals; Bass; Eggs; Environmental Exposure; Female; Fish Diseases; Florida; Hydrocarbons, Chlorinated; Maternal Exposure; Mortality; Ovum; Pesticides; Reproduction; Thiamine; Thiamine Deficiency; Water Pollutants, Chemical

New antiprotozoals active against Philasterides dicentrarchi, the causative agent of scuticociliatosis in farmed turbot and Black Sea bass-bream, have been synthesised and tested. The most active compounds posses a piperazine ring, generally N-bonded to the heterocycle, and are the 1,8-naphthyridines, 2f and 5o, the pyridothienopyrimidine (7), and the pyridothienotriazines, 8, 9, 12d, 12f, 12h, 12m and 12k. Pyridothienotriazine (12k) presents the same activity (Lethal Dose, LD=0.8/1.5 mg L(-1)) as the well-known antiparasitics niclosamide and oxyclozanide.

Topics: Animals; Antiprotozoal Agents; Bass; Ciliophora Infections; Culture Media; Fish Diseases; Flatfishes; Heterocyclic Compounds; Indicators and Reagents; Lethal Dose 50; Magnetic Resonance Spectroscopy; Mass Spectrometry; Naphthyridines; Oligohymenophorea; Pyrimidines; Spectrophotometry, Infrared; Structure-Activity Relationship

Slowly growing, non-pigmented mycobacteria were isolated from striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. Growth characteristics, acid-fastness and results of 16S rRNA gene sequencing were consistent with those of the genus Mycobacterium. A unique profile of biochemical reactions was observed among the 21 isolates. A single cluster of eight peaks identified by analysis of mycolic acids (HPLC) resembled those of reference patterns but differed in peak elution times from profiles of reference species of the Mycobacterium tuberculosis complex. One isolate (M1 75T) was placed within the slowly growing mycobacteria by analysis of aligned 168S rRNA gene sequences and was proximate in phylogeny to Mycobacterium ulcerans and Mycobacterium marinum. However, distinct nucleotide differences were detected in the 16S rRNA gene sequence among M175T, M. ulcerans and M. marinum (99.2% similarity). Isolate M175T could be differentiated from other slowly growing, non-pigmented mycobacteria by its inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase and resistance to isoniazid (1 microg ml(-1)), thiacetazone and thiophene-2-carboxylic hydrazide. Based upon these genetic and phenotypic differences, isolate M175T (=ATCC 700981T =NCTC 13215T) is proposed as the type strain of a novel species, Mycobacterium shottsii sp. nov.

Topics: Animals; Bass; Fish Diseases; Molecular Sequence Data; Mycobacterium; Mycobacterium Infections; Phenotype; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA

Four synthetic peptides of 15 amino acids (aa), corresponding to sequences of the nodavirus DIEV RNA(2) protein, were chosen to test their potential immunogenicity in sea bass. Two of these included the N or C terminal regions (N-ter or C-ter) and the sequences of the others contained a potential external site (aa 127-140: Lp1 and as 266-279: Lp2). Two heat inactivated strains of nodavirus (HI Sb1 and HI Sb2), were used as positive controls and the carrier (KLH) as a negative control. ELISAs were performed to quantify serum antibodies specific to nodavirus, to peptides, and to the carrier in order to monitor their immunogenicity. All the fish reacted to the peptides C-Ter, Lp1 and Lp2 but only 55% of animals injected with N-ter produced specific antibodies. The proportion of fish that produced antibodies that cross reacted with nodavirus was very different with regard to the antigen injected: HI Sb1=88%; HI Sb2=85%; N-ter=38%; C-ter=27%. Protection against nodavirus was investigated by challenging the fish with a virulent viral suspension. The results showed that heat-inactivated Nodavirus protect fish and the N-ter peptide is a potential protective peptide. This initial approach showed that although vaccinating fish with peptides is possible, the tools and strategies of the research used in this field still need to be adapted to fish.

Topics: Animals; Antibodies, Viral; Bass; Brain; Capsid; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunization; Immunohistochemistry; Nodaviridae; Peptides; RNA Virus Infections; Vaccines, Subunit; Viral Vaccines

A polymerase chain reaction (PCR) method was developed for largemouth bass virus (LMBV). This iridovirus can cause a lethal disease of largemouth bass Micropterus salmoides, but also subclinically infects largemouth bass and other species of fishes. Oligonucleotide primers were designed to specifically amplify the major capsid protein gene of LMBV. The protocol for sample processing and PCR provided a method that was more sensitive than cell culture for detection of LMBV in fish. The specific amplification of LMBV also provided an improved method for confirming the identity of cell-culture isolates presumptively identified as LMBV.

Topics: Animals; Base Sequence; Bass; Capsid; Capsid Proteins; DNA Virus Infections; DNA, Viral; Fish Diseases; Gene Amplification; Iridovirus; Molecular Sequence Data; Polymerase Chain Reaction; Sensitivity and Specificity

Striped bass Morone saxatilis were infected intraperitoneally with approximately 10(5) Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21 degrees C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 10(9) colony-forming units g(-1). Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii- and M. gordonae-infected fish resolved over time, and no reactivation of disease was observed.

Topics: Animals; Bass; Colony Count, Microbial; Fish Diseases; Granuloma; Immunohistochemistry; Injections, Intraperitoneal; Kidney; Liver; Mycobacterium; Mycobacterium Infections; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Spleen; Time Factors; Virulence

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel

Topics: Animals; Apoptosis; Ascitic Fluid; Bacterial Proteins; Bass; DNA Fragmentation; DNA, Bacterial; Electrophoresis, Agar Gel; Fish Diseases; In Situ Nick-End Labeling; Macrophages, Peritoneal; Microscopy, Electron; Microscopy, Fluorescence; Neutrophils; Pasteurella Infections; Photobacterium; Virulence

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.

Topics: Animals; Bacterial Typing Techniques; Bass; Carrier State; DNA, Bacterial; Fish Diseases; Fisheries; Perciformes; Phenotype; Random Amplified Polymorphic DNA Technique; Ribotyping; Sea Bream; Vibrio; Vibrio Infections; Virulence

Topics: Animals; Bass; Brain; Brain Diseases; Central Nervous System Viral Diseases; Disease Outbreaks; Eye Infections, Viral; Fish Diseases; Fisheries; Fresh Water; Greece; Nodaviridae; Polymerase Chain Reaction; Retina; Retinal Diseases; RNA Virus Infections; RNA, Viral; Spinal Cord

Cryptosporidium molnari was experimentally transmitted to gilthead sea bream (Sparus aurata) and European sea bass (Dicentrachus labrax) by oral infection with infected stomach scrapings. The infection was also cross-transmitted from infected gilthead sea bream to sea bass by cohabitation. The course of the infection was assessed after necropsy by three microscopic diagnostic methods and their sensitivity was compared. At the end of all the experiments the prevalence of infection reached 100%. In the oral experiments, both fish hosts appeared infected as early as 7 days post exposure (p.e.), but gilthead sea bream exhibited a higher intensity of infection and infection proceeded at a faster rate than in sea bass. The cellular host reaction was stronger in sea bass than in sea bream, whereas the histopathological effect was lower in the former. Transmission could be favoured by cannibalism among cohabiting fish. This is the first report on piscine Cryptosporidium transmission. The implications for the aquaculture industry are discussed.

Topics: Animals; Aquaculture; Bass; Cryptosporidiosis; Cryptosporidium; Fish Diseases; Sea Bream; Stomach

The effects of the fish parasitic isopod, Ceratothoa oestroides (Risso), on haematological parameters of its cage-cultured sea bass host, Dicentrarchus labrax (L.), were studied. Analyses of blood parameters (cell counts, haemoglobin content and haematocrit) were carried out on parasitized and unparasitized sea bass from a fish farm in Turkey. Parasitized fish had significantly lowered erythrocyte counts, haematocrit and haemoglobin values and significantly increased leucocyte counts. Blood feeding by C. oestroides thus produces a post-haemorrhagic anaemia and the fish appear to mount an immune response to the presence of parasites.

Topics: Anemia; Animals; Bass; Fish Diseases; Hematocrit; Isopoda; Leukocyte Count; Parasitic Diseases, Animal

Bacterial cells of the marine fish pathogen Photobacterium damsela subsp. piscicida were grown in novel culture media. A mixture of whole cells and extracellular components was inactivated and used in bath, intraperitoneal (i.p.) and oral vaccination of sea bass, Dicentrarchus labrax, employing two sizes of fish. A commercial vaccine was used for comparative purposes. Control and immunized fish were either bath or intraperitoneally challenged 6 and 12 weeks post-vaccination. Small fish had significantly higher relative percentage survival with the novel vaccine mixture both at 6 and 12 weeks post-vaccination by bath, in comparison with the commercial vaccine. No protection was afforded at 6 or 12 weeks post-immunization by either vaccine after challenge via i.p. injection. Sea bass (1.5-2 g) intraperitoneally vaccinated with various adjuvanted vaccine mixtures were not protected against pasteurellosis. In contrast, larger sea bass (20 g) benefited from vaccination with the novel vaccine mixtures. Intraperitoneal challenge with the pathogen resulted in protection in both fish groups vaccinated with novel vaccine mixtures, whereas control fish suffered high mortalities (> 80%). Orally vaccinated fish were immersion challenged with the pathogen. At 6 and 12 weeks post-vaccination the control fish had a high mortality and the fish vaccinated with the novel vaccine mixture achieved good protection.

Topics: Administration, Oral; Animals; Aquaculture; Bacterial Vaccines; Bass; Fish Diseases; Gram-Negative Bacterial Infections; Injections, Intraperitoneal; Photobacterium; Vaccination

Topics: Animals; Artemia; Bacteriophages; Bass; Fish Diseases; Hemolysis; Oncorhynchus mykiss; Penaeidae; Salmo salar; Vibrio; Virulence

Striped bass (Morone saxatilis) and hybrid tilapia (Oreochromis spp.) were experimentally infected with Mycobacterium marinum. Splenic mononuclear cell transforming growth factor-beta (TGF-beta) mRNA was measured by reverse transcription quantitative-competitive PCR (RT-qcPCR). In histologic sections of liver and anterior kidney, the area of each section that was occupied by granulomas and the total area of each section were measured by computer-assisted image analysis and compared as a proportion (the granuloma proportion). Infected striped bass splenic mononuclear cell TGF-beta mRNA expression was significantly lower than uninfected controls, while for tilapia there was no significant difference between infected and control fish. Mycobacterial granuloma proportion of liver and anterior kidney sections was significantly greater for infected striped bass than tilapia. Three (of 10) infected tilapia with the most pronounced inflammatory response displayed a decrease in TGF-beta mRNA expression, similar to the overall striped bass response to mycobacterium challenge. Downregulation of TGF-beta and failure to modulate the immune response may be related to excessive inflammatory damage to organs observed in mycobacteria-sensitive fish species.

Topics: Animals; Bass; Fish Diseases; Gene Expression Regulation; Genetic Predisposition to Disease; Granuloma; Image Processing, Computer-Assisted; Kidney; Liver; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tilapia; Transforming Growth Factor beta

A recombinant vaccine was constructed for piscine mycobacteriosis utilizing a Brucella abortus strain RB51 vector expressing a mammalian Mycobacterium sp. 85A antigen. Juvenile striped bass were inoculated with the resulting construct at doses equivalent to 10(6), 10(7), 10(8), 10(9), and 10(10) colony-forming units/fish. Blood and tissue samples from these fish demonstrated significant specific humoral and cell-mediated immune responses towards the 85A antigen in a dose-dependent manner. However, survival studies determined that inoculated fish failed to demonstrate cross-protective responses after live Mycobacterium marinum challenge 70 days post-inoculation.

Topics: Amino Acid Sequence; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Vaccines; Base Sequence; Bass; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunization; Molecular Sequence Data; Mycobacterium; Mycobacterium Infections; Recombinant Proteins; Sequence Alignment; Survival Analysis; Vaccines, Synthetic

Topics: Animals; Bass; Fish Diseases; Fisheries; Fishes; Meat; Perciformes; Trout

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.

Topics: 3-Phosphoshikimate 1-Carboxyvinyltransferase; Alkyl and Aryl Transferases; Animals; Base Sequence; Bass; Electrophoresis, Agar Gel; Fish Diseases; Gram-Negative Bacterial Infections; Kanamycin Resistance; Molecular Sequence Data; Photobacterium; Plasmids; Sequence Analysis, DNA; Vaccines

Ceratothoa oestroides (Risso, 1826) (Isopoda: Cymothoida) is a protandric hermaphrodite parasite on a wide range of wild fish species. In recent years it has become a threat to cage-reared fish facilities, where high fish density provides optimal conditions for transmission. Its impact on fish health and economical gain is significant, varying from growth retardation and decreased immunocompetency to direct loss due to mass mortalities of juvenile fishes. Because of the sheltered location of the parasite in the buccal cavity of fishes, chemotherapeutics are ineffective. An understanding of the C. oestroides life cycle and its behavioral mechanisms could prove constructive tools for the prevention and control of infection. This study describes the reproductive cycle of C. oestroides experimentally induced in different fish hosts and temperature regimes. Sea bream larvae Sparus aurata and 1 yr annular sea bream Diplodus annularis were chosen as experimental models, and were held at 22 and 19.5 degrees C, respectively. The reproductive cycle of S. aurata was not completed within 4 mo (at which point the last larva died of severe anemia and respiratory distress), while that of the annular sea bream was completed successfully after 1 mo.

Topics: Animals; Aquaculture; Bass; Body Weights and Measures; Fish Diseases; Host-Parasite Interactions; Isopoda; Parasitic Diseases, Animal; Reproduction; Sea Bream; Temperature

Twenty sea bass Dicentrarchus labrax L. from a fish farm (floating cage) in Greece were examined for the presence of parasites. The gills of 7 (35%) fish were infected with adult female specimens of the parasitic copepod Lernanthropus kroyeri van Beneden, 1851, and the intensity of infection ranged from 1 to 24 parasites per host. The most infected portion of the gills appeared to be the primary lamellae. Erosion, desquamation and necrosis of the secondary lamellae were noticed near the site of copepod attachment; furthermore, the terminal claw of the second antennae lacerated tissue and vessels of infected gill. Parasitism by L. kroyeri affected the host's condition factor (mean +/- SE in uninfected vs parasitized; 1.88 +/- 0.04 vs 1.66 +/- 0.12; p < 0.05).

Topics: Animals; Aquaculture; Bass; Body Weights and Measures; Copepoda; Fish Diseases; Gills; Histological Techniques; Parasitic Diseases, Animal; Seawater

The histophagous ciliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus and sea bass Dicentrarchus labrax. The present study screened 52 candidate antiprotozoals for activity against this pathogen in vitro. Of these compounds, 14 were effective (i.e. killed all ciliates within a 24 h assay period). In descending order of efficacy (minimum lethal concentration 100 to 0.8 ppm), these were niclosamide, oxyclozanide, bithionol sulfoxide, toltrazuril, N-(2'-hydroxy-5'-chloro-benzoyl) 2-chloro-4-nitroaniline, furaltadone, doxycycline hyclate, formalin, albendazole, carnidazole, pyrimethamine, quinacrine hydrochloride and quinine sulfate. Administration in filtered seawater rather than phosphate-buffered saline inactivated doxycycline hyclate and albendazole, and markedly reduced that of bithionol sulfoxide and toltrazuril, suggesting that these compounds may not be effective in bath administration. In view of these findings, we discuss the potential utility of chemotherapy as a strategy for the control of scuticociliatosis in farmed turbot and sea bass.

Topics: Animals; Antiprotozoal Agents; Bass; Ciliophora; Ciliophora Infections; Dose-Response Relationship, Drug; Fish Diseases; Fisheries; Flatfishes; In Vitro Techniques; Treatment Outcome

An adult black sea bass was examined because of abdominal distention and decreased appetite. A large abdominal swelling was evident and was firm on palpation. Differential diagnoses included neoplasia, abscess or granuloma, hematoma, or swim bladder abnormality. Diagnostic tests included survey radiography, positive-contrast radiography, and computed tomography. The sea bass was anesthetized with tricaine methanesulfonate. A ventral midline abdominal incision was made, and adhesions to the mass were gently dissected. The fish recovered without complications. Radiography was repeated 8 weeks after surgery, and there was no evidence of mass regrowth. To the authors' knowledge, this is the first report of a barium enema being performed in a fish. Although surgical procedures are performed more commonly on fish for research, a few reports of clinical surgical cases have been described. Our experience supports the conclusions of other reports that certain surgical procedures can be performed safely in fish.

Topics: Abdomen; Animals; Barium Sulfate; Bass; Contrast Media; Diagnosis, Differential; Enema; Fish Diseases; Male; Radiography, Abdominal; Seminoma; Testicular Neoplasms; Tomography, X-Ray Computed

The first reported fish kill caused by largemouth bass virus (LMBV) occurred in 1995 in Santee-Cooper Reservoir, South Carolina, USA. Subsequently, this iridovirus has been implicated in additional fish kills and has also been found in clinically healthy fish in numerous locations in the southeastern USA. We compared the virus from Santee-Cooper Reservoir with a virus isolated in 1991 from large-mouth bass, Micropterus salmoides, from Lake Weir, Florida. Restriction fragment length polymorphisms and the DNA sequence of a portion of the major capsid protein gene were identical for the South Carolina and Florida isolates. These results establish that LMBV was first found in Florida, rather than South Carolina. We propose that the name largemouth bass virus continue to be used for this virus, rather than alternative names based on geographical origin.

Topics: Animals; Base Sequence; Bass; Capsid Proteins; DNA Virus Infections; DNA, Viral; Fish Diseases; Florida; Gene Amplification; Iridovirus; Phylogeny; Polymorphism, Restriction Fragment Length; South Carolina; Viral Proteins

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.

Topics: Animals; Bass; Carboxylic Ester Hydrolases; Fish Diseases; Histocytochemistry; Injections, Intraperitoneal; Macrophages; Neutrophils; Peritoneal Cavity; Peritonitis; Peroxidase; Phagocytes; Phagocytosis; Photobacterium; Staining and Labeling

There is a need to develop simple, rapid, and accurate methods for assessing health in fish populations. In this study we demonstrate that use of fluorescein, a nontoxic fluorescent dye, can rapidly and easily detect the presence of skin ulcers in all fish tested, including rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), goldfish (Carassius auratus), and hybrid striped bass (Morone saxatilis male X M. chrysops female). Exposure of fish to as little as 0.10 mg fluorescein per milliliter of water for 3 minutes was sufficient to identify experimentally induced lesions, even pinpoint ulcerations. Such lesions were not visible to the naked eye but were clearly demarcated with fluorescein treatment. Examination of fish that appeared clinically normal often revealed the presence of focal ulcerations, which might have been a consequence of damage during capture, but it also might suggest that skin ulceration may be common even in "clinically normal" fish. Exposure of either nonulcerated or experimentally ulcerated hybrid striped bass to an excessively high concentration of fluorescein had no apparent effect on health or survival. Our studies suggest that fluorescein may be a highly useful tool for rapid health screening in fish populations.

Topics: Animals; Aquaculture; Bass; Female; Fish Diseases; Fluorescein; Fluorescent Dyes; Goldfish; Ictaluridae; Male; Oncorhynchus mykiss; Photography; Skin Ulcer; Stress, Physiological

Histone-like proteins (HLPs) are broad-spectrum, endogenously produced antibiotics which we have isolated from tissues of rainbow trout Oncorhynchus mykiss and hybrid striped bass (Morone saxatilis male x M. chrysops female). Here, we show that HLP-1, which has high sequence homology to histone H2B, equally inhibited both young and mature trophonts of the important ectoparasite Amyloodinium ocellatum. In addition to direct killing of Amyloodinium trophonts, there was evidence that HLP-1 from both rainbow trout and hybrid striped bass caused severe developmental abnormalities, including delayed development, in both the parasitic trophont stage as well as the reproductive tomont stage. The deleterious effects of HLP-1 also were manifested in what appeared to be 'delayed mortality', where parasites of normal appearance would die later in development. Similar serious damage was also seen with calf histone H2B and the unrelated peptide antibiotic magainin 2. A comparison of the antibiotic activity in mucus versus epidermis compartments of the skin of hybrid striped bass suggested that the majority of antibiotic (including HLP-1) activity resided in the epidermis, although some activity was present in the mucus. These data suggest that normal, nonimmune fish skin contains potent defenses against protozoan ectoparasites and that the effects of these defenses may extend beyond their transient interactions with the parasites, which has important implications for this host-parasite relationship.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Dinoflagellida; Epidermis; Female; Fish Diseases; Histones; Host-Parasite Interactions; Male; Mucus; Oncorhynchus mykiss; Skin

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.

Topics: Animals; Bass; DNA, Bacterial; Fish Diseases; Gene Amplification; Genetic Variation; Genotype; Gram-Negative Bacterial Infections; Photobacterium; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Ribosomal, 16S; Sea Bream; Sensitivity and Specificity; Species Specificity

Thirteen wild sea bass from the Oslo fjord in south-eastern Norway were examined for parasites. Nineteen species were found, comprising 5 protozoans, 1 monogenean, 8 digeneans, 1 cestode, 2 nematodes and 2 crustaceans. Based on the similarity to the parasitic fauna of Mediterranean sea bass, it is predicted that sea bass farmers in Northern Europe will experience the same parasite problems as sea bass farmers in warmer regions.

Topics: Animals; Aquaculture; Bass; Fish Diseases; Gills; Microscopy, Electron, Scanning; Norway; Parasites; Parasitic Diseases, Animal

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.

Topics: Animals; Bass; Cryptosporidiidae; Cryptosporidiosis; Fish Diseases; Host-Parasite Interactions; Oceans and Seas; Phylogeny; Prevalence; Sea Bream; Spain; Stomach

Investigation into recent declines in striped bass health in the Chesapeake Bay in Maryland resulted in the isolation of a putative new species of Mycobacterium. This isolate was obtained from fish showing skin ulcers and internal granulomas in various organs. The isolate was slow growing at 28 degrees C; was nonchromogenic; showed no activities of nitrate reduction, catalase activity, Tween 80 hydrolysis, tellurite reduction, or arylsulfatase reduction; grew best at low salt concentrations; and was urease and pyrazinamidase positive. By PCR a unique insertional sequence was identified which matched nothing in any database. Analysis of the nearly complete 16S rRNA gene sequence also indicated a unique sequence which had 87.7% sequence homology to Mycobacterium ulcerans, 87.6% homology to Mycobacterium tuberculosis, and 85.9% homology to Mycobacterium marinum. Phylogenetic analysis placed the organism close to the tuberculosis complex. These data support the conclusion that the isolate probably represents a new mycobacterial species.

Topics: Animals; Base Sequence; Bass; DNA Primers; DNA, Bacterial; DNA, Ribosomal; Fish Diseases; Granuloma; Maryland; Molecular Sequence Data; Mycobacterium; Mycobacterium Infections; Phylogeny; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Seawater; Sequence Alignment; Sequence Homology, Nucleic Acid; Skin Diseases, Bacterial

To determine the prevalence of Streptococcus iniae in tilapia (Oreochromis spp), hybrid striped bass (Morone chrysops X M saxatilis), and channel catfish (Ictalurus punctatus) on commercial fish farms in the United States.. 1,543 fish (970 tilapia, 415 hybrid striped bass, and 158 channel catfish).. The dry-swab technique was used for collection of specimens for streptococcal isolation. Specimens were shipped by overnight delivery and processed by use of standard bacteriologic techniques.. Streptococcus iniae was not isolated from market-size channel catfish. Prevalence in tilapia and hybrid striped bass was 37 of 970 (3.81%) and 30 of 415 (7.23%), respectively. Prevalence by farm ranged from 0.0 to 27.4% for tilapia and 0.0 to 21.6% for hybrid striped bass. In tilapia, prevalence was lowest in market-size and nursery fish (4 of 239 [1.67%] and 3 of 339 [0.88%], respectively), with an increase in prevalence for fish in the grow-out stage (30 of 337 [7.96%]). For hybrid striped bass, prevalence was lowest in nursery and market-size fish (3 of 96 [3.12%] and 1 of 47 [2.12%], respectively) and highest in fish in the grow-out stage (26 of 272 [9.56%]). Prevalence in market-size tilapia and hybrid striped bass was 5 of 286 (1.75%).. Results of this study do not support the contention that S iniae is a serious public health threat associated with commercially raised fish; rather, it represents a limited risk for older or immunocompromised people who incur puncture wounds while handling and preparing fish.

Topics: Animals; Bass; Colony Count, Microbial; Fish Diseases; Fisheries; Fishes; Ictaluridae; Prevalence; Streptococcal Infections; Streptococcus; Tilapia; United States

To assess the immunogenic and immunoprotective role of the extracellular lectin from Aeromonas veronii (MCBP), which has affinity for mucosal constituents such as mucin, lactoferrin, immunoglobulins and collagen, spotted sand bass (Paralabrax maculatofasciatus) were orally immunised either with soluble MCBP, adjuvant-conjugated MCBP or immobilised MCBP on latex microspheres. The results suggest that the MCBP is capable of eliciting protective immunity against A. veronii infections when administered orally. The highest mucosal immune response was elicited in fish immunised with MCBP covalently linked to cholera toxin B subunit (CTB) or to Escherichia coli heat-labile toxin (hLT). MCBP-CTB was found to elicit immunoprotection against a challenge with live Aeromonas cells with a relative percent survival of almost 70% and without the expression of the severe histopathological alterations induced by A. veronii.

Topics: Aeromonas; Animals; Bacterial Toxins; Bass; Cholera Toxin; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Proteins; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Mucosal; Lectins; Vaccination

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).

Topics: Animals; Aquaculture; Bass; Brain Diseases; Cell Line; Enzyme-Linked Immunosorbent Assay; Fish Diseases; France; Immunohistochemistry; Larva; RNA Virus Infections; RNA Viruses; Sensitivity and Specificity

Antimicrobial proteins were purified from acid extracts of rainbow trout (Oncorhynchus mykiss) and sunshine bass (Morone saxatilis male x M. chrysops female) skin, gill and spleen by reverse-phase HPLC. Mass spectrometry and amino acid sequence data suggest that these proteins are closely related to histone H2B and histone H1 and thus they were designated histone-like proteins (HLPs). These proteins were lethal to Amyloodinium ocellatum, which is one of the most important parasitic agents affecting fish. Antibiotic concentrations as low as 12.5 microg/ml were inhibitory. Activity was directed against the trophont (feeding) stage of the parasite, while the disseminative (dinospore) stage was unaffected. Thus, HLPs act unlike typical drugs used to treat amyloodiniosis, which usually target the dinospore. Both the ability of the parasite to infect host cells, as well as the ability to grow and differentiate after infection were severely inhibited. This is in contrast to magainin 2, which was similarly toxic to both the dinospore and trophont stages. These findings provide further evidence that histone-like proteins may be important defensive molecules in fish.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; Bass; Chromatography, High Pressure Liquid; Dinoflagellida; Electrophoresis, Polyacrylamide Gel; Female; Fish Diseases; Histones; Male; Oncorhynchus mykiss; Peptides; Sequence Analysis, Protein

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus. Nevertheless, the sea bream appeared to be a potential carrier of nodavirus, as juvenile sea bass became infected when maintained in a tank containing experimentally contaminated sea bream.

Topics: Animals; Aquaculture; Base Sequence; Bass; Carrier State; Disease Transmission, Infectious; Fish Diseases; Fluorescent Antibody Technique; Molecular Sequence Data; Nodaviridae; Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sea Bream; Sequence Alignment; Sequence Analysis; Virulence

Juvenile largemouth bass Micropterus salmoides were intraperitoneally injected with largemouth bass virus (LMBV), a member of the genus Ranavirus, family Iridoviridae. Moribund fish which had been injected with 10(6.2) tissue culture infectious doses, 50% endpoint (TCID50), were sampled 4 d after injection; other largemouth bass injected with this dose died between 3 and 5 d after injection. Fish injected with 10(2.8) TCID50 of LMBV were also examined after 4 d and had lesions similar to those of fish injected with the high dose. Clinical signs included darker pigmentation, inflammation and necrosis at the site of injection, distended abdomen, corkscrew swimming, and lateral recumbency. Internally, fish had focally pale livers, bright red spleens and reddened intestinal ceca. Histologically acute fibrinous peritonitis affected the surface of all organs in the peritoneal cavity, but deeper portions of organs appeared normal. There was also necrosis of the gastrointestinal mucosa. Except for the injection site, lesions were confined to the peritoneal cavity.

Topics: Animals; Bass; DNA Virus Infections; Fish Diseases; Injections, Intraperitoneal; Intestines; Liver; Ranavirus

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

Topics: Aeromonas; Animals; Antibodies; Bacterial Adhesion; Bass; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Fish Diseases; Fishes; Glycoconjugates; Gram-Negative Bacterial Infections; Kinetics; Lactoferrin; Lectins; Time Factors

The immune response of European sea bass after intracaelomic immunization with Sphaerospora dicentrarchi was studied. Fish were injected with S. dicentrarchi spores (DIC), with spores plus adjuvant (DIC + FCA), with adjuvant alone (FCA) or with PBS. Several parameters of the immune response were measured. Serum lysozyme increased significantly in DIC fish 1 week after immunization (p.i.) and it remained significantly higher in DIC + FCA fish 4 weeks p.i., and in DIC fish 8 weeks p.i. than in PBS-injected fish. The number of nitroblue tetrazolium-positive blood cells was significantly higher in DIC + FCA fish 1, 4 and 8 weeks p.i, but the highest values were detected 1 week p.i. The highest stimulation index was detected in phagocytes from DIC + FCA fish. The number of S. dicentrarchi antibody-secreting cells was significantly higher in DIC + FCA fish than in DIC fish. Serum from DIC and DIC + FCA fish, stained the polar capsules and the valves of S. dicentrarchi spores in immunohistochemistry. Serum antibodies could not be detected using immunoblot assay. All these results show that immunization with S. dicentrarchi resulted in the activation of the non-specific immune response, mainly 7 days p.i. A specific humoral response against the parasite was also demonstrated but it had a low magnitude.

Topics: Animals; Antibodies, Protozoan; Antibody-Producing Cells; Bass; Complement Pathway, Alternative; Eukaryota; Fish Diseases; Immunity, Cellular; Immunization; Muramidase; Nitroblue Tetrazolium; Protozoan Infections, Animal; Respiratory Burst

Because of the affinity of certain bacterial species for sulphated glycoconjugates exposed on the epithelial cells of susceptible hosts, we hypothesized that sulphated exopolysaccharides of microalgae can be used in anti-adhesive therapies against bacterial infections, both in cold- and warm-blooded animals. In this study we found that adhesion of the human pathogen Helicobacter pylori to the HeLa S3 cell line, and adhesion of the fish pathogens Vibrio campbellii, V. ordalii, Streptococcus saprophyticus, and Aeromonas veronii to spotted sand bass primary tissue culture cells, can be effectively blocked with the various sulphated exopolysaccharides used.

Topics: Aeromonas; Animals; Anti-Bacterial Agents; Bacterial Adhesion; Bass; Cells, Cultured; Eukaryota; Fish Diseases; HeLa Cells; Helicobacter pylori; Humans; Polysaccharides; Skin Diseases, Bacterial; Streptococcus; Vibrio; Water Microbiology

The existence and localisation of carbohydrates in four myxosporean parasites was investigated at transmission electron microscope, using lectin histochemistry techniques. The Myxosporea studied showed different lectin binding patterns. N-acetyl-glucosamine or its polymers were predominant in the valves of Leptotheca sp. and were also present in Sphaerospora dicentrarchi and Polysporoplasma sparis ones. Mannose and/or glucose terminals were mainly detected in S. dicentrarchi and P. sparis valves. Polar capsule walls were intensively recognised by Con-A is S. dicentrarchi and with medium intensity in P. sparis and Leptotheca sp. The polar filament was scarcely labelled except in Leptotheca sp. with BS-I. The sporoplasms of the studied parasites were stained with all the lectins tested with each myxosporea, except those of Leptotheca sp. with BS-I. Some structures of the developmental stages of Zschokkella mugilis and Leptotheca sp. were stained with BS-I. The possible role of these carbohydrate terminals found in the studied Myxosporea is discussed.

Topics: Animals; Bass; Carbohydrates; Eukaryota; Fish Diseases; Gills; Lectins; Microscopy, Electron; Protozoan Infections, Animal; Spores

Several in vitro studies were performed to study the cellular reaction of European sea bass (Dicentrarchus labrax L.) against Sphaerospora dicentrarchi. Head kidney phagocytes were obtained from parasitised (P) and non-parasitised (NP) fish. The production of superoxide anion (O2-), tested by the NBT method, was higher in P than in NP fish. The addition of increasing amounts of sea bass serum (SBS) produced a gradual increment of the respiratory burst with SBS from parasitised animals (P-SBS), whereas this increment reached a plateau at lower concentration with SBS from nonparasitised ones (NP-SBS). O2- production was higher when adding NP-SBS than with P-SBS or fetal bovine serum. Heat inactivation of NP-SBS and FBS reduced the respiratory burst significantly, whereas it did not change the effect of P-SBS. The number of NBT-positive cells after particulate stimulation was significantly higher using S. dicentrarchi spores than using SRBC, but lower than with phorbol myristate acetate. Phagocytes primed overnight with spore extracts produced higher amounts of O2- than those LPS-primed or non-primed ones. A similar percentage of phagocytosis was detected using glutaraldehyde fixed spores and SRBC. Most of the phagocytes engulfed three or more SRBC, whereas most of the phagocytes engulfed only one spore. Complement mediated opsonisation by NP-SBS may occur, as the phagocytic index was reduced when the serum was heat inactivated.

Topics: Animals; Bass; Eukaryota; Fish Diseases; Hot Temperature; In Vitro Techniques; Phagocytes; Phagocytosis; Protozoan Infections, Animal; Respiratory Burst; Superoxides

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 microm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20 degrees C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13 degrees C, up to 7 d at 20 degrees C, but it was inactivated at 37 and 56 degrees C within 24 and 1 h, respectively. Freezing at -20 degrees C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20 degrees C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10(2.5)-50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.

Topics: Animals; Aquaculture; Bass; California; Fish Diseases; Microscopy, Fluorescence; Oncorhynchus kisutch; Rickettsia; Rickettsia Infections; Virulence

Although amoebic gill disease (AGD) has emerged as one of the most severe health problems in the fish industry, proof of the identity of AGD agents from various localities is still missing. Six strains of amoebae designated until recently as Paramoeba species (the agents of AGD) were studied in cultures by light and electron microscopy. Although they were isolated from gills of different hosts (Dicentrarchus labrax and Scophthalmus maximus) and from distant localities, their morphology was identical. The strains differed from Paramoeba eilhardi, the type species of the genus, in that they lacked the boat-shaped microscales on the cell surface but could be safely identified as belonging to the genus Neoparamoeba Page, 1987. Transmission electron microscopy revealed the presence of a symbiotic organism, Perkinsiella amoebae Hollande, 1980, in all strains under study. The only difference among the strains examined was found in the size of trophozoites, which could be attributed to the different origins of the strains, but until more refined diagnostic methods are available, in addition to N. pemaquidensis, the closely related species N. aestuarina also has to be taken into consideration as the agent of AGD.

Topics: Amebiasis; Amoeba; Animals; Bass; Fish Diseases; Flatfishes; Gills; Histocytochemistry; Microscopy, Electron

Morphological characteristics of a metacercaria from muscle of 15 sea bass Caprodon longimanus (Serranidae) collected near Alejandro Selkirk Island (Chile) in the south-eastern Pacific Ocean indicate that it belongs to the genus Manteria (Acanthocolpidae). All metacercarie were encapsulated with connective tissue. The prevalence of infection was 100%, with 64% of metacercariae were located between dorsal pterygiophores and dorsal fin base. Mean intensities of metacercariae did not differ significantly with respect to sex of the fish. There was no significant correlation with intensities of infection and condition in the host.

Topics: Animals; Bass; Chile; Female; Fish Diseases; Male; Pacific Ocean; Prevalence; Trematoda; Trematode Infections

Topics: Animals; Ataxia; Bass; Behavior, Animal; Fish Diseases; Insecticides; Permethrin; Pesticide Synergists; Piperonyl Butoxide; Pyrethrins; Toxicity Tests

Immunological staining with rabbit antibodies raised against Sphaerospora testicularis and Ceratomyxa labracis was used to characterise their specificity and their reactivity towards other fish parasites. Polar capsules and valves of S. testicularis and C. labracis were labelled with their homologous antibody and cross reaction was observed with all the myxosporean parasites assayed from marine and freshwater fish hosts. All polar capsules were stained with both antibodies, except those of Zschokkella mugilis, which were not labelled with anti-S. testicularis serum. These observations suggest that polar capsules may be very conserved structures in myxosporean parasites from different hosts.

Topics: Animals; Antibodies, Protozoan; Antibody Specificity; Antigens, Helminth; Bass; Cross Reactions; Epitopes; Eukaryota; Fish Diseases; Fishes; Host-Parasite Interactions; Immunohistochemistry; Protozoan Infections, Animal; Rabbits; Seawater

The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bass (3 g) Dicentrarchus labrax by using cell culture supernatant (SSN-1 cell line) containing nodavirus. Five methods of infection were tested: intramuscular injection (IM), intraperitoneal injection (IP), oral infection, bath exposure and cohabitation of healthy fish with infected fish. Some differences were observed in time of disease onset and severity of symptoms depending on the mode of infection used. Clinical symptoms such as whirling swimming and lethargic or hyperactive behaviour were generally reproduced, except for fish infected via oral and IP infection. First mortalities occurred 3 d after IM and IP infection and 6 d after for the other modes of infection. Cumulative mortalities were also variable: 100% after IM infection, 10% after IP infection, 32% for bath exposure, 43% after cohabitation and 24% via oral infection. Histopathologically, vacuolation was observed in the central nervous tissues and in the retina. The observed lesions were more or less severe depending on the mode of infection, the sampling time and the organs: lesions on the surviving fish (42 days post infection, d p.i.) seemed to be generally more conspicuous in the retina than in the brain of the same fish. In most cases, the presence of nodavirus was confirmed in the same samples of brain and retina by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The virus was not detected in other organs examined. The present results suggest that 2 forms of VER can be induced: IM injection leads to an acute form (severe nervous disorders with high and fast mortality) whereas oral infection, bath exposure and cohabitation induce a subacute form (less severe disorders and weak daily mortality). This experiment demonstrates experimentally induced horizontal transmission of VER in sea bass for the first time.

Topics: Administration, Oral; Animals; Antibodies, Viral; Bass; Brain; Brain Diseases; Cytopathogenic Effect, Viral; Disease Transmission, Infectious; DNA Primers; Electrophoresis, Agar Gel; Fish Diseases; Fluorescent Antibody Technique, Indirect; Immunohistochemistry; Injections, Intramuscular; Injections, Intraperitoneal; Random Allocation; Retina; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA Viruses; RNA, Viral; Spinal Cord

Immunoglobulins (Ig) in serum from barramundi vaccinated with bovine serum albumin (BSA) were purified by ammonium sulphate precipitation and affinity chromatography using BSA as the ligand. The BSA-binding activity of eluted putative Ig fractions was assessed by enzyme-linked immunosorbent assay (ELISA) before being pooled and characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Double affinity purification did not improve the purity of the Ig preparation compared to single affinity purification. Barramundi Ig were injected into sheep to produce anti-Ig antisera which were assessed in an indirect ELISA as the secondary antibody to detect serum Ig in barramundi vaccinated with Cryptocaryon irritans theronts. Affinity-purified Ig induced a more specific reagent for use as secondary antibody in ELISA than did normal whole-barramundi sera. The heavy (H) chain of barramundi Ig had an apparent molecular weight of 70 kDa while that of the light (L) chain was 27 kDa in SDS-PAGE studies. Under non-reducing conditions 2 putative populations of Ig were identified, at 768 and 210 kDa. The N-terminal sequence of the barramundi Ig H chain showed 78% homology with channel catfish Ictalurus punctatus Ig H chain sequence.

Topics: Amino Acid Sequence; Animals; Antibodies, Protozoan; Antigens, Protozoan; Bass; Chromatography, Affinity; Ciliophora; Ciliophora Infections; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immune Sera; Immunoglobulins; Molecular Sequence Data; Sequence Analysis; Sequence Homology, Amino Acid; Serum Albumin, Bovine; Sheep; Vaccination

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

Topics: Animals; Bass; DNA Primers; DNA, Bacterial; DNA, Ribosomal; Fish Diseases; Fishes; Genes, rRNA; Gram-Negative Bacterial Infections; Humans; Molecular Sequence Data; Photobacterium; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sensitivity and Specificity; Sequence Analysis, DNA

The parasites of 541 largemouth bass, Micropterus salmoides, were studied over a period of 12 months. The results showed that the bass from Lake Naivasha are paratenic hosts of Contracaecum sp. larva and final hosts for the acanthocephalan Polyacanthorhynchus kenyensis. The nematode occurred in large numbers in fish caught in the more saline Oloidien Bay but only in small numbers in those in the main lake. Bass in the main lake, however, were more heavily infected with acanthocephalans than those in Oloidien Bay. One of the major pathological effects of the acanthocephalan was perforation of the liver by the spiny proboscis. Seasonal variation was not apparent for either of the parasites. The intensity of infection by Contracaecum sp. larva increased with the size of the host and female fish were more heavily infected than males.

Topics: Acanthocephala; Animals; Bass; Data Interpretation, Statistical; Female; Fish Diseases; Helminthiasis, Animal; Host-Parasite Interactions; Kenya; Male; Prevalence

An iridovirus, isolated from largemouth bass Micropterus salmoides following a die-off among adult fish and provisionally designated largemouth bass virus (LMBV), was characterized by analysis of viral protein synthesis in infected cells, viral DNA restriction fragment length polymorphisms (RFLP), and sequence determination of the major capsid protein and viral DNA methyltransferase genes. All 3 approaches yielded results consistent with the suggestion that LMBV was a member of the genus Ranavirus. Moreover, LMBV was nearly identical to 2 isolates from Southeast Asia which had been previously detected in imported ornamental fish. It remains to be determined whether infection of largemouth bass resulted from exposure to an imported virus, or whether the presence of similar viruses in southeast Asia and the southeastern United States indicates that iridovirus species are not geographically limited as suggested earlier, but rather globally distributed.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; Capsid; Cell Line; DNA Modification Methylases; DNA, Viral; Fish Diseases; Fishes; Molecular Sequence Data; Polymorphism, Restriction Fragment Length; Ranavirus; RNA Virus Infections; Sequence Alignment; Viral Proteins

In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.

Topics: Animals; Bass; Brain; Encephalitis Viruses; Encephalitis, Arbovirus; Fish Diseases; Polymerase Chain Reaction; Retina; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.

Topics: Animals; Bass; Encephalitis Viruses; Encephalitis, Arbovirus; Fish Diseases; Humans; Immunohistochemistry; Microscopy, Electron

This preliminary study elucidates the in vitro and in vivo effects of temperature on grouper nervous necrosis virus (GNNV) infection. A novel continuous cell line derived from the fin tissue of a grouper (Epinephelus coioides, Hamilton), named as GF-1 cell line, was used. Cytopathic effect was observed in GNNV-infected GF-1 cells incubated at 24-32 degrees C after viral adsorption, but not at 20 degrees C or 37 degrees C even though the viral adsorption temperature was 28 degrees C. Viral protein could be detected in the pellets of GNNV-infected GF-1 cells cultured at 20-32 degrees C, but not at 37 degrees C. In a challenge test, GNNV-challenged larvae which were maintained at a constant 28 degrees C began to die 1 day post challenge (p.c.) with a death rate of 80%. Mortality reached 100% by 50 h p.c., while the mortality of negative control fish was only 5%. The cumulative mortality of GNNV-challenged larvae at ambient temperature, i.e. 28 degrees C at noon and 24 degrees C at midnight, was 10% 1 day p.c., and increased to 100% by 80 h p.c. Based on the results, we concluded that temperature plays an important role in GNNV infection and pathogenicity.

Topics: Animals; Bass; Capsid; Cell Line; Fish Diseases; Gene Expression; Larva; Microscopy, Electron; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA Viruses; Seawater; Temperature

In the present study, attempts were made to clarify the effect of heavy metal stressors and salinity shock on the disease susceptibility of grouper fry (Epinephelus sp.) to infectious pancreatic necrosis virus (IPNV) infection. Zinc, cadmium and copper (5 ppm ZnCl2, 3 ppm CdCl2 and 1 ppm CuCl2) were used to treat groupers before and after virus infection. Cumulative mortalities in the experimental groups were 96-100% within 42 days. Only 5-15% mortalities were observed in most of the groups that were exposed to either heavy metals or virus infection alone. Subsequently, virus was re-isolated from the experimentally infected groupers, and copper concentration was measured in fish that had been exposed to CuCl2. We also investigated the effect of salinity shock (i.e. an abrupt change of salinity level from 33 ppt to either 40 ppt or 20 ppt) on susceptibility of grouper to IPNV. Similar results were obtained, mortalities of groupers in the experimental groups reached 80-100%. The results of the present study suggest that an IPN virus with only low pathogenicity could cause high mortality in groupers when combined with environmental stress.

Topics: Animals; Bass; Birnaviridae Infections; Cadmium Chloride; Chlorides; Copper; Disease Susceptibility; Fish Diseases; Infectious pancreatic necrosis virus; Metals, Heavy; Sodium Chloride; Zinc Compounds

The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (10(3)-10(4) TCID50 IPNV per ml, 10(6)-10(7) colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (10(3)-10(4) TCID50 IPNV per g fish or 10(7) CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers.

Topics: Animals; Bass; Birnaviridae Infections; Fish Diseases; Infectious pancreatic necrosis virus; Vibrio; Vibrio Infections; Water Microbiology

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

Topics: Animals; Bass; Brain; Encephalitis Viruses; Encephalitis, Arbovirus; Fish Diseases; Fisheries; Italy; Microscopy, Electron; Retina; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral

Twenty striped bass Morone saxatilis and 20 hybrid tilapia Oreochromis niloticus x O. mossambicus x O. aureus each received a single intramuscular injection of 1.6 x 10(6) colony forming units per gram body weight of Mycobacterium marinum. Striped bass manifested significantly greater clinical and microscopic disease compared to tilapia. Whereas all the striped bass had died or were clinically ill by Day 8 post-infection, there was no apparent disruption of normal behaviour, physical appearance, or growth in any of the sacrificed or surviving tilapia. Histologically, granulomas in striped bass were generally larger and less discrete, with a higher proportion of heavily vacuolated macrophages, and large cores of necrotic cells. Visceral granulomas in tilapia were smaller, with a higher proportion of epithelioid macrophages, more pigment-containing cells, more peripheral lymphocytes, and virtually no central necrosis. Visceral granulomas were 18-fold more numerous in striped bass than in tilapia. Based upon histomorphometric data, mean proportions of acid-fast bacteria within pronephros granulomas were 4-fold greater in striped bass than tilapia, and striped bass granulomas averaged more than twice as large as tilapia granulomas. In the anterior kidney of striped bass, a positive correlation existed between mean mycobacterial proportions and mean necrosis scores. In tilapia, mean mycobacterial proportions correlated negatively with mean granuloma numbers, whereas there was no correlation between these parameters in striped bass. Results suggest that intrinsic functional differences in the immunologic systems of striped bass and hybrid tilapia may contribute to inter-species variation in mycobacteriosis susceptibility.

Topics: Animals; Bass; Disease Susceptibility; Fish Diseases; Granuloma; Image Processing, Computer-Assisted; Injections, Intramuscular; Kidney; Liver; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Pilot Projects; Spleen; Statistics, Nonparametric; Tilapia; Virulence

For the first time, Emetha audouini (Milne Edwards, 1840), a cymothoid isopod, is reported parasitising cage cultured sea bass Dicentrarchus labrax L., 1758 in Greece. The specimens observed are larvae (Pulli II). They were found in great numbers in the buccal and branchial cavity of young (3.5 m.o. old) sea bass, in an intensive cage farm facility. This parasite is certainly transferred to sea bass from wild populations of Sparidae or Centracanthidae. Serious lesions were visible and typical of a crustacean infection, with extensive and deep skin damage in the head area. The cumulative mortality, over a 2 wk period, was 10.75%. The parasitic problem was successfully dealt with by optimization of management practices rather than use of costly and dangerous chemotherapeutants.

Topics: Animals; Aquaculture; Bass; Cheek; Crustacea; Disease Outbreaks; Fish Diseases; Gills; Greece; Parasitic Diseases, Animal; Prevalence; Skin

Adult, gravid female Neoergasilus japonicus Harada, 1930 are described from several species of fish from Lee County, Alabama. Samples of bluegill Lepomis macrochirus Rafinesque (n = 25), largemouth bass Micropterus salmoides Lacépéde (n = 6), redear sunfish Lepomis microlophus Gunther (n = 5), and channel catfish Ictalurus punctatus Rafinesque (n = 1) were collected between November 1993 and May 1995. Prevalence of infection was 100% in all fish collected. The dorsal fin was the site of infection containing the highest number of parasites and the anal fin showed the highest frequency of infection. Morphological comparisons are made between this report and previous descriptions, and disparities among them are indicated. New hosts for N. japonicus include largemouth bass, channel catfish, and redear sunfish. This report is the first North American record for the genus Neoergasilus.

Topics: Alabama; Animals; Bass; Crustacea; Ectoparasitic Infestations; Female; Fish Diseases; Fresh Water; Gills; Ictaluridae; Perciformes; Prevalence; Skin

Exposure of striped bass (Morone saxatilis) and hybrid bass (M. saxatilis female x Morone chrysops male) to an acute (2-hour) confinement stress caused skin ulceration on the fins but not on the body of all confined fish. Striped bass displayed more severe lesions than did hybrid bass. Histologically, lesions had varying degrees of epithelial erosion and ulceration, which was most severe at the distal portion of the fins. Ulceration was associated with dermal and hypodermal edema and necrosis of the remaining stromal tissue and tips of bone in the fin rays. No hemorrhage or thrombosis was present to suggest any obvious vascular derangement. No evidence was found for either trauma or an infectious agent initiating the lesions. Injecting fish with epinephrine caused a similar response, although the degree of ulceration was less severe. These findings may explain why many opportunistic skin pathogens can rapidly develop into serious infections in fish.

Topics: Animals; Bass; Colony Count, Microbial; Crosses, Genetic; Crowding; Epinephrine; Fish Diseases; Immunity, Innate; Incidence; Opportunistic Infections; Skin Ulcer; Stress, Physiological

Streptococcus iniae was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single S. iniae clone. Wild fish are therefore potential amplifiers of pathogenic S. iniae strains.

Topics: Animals; Animals, Domestic; Animals, Wild; Bass; Fish Diseases; Perciformes; Polymerase Chain Reaction; Sensitivity and Specificity; Streptococcal Infections; Streptococcus

Rabbit antibody was raised against Sphaerospora dicentrarchi, a histozoic parasite of sea bass (Dicentrarchus labrax L.). Light and electron immunohistological staining were used to characterize its specificity and possible reactivity toward other fish parasites. In light immunohistochemistry the polar capsules and valves of the S. dicentrarchi spores appeared strongly stained, whereas developmental stages were not. Electron microscopic histochemistry revealed intense labeling in valves and some developmental stages. Cross-reaction was observed with all the myxosporean parasites assayed, even with those belonging to other genera. Polar capsules of all the myxosporean species except Polysporoplasma sparis were the main structures stained by the polyclonal antibody. These observations could reveal the existence of conserved antigenic epitopes in polar capsules of different Myxosporea.

Topics: Animals; Antibodies, Protozoan; Bass; Eukaryota; Fish Diseases; Immunohistochemistry; Microscopy, Electron; Parasitic Diseases, Animal; Rabbits; Spores

The first epizootic of edwardsiellosis, caused by Edwardsiella tarda, is described. The epizootic occurred in the Chesapeake Bay, Maryland (USA) during the summer and autumn of 1994, and affected wild adult striped bass (Morone saxatilis). Clinical signs included numerous irregular coalescing hemorrhagic ulcers on the body and fins that were distinctly malodorous. Internally, the body cavity was filled with abundant yellowish or sanguinous mucoid fluid, and the visceral organs had multiple tiny white foci. The intestines contained thick white opaque mucus. Histopathological lesions included ulcerative dermatitis, cardiac endothelial hyperplasia, and necrotic foci and granulomata in multiple organs. A bacterium isolated in pure culture was characterized taxonomically and serologically as the wild-type or classical biotype of E. tarda: In infectivity trials, it was pathogenic for striped bass, gilthead seabream (Sparus aurata), and turbot (Scophthalmus maximus) with an LD50 of about 10(5) cells; however, the isolate was non-virulent for mice (LD50 > 10(8) cells). The isolate also was resistant to the bacteriolytic activity of normal fish skin mucus.

Topics: Animals; Bass; Disease Outbreaks; Fish Diseases; Flatfishes; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Lethal Dose 50; Maryland; Mice; Mice, Inbred BALB C; Perciformes; Phenotype; Skin; Virulence

To test the hypothesis that, compared with pure striped bass, hybrid striped bass have reduced secondary physiologic responses to handling stress. A secondary objective was to determine whether not feeding fish for a 3-day period affected responses.. Hatchery-reared adult striped bass (Morone saxatilis) and adult hybrids of striped bass with white bass (M chrysops), with mean length of 27.9 cm and mean weight of 487 g.. Fed and 3-day nonfed fish, in groups of 6, were held in dip nets above water for 3 minutes. Severity of response to handling was determined by measuring plasma glucose and chloride and blood lactic acid, sodium, and potassium concentrations. Terminal samples were taken from fish before handling (control), immediately after handling, and after 12, 24, and 48 hours of recovery.. Striped bass were hyperglycemic and lactacidemic after stress and for 12 to 48 hours afterward, whereas glucose and lactic acid values in hybrids were essentially unchanged. Blood sodium and chloride concentrations of hybrids decreased after stress, then returned to control values within 24 hours. Striped bass, however, had a greater decrease in values for these electrolytes and failed to recover in 48 hours. Blood potassium concentration remained unchanged in all test groups. Nonfeeding for 3 days before handling did not appear to affect stress response in striped bass or hybrids.. Striped bass have an appreciably greater response to acute handling stresses, such as those that may be experienced in hatcheries and experimental laboratories, than do hybrid bass. Thus, pure striped bass require more care in handling. The usual practice of not feeding before handling does not affect physiologic responses.

Topics: Analysis of Variance; Animal Welfare; Animals; Bass; Blood Glucose; Chlorides; Fish Diseases; Handling, Psychological; Lactates; Potassium; Sodium; Stress, Physiological; Time Factors

Two isolates of Cryptocaryon irritans obtained from Acanthopagrus australis from Moreton Bay (isolate C1) and Gymnocranius audleyi from Heron Island (isolate C2) were passaged on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C under identical laboratory conditions. There were significant differences between isolates in the diameter of trophonts and tomonts, the incubation period of tomonts, and the length of theronts. Trophonts of C1 were significantly larger on L. calcarifer than on M. novemaculeata and showed marked size variation with temperature, whereas trophonts of C2 developed equally well on both species and showed little size variation with temperature. Tomonts of C1 were significantly larger than those of C2 when grown on L. calcarifer, whereas on M. novemaculeata tomonts from C1 were significantly smaller than C2 tomonts. The incubation period of tomonts from C1 was significantly shorter than that for tomonts of C2, and theronts of C1 were significantly larger than theronts of C2 under all host/temperature conditions. The differences in the development of these isolates are of biological and epidemiological importance. This indicates that distinct intraspecific variants of C. irritans occur along the coast of southeast Queensland.

Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Fishes

In a previous paper (H.J. Nelis, P. Léger, P. Sorgeloos, and A. P. De Leenheer, Antimicrob. Agents Chemother. 35:2486-2489, 1991) it was reported that two selected antibacterial agents, i.e., trimethoprim and sulfamethoxazole, can be efficiently bioencapsulated in nauplii of the brine shrimp Artemia franciscana for administration to fish. This follow-up study showed that larvae of the sea bass and the turbot as well as postlarvae of the white shrimp accumulate the therapeutic agents in high quantities when fed medicated A. franciscana. To monitor their levels as a function of time, the liquid chromatographic method originally developed for the analysis of A. franciscana was modified with respect to chromatography, internal standardization, and sample pretreatment. The levels of trimethoprim ranged from 1 to 7 micrograms/g (sea bass), 1 to 13 micrograms/g (turbot), and 4 to 38 micrograms/g (white shrimp). The corresponding values for sulfamethoxazole were 0.3 to 4 micrograms/g (sea bass), 1 to 42 micrograms/g (turbot), and 4 to 35 micrograms/g (white shrimp). Only the two fish species, unlike the shrimp, metabolized the latter to N-acetylsulfamethoxazole (concentration range, 1 to 10 micrograms/g). These data suggest the potential of the bioencapsulation of therapeutic agents in live food as a tool to control infectious diseases in aquaculture. A preliminary challenge test also confirmed the in vivo efficacy of this approach.

Topics: Animals; Anti-Infective Agents; Artemia; Bass; Chromatography, High Pressure Liquid; Chromatography, Liquid; Decapoda; Fish Diseases; Flatfishes; Reproducibility of Results; Sulfamethoxazole; Trimethoprim; Vibrio Infections

A virus causing a vacuolating encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax, was isolated from brain tissue in a fish cell line (SSN-1) derived from striped snakehead, Channa striatus. The isometric, non-enveloped, 30 nm diameter virus particles were resistant to pH 2-9 and heating at 56 degrees C for 30 min. Infectious particles had a buoyant density of approximately 1.31 g/cm3 in CsCl. Two structural polypeptides of molecular mass 40 and 42 kDa were identified and the ssRNA consisted of two fragments of molecular mass 1.10 and 0.51 x 10(6) Da. From these characteristics the virus was identified as a nodavirus. Due to the broad range of susceptible fish hosts and the consistent neuropathology of the disease condition, the generic term piscine neuropathy nodavirus (PNN) is proposed for this infectious agent.

Topics: Animals; Bass; Brain Diseases; Cell Line; Fish Diseases; RNA Viruses; RNA, Viral; Viral Structural Proteins; Virus Diseases

The degree to which host suitability is a reflection of host community structure in generalist parasites was studied experimentally in the common fish acanthocephalan Leptorhynchoides thecatus. Previous study has shown that green sunfish (Lepomis cyanellus) are required, and largemouth bass (Micropterus salmoides) are suitable (but not required) hosts, where they occur sympatrically in natural communities. The present study examined populations of L. cyanellus and M. salmoides held separately in mesocosms and exposed to L. thecatus cystacanths via laboratory-infected Hyalella azteca (Amphipoda). Recruitment, maturation, and transmission of worms were examined over a 17-wk period and compared between fish species. Infections with L. thecatus were found as early as 2 wk after the introduction of cystacanth-infected amphipods, and by week 11 fishes of both species harbored gravid worms. Immature worms were observed in both host species by week 17 and were presumed to be a result of natural egg production and release resulting in infections of amphipods and the subsequent reinfection of fish. No significant difference in the prevalence, abundance, percentage of worms gravid, or time of parasite maturation was found between host populations. Results indicate that the different roles played by these host species in the maintenance of L. thecatus supra-populations in natural systems are not due to intrinsic factors but rather to differences in host autecology and community structure.

Topics: Acanthocephala; Animals; Bass; Female; Fish Diseases; Helminthiasis; Helminthiasis, Animal; Male; Nebraska; Perciformes; Prevalence; Species Specificity