muramidase and Fish-Diseases

In recent years, the deterioration of the aquaculture ecological environment has led to a high incidence of fish diseases. Lysozymes, important antimicrobial enzymes, play an important role in the innate immune system of fish. The studies of fish lysozymes benefit the control of fish infections caused by pathogens. In this review, we reviewed recent progress in fish lysozymes, including their classification, structural characteristics, biological functions and mechanisms, tissue distributions, and properties of their recombinant proteins, which will help us to systematically understand the fish lysozymes and facilitate their applications in the fields of food and agriculture.

Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents; Fish Diseases; Fishes; Muramidase

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (10

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Vaccines; Cytokines; Edwardsiella tarda; Fish Diseases; Flounder; Gene Expression Profiling; Gene Expression Regulation; HSP72 Heat-Shock Proteins; Immunization; Immunoglobulin M; Inflammation Mediators; Muramidase; Vaccines, Attenuated

Maternal immunity is of paramount importance for protection of young ones at early stage of life since the immune factors of an immunocompetent female are transferred transplacentally or through colostrum, milk or yolk to an immunologically naive neonate. Both innate and adaptive type of immunity are transferred of from mother to offspring in fishes. These factors include immunoglobulin (Ig)/antibody, complement factors, lysozymes, protease inhibitors like alpha macroglobulin, different types of lectins and serine proteases like molecules. Among different types of Ig viz. IgM, IgD, IgT/IgZ and IgM-IgZ chimera types, IgM is present in most of the teleostean fishes. In teleosts, IgM either as a reduced/breakdown product or monomeric form is usually transferred to the offsprings. The maternally derived IgM usually persists for a limited duration, exhausts within the completion of yolk absorption process, and completely disappears thereafter during larval stages. Maternal transfer of immunity which provides defense to embryo and larvae depends upon the health as well as the immune status of brood fish. The overall health status of brood fish can affect breeding performances, quality seed production and protection of offsprings. However, factors such as age, maturation, reproductive behaviour and nutrition (micro and macro-nutrients) may affect the immunity in brood fishes. Besides these, seasonal changes such as photoperiods, temperature, adverse environmental conditions, and stress conditions like handling, crowding, and water pollution/contamination can also affect the immunity of brood fishes. The maintenance of the brood stock immunity at high level during vitellogenesis and oogenesis, is utmost important for reducing mortalities at larval/post larval stages through maximum/optimum transfer of maternal immunity. Brood stock immunization prior to breeding as well as selective breeding among the disease resistant families might be the ideal criteria for producing quality seed.

Topics: Animal Nutritional Physiological Phenomena; Animals; Complement System Proteins; Environment; Fish Diseases; Fishes; Immunity; Immunoglobulins; Lectins; Muramidase; Reproduction

With the progression of aquaculture industry, there has been a spurt in dietary supplementation with economically viable medicinal herbs having enough immunostimulatory potential. This also aids in avoidance of environmentally undesirable therapeutics that are almost inevitable to safeguard fish against an array of diseases in aquaculture practices. The study aims to determine the optimal dose of herbs that can stimulate substantial immune response in fish for reclamation of aquaculture. Immunostimulatory potential of the two medicinal herbs- Asparagus racemosus (Shatavari), Withania somnifera (Ashwagandha), individually, and in combination, with a basal diet was screened up to 60 days in Channa punctatus. 300 laboratory acclimatized healthy fish (14 ± 1 g; 11 ± 1 cm) were divided into ten groups- C, S1, S2, S3, A1, A2, A3, AS1, AS2, and AS3, based on the composition of dietary supplementation, in triplicates, with 10 specimens per group. The hematological index, total protein and lysozyme enzyme activity were performed after 30 and 60 days, while qRT-PCR analysis of lysozyme expression was done after 60 days of the feeding trial. The significant (P < 0.05) increments in hematological indices- (TEC, TLC, DLC, Hb, Hct, MCV, MCH and MCHC), total protein content and serum lysozyme activity, after 30 and 60 days; whereas upregulation of lysozyme transcript levels, both in liver and muscle tissues after 60 days of the feeding trial were recorded in groups- AS1, AS2, and AS3. The maximal increment in lysozyme expression was recorded in AS3, both in liver and muscle tissues, with 3.75 ± 0.13 and 3.21 ± 0.18-folds, respectively. However, increments were non-significant (P > 0.05) for MCV in AS2 and AS3 after 30 days; and for MCHC in AS1 for both the durations; whereas in AS2 and AS3, after 60 days of the feeding trial. A positive correlation (P < 0.05) among lysozyme expression, MCH, lymphocytes, neutrophils, total protein content, and serum lysozyme activity in AS3, after 60 days, conclusively, evinces that a 3% dietary supplementation with both A. racemosus and W. somnifera enhances immunity and health profile of the fish, C. punctatus. The study, thus finds ample scope in augmentation of aquaculture production and also paves the way for more researches for biological screenings of potential immunostimulatory medicinal herbs that can be appropriately incorporated in the fish diet.

Topics: Animal Feed; Animals; Diet; Dietary Supplements; Fish Diseases; Fishes; Muramidase; Withania

This study evaluated the effects of microencapsulation of L. plantarum (as a probiotic) with chitosan/alginate biopolymers (MLCA) on innate immune response, disease resistance, and growth performance of Nile tilapia (Oreochromis niloticus). Four hundred and eighty fish were randomly distributed in glass tanks (150 L) and fed with diets including diet 1: control; diet 2: 10 g kg

Topics: Alginates; Animal Feed; Animals; Capsules; Chitosan; Cichlids; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Immunity, Innate; Lactobacillus plantarum; Muramidase

Topics: Aeromonas caviae; Animals; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunoglobulin M; Interleukin-10; Mammals; Muramidase; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

Several studies have looked into the use of basil, Ocimum basilicum (L.) in aquaculture as a dietary additive; however, more research is needed to see the possibility of it's including in nanocarriers in aquafeeds. An experiment was undertaken to highlight the efficacy chitosan-Ocimum basilicum nanocomposite (COBN), for the first time, on Nile tilapia (Oreochromis niloticus) growth, stress and antioxidant status, immune-related parameters, and gene expression. For 60 days, fish (average weight: 23.55 ± 0.08 g) were fed diets provided with different concentrations of COBN (g/kg): 0 g [COBN0], 1 g [COBN1], 2 g [COBN2], and 3 g [COBN3], where COBN0 was kept as control diet. Following the trial, the fish were challenged with pathogenic bacteria (Aeromonas sobria) and yeast (Candida albicans) infection. In comparison to the control (COBN0), a notable increase in growth parameters (weight gain, feed intake, and specific growth rate) and intestinal morphometric indices (average intestinal goblet cells count, villous width, and length) in all COBN groups was observed, where COBN2 and COBN3 groups had the highest values. The COBN diets significantly (p < 0.05) declined levels of serum triglycerides, glucose, cholesterol, and hepatic malondialdehyde. Moreover, the higher levels of serum biochemical biomarkers (growth hormone, total protein, globulin, and albumin), immunological parameters (phagocytic activity%, nitric oxide, and lysozyme), and hepatic antioxidant parameters (superoxide dismutase, total antioxidant capacity, and glutathione peroxidase) were obvious in the COBN2 and COBN3 groups followed by COBN1. The immune-antioxidant genes (TNF-α, IL-10, IL-1β, TGF-β, GPx, and SOD) were found to be considerably up-regulated in all COBN groups (COBN2 and COBN3 followed by COBN1). Fifteen days post-challenge with A. sobria and C. albicans, the highest survival rate was recorded in the COBN2 group (83.33 and 91.67%) followed by the COBN3 group (75 and 83.33%), respectively. The findings showed that a dietary intervention with COBN can promote growth, intestinal architecture, immunity, and antioxidant markers as well as protect O. niloticus against A. sobria and C. albicans infection. As a result, the COBN at a dose of 2 g/kg could be used as a food additive for the sustainable aquaculture industry.

Topics: Albumins; Animal Feed; Animals; Antioxidants; Chitosan; Cichlids; Diet; Dietary Supplements; Fish Diseases; Food Additives; Gene Expression; Glucose; Glutathione Peroxidase; Gram-Negative Bacterial Infections; Growth Hormone; Head Kidney; Interleukin-10; Malondialdehyde; Muramidase; Nanocomposites; Nitric Oxide; Ocimum basilicum; Superoxide Dismutase; Transforming Growth Factor beta; Triglycerides; Tumor Necrosis Factor-alpha

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRβ, IL-1β, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Catalase; Cichlids; Fish Diseases; Fish Proteins; Liver; Mannitol; Muramidase; Peroxidase; Spleen; Streptococcal Infections; Streptococcal Vaccines; Streptococcus agalactiae

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg

Topics: Aeromonas hydrophila; Amylases; Animals; Blood Cell Count; Carps; Cistus; Cytokines; Ethanol; Fish Diseases; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Immunologic Factors; Lipase; Muramidase; Plant Extracts; Plant Leaves; Solvents; Superoxides; Trypsin

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg

Topics: Animals; Cyprinidae; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gene Expression Regulation; Glutathione; Glutathione Peroxidase; Head Kidney; Immunoglobulin M; Immunologic Factors; Malondialdehyde; Muramidase; Polysaccharides; Superoxide Dismutase; Weight Gain

The association of vaccines with immunostimulants such as β-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between β-glucan types and trials make it difficult to understand β-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed β-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated β-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1β, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + β-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + β-glucan fish. The results suggest that β-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; beta-Glucans; Cichlids; Cytokines; Fish Diseases; Fish Proteins; Gene Expression; Head Kidney; HSP70 Heat-Shock Proteins; Muramidase; Signal Transduction; Spleen; Streptococcal Infections; Streptococcal Vaccines; Streptococcus agalactiae

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Topics: Animals; Citrus; Dietary Supplements; Disease Susceptibility; Fermentation; Fish Diseases; Fruit; Gram-Negative Bacterial Infections; Liver; Malondialdehyde; Muramidase; Perciformes; Photobacterium

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 μl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1β gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.

Topics: Adjuvants, Immunologic; Administration, Oral; Aeromonas hydrophila; Animals; Complex Mixtures; Cytokines; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Oncorhynchus mykiss; Peroxidase; Sargassum

The effect of diet enriched with dried lemon (Citrus limon) peel was fed to Labeo rohita at three different levels (0, 1, 2.5, and 5 g kg

Topics: Aeromonas; Animals; Antioxidants; Catalase; Citrus; Complement C3; Cyprinidae; Cytokines; Diet; Fish Diseases; Fish Proteins; Fruit; Glutathione; Glutathione Peroxidase; Gram-Negative Bacterial Infections; Head Kidney; Immunoglobulin M; Muramidase; Superoxide Dismutase

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

The efficacy of immersion vaccination Yersinia ruckeri bacterin containing Montanide™ IMS 1312 VG was evaluated in 100-120 g rainbow trout against yersiniosis. Healthy fish were vaccinated by immersion vaccination with inactivated whole cells (1 × 10(8) cells/ml) of a virulent strain of Y. ruckeri biotype I with and without Montanide (1:1; Montanide/antigen) for 2 min at 12-14 °C. Control group was immersed in sterile PBS. Leukocyte counts, serum lysozyme assay, alternative hemolytic complement (ACH50) assay, antibody titration and relative percent survival (RPS) were measured on 2-10 weeks post-immunization. No significant difference was seen in leucocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P > 0.05), while leucocyte and heterophil populations in control group were significantly lower and higher, respectively, than both immunized groups (P < 0.05). In addition, there was no significant difference in lymphocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P > 0.05), while lymphocyte population in control group was significantly lower than both immunized groups (P < 0.05). Lysozyme activity in immunized fish with Y. ruckeri containing Montanide was higher than the immunized fish with Y. ruckeri antigen alone during 8 weeks post-immunization ((P < 0.05). Also, level of lysozyme in control fish was generally lower than both immunized groups (P < 0.05). The level of ACH50 between both immunized groups was insignificant (P > 0.05) but these were significantly higher than control group through the experiment (P < 0.05). The lowest anti-Y. ruckeri antibody titers in both immersion vaccination groups were significantly higher through 2-8 weeks post-vaccination compared to the control group (P < 0.05). In the group immersion vaccinated with Y. ruckeri bacterin plus Montanide the titers 2-8 weeks post-vaccination were significantly higher the titer in the immersion vaccinated with Y. ruckeri bacterin (P < 0.05). Fish vaccinated with antigen without Montanide resulted in RPS of 80-82% on 2-10 weeks post-vaccination, while those for antigen containing montanide gave RPSs of 93.8-100% 2-10 weeks post-immunization (P < 0.05).

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Antibodies, Bacterial; Bacterial Vaccines; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Leukocyte Count; Muramidase; Oncorhynchus mykiss; Yersinia Infections; Yersinia ruckeri

The effects of dietary Bacillus coagulans (MTCC 9872), Bacillus licheniformis (MTCC 6824) and Paenibacillus polymyxa (MTCC 122) supplementation on growth performance, non-specific immunity and protection against Aeromonas hydrophila infection were evaluated in common carp, Cyprinus carpio fry. Laboratory maintained B. coagulans, B. licheniformis and P. polymyxa were used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy fish fry were challenged by this bacterium for determination of its safety. Fish were fed for 80 days with control basal diet (B0) and experimental diets containing B. coagulans (B1), B. licheniformis (B2) and P. polymyxa (B3) at 10(9) CFU/g diet. Fish fry (mean weight 0.329 ± 0.01 g) were fed these diets and growth performance, various non-specific immune parameters and disease resistance study were conducted at 80 days post-feeding. The antagonism study showed inhibition zone against A. hydrophila and Vibrio harveyi. All the probiotic bacterial strains were harmless to fish fry as neither mortality nor morbidities were observed of the challenge. The growth-promoting influences of probiotic supplemented dietary treatments were observed with fish fry and the optimum survival, growth and feed utilization were obtained with P. polymyxa (B3) supplemented diet. Study of different non-specific innate immunological parameters viz. lysozyme activity, respiratory burst assay and myeloperoxidase content showed significant (p < 0.05) higher values in fish fry fed B3 diet at 10(9) CFU/g. The challenge test showed dietary supplementation of B. coagulans, B. licheniformis and P. polymyxa significantly (p < 0.05) enhanced the resistance of fish fry against bacterial challenge. These results collectively suggests that P. polymyxa is a potential probiotic species and can be used in aquaculture to improve growth, feed utilization, non-specific immune responses and disease resistance of fry common carp, C. carpio.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Aquaculture; Bacillus; Carps; Dietary Supplements; Disease Resistance; Disk Diffusion Antimicrobial Tests; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Peroxidase; Probiotics; Respiratory Burst

A feeding study was conducted to determine the effect of dietary nucleotides (NT) on growth performance, immune responses and resistance of rainbow trout fingerlings to Streptococcus iniae. A basal diet supplemented with 0 (control), 0.5, 1, 1.5 and 2 g NT kg(-1) to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 23 g. After 8 weeks of feeding trial, fish fed diets with 1.5-2 g NT kg(-1) had the highest final weight and SGR, followed by groups fed the diets with 0.5-1 g NT kg(-1), and lowest in fish fed the NT-free control diet. The feed conversion ratio (FCR) was better when the fish were fed diet with 2 g NT kg(-1). The ACH(50) level and lysozyme activity in serum were found to be significantly (P < 0.05) greater in fish fed nucleotides at 1.5-2 g kg(-1). The fish fed with 2 g NT kg(-1) had higher IgM level. The challenge experiment showed that relative percent survival (RPS) increased significantly (P < 0.05) with increasing levels of nucleotides from 0.5 to 2 g kg(-1) and was lowest in the control group. We therefore recommend dietary nucleotide administration at 1.5-2 g kg(-1) to promote growth and enhance immunity and resistance against S. iniae.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Muramidase; Nucleotides; Oncorhynchus mykiss; Streptococcal Infections

A 60-day feeding trial was conducted to study the immuno-protective effect of microbial levan on Labeo rohita juveniles challenged with Aeromonas hydrophila. Six purified diets were prepared with different levels of microbial levan: control (no levan), T1 (Basal + 0.25%), T2 (Basal + 0.50%), T3 (Basal + 0.75%), T4 (Basal + 1%) and T5 (Basal + 1.25%), fed to six groups of fish in triplicate. Among the treatment groups the haemoglobin content and total leucocyte count were increased with a dietary supplementation of levan at 1% or more. An increasing trend for total erythrocyte count was observed with increasing level of dietary levan. Lower levan-supplemented groups showed a higher albumin/globulin ratio. As the levan supplementation was increased, there was a gradual increase in serum lysozyme activity and respiratory burst activity [nitroblue tetrazolium (NBT) assay] reduction values. The highest lysozyme activity and NBT were observed in the T5 group although this was similar to the T4 group (P > 0.05). No significant histo-architectural changes were associated with dietary levan levels. After challenge with A. hydrophila, moderately degenerated hepatocytes, oedema and leucocytic infiltration in parenchymatous tissues, and extensive haemorrhage and haemosiderosis in the kidney were observed in the control group. However, the T5 group supplemented with 1.25% levan showed infiltrating leucocytes in the liver while the kidney showed only moderate degeneration of renal tubules. The relative survival per cent of juveniles after challenge with A. hydrophila was the highest in the T5 group followed by T4. This suggests that microbial levan at 1.25% can be used as dietary immunostimulant for L. rohita juveniles.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Diet; Fish Diseases; Fructans; Gram-Negative Bacterial Infections; Hepatocytes; Immunity, Innate; Kidney; Liver; Muramidase; Probiotics; Respiratory Burst; Survival Analysis

A preliminary study with 60 days feeding was conducted to study the immunomodulatory role of different immunostimulants like beta-carotene, omega-3 fatty acid and yeast-RNA in Catla catla fingerlings. Two hundred and sixty four fingerlings were randomly distributed into eight treatment groups with each of three replicates. Eight isonitrogenous (crude protein 34.12-35.40%) and isocaloric (458.41-461.48 kcal/100g) purified diets were prepared with graded level of beta-carotene, omega-3 fatty acid and yeast-RNA viz., Control (basal diet), T1 (Basal + 1% omega-3 fatty acid), T2 (Basal + 3% omega-3 fatty acid), T3 (Basal + beta-carotene), T4 (T1 + beta-carotene), T5 (T2 + beta-carotene), T6 (Basal + 0.4% yeast-RNA) and T7 (Basal + 0.8% yeast-RNA). The immunomodulatory effects of dietary immunostimulants were studied in terms of respiratory burst activity (NBT) of blood phagocytes, total leukocyte count, serum total protein, serum globulin, A/G ratio (A/G) and serum lysozyme activity. The respiratory burst activity of T7 group was significantly higher (p<0.05) than the other groups. Haemoglobin content, total erythrocyte count and serum albumin content did not vary among the treatment groups, whereas total leukocyte count, serum globulin content and serum lysozyme activity were found to be highest in T7 group. Relative survival percent after challenge with Aeromonas hydrophila was also highest in T7 (88.88%) group followed by T6 (75.06%) and T4 (66.66%) and the lowest in T2 group. It was observed that total leucocyte count, NBT and lysozyme activity of T2 group fed with high omega-3 fatty acid (3%) was less than (p<0.05) its lower counterparts T1 (1%) and control group. Based on the results of the present study, it concludes that supplementation of yeast-RNA at 0.8% registered higher immunological responses in C. catla juveniles. It is also observed that higher supplementation of omega-3 fatty acid (3%) in the diet causes immunosuppression in C. catla juveniles.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta Carotene; Blood Proteins; Cyprinidae; Dietary Supplements; Eating; Erythrocyte Count; Fatty Acids, Omega-3; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Leukocyte Count; Muramidase; Random Allocation; Respiratory Burst; RNA, Fungal

The effects of including bovine lactoferrin (Lf) in the diet of the Asian catfish (Clarias batrachus) on specific and non-specific immunity as well as disease resistance were investigated. The catfish were fed four different diets for 2 weeks: a commercial diet as control and the same diet supplemented with 50, 100 and 200mg bovine Lf/kg feed. After 1 and 2 weeks, serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity; natural serum haemolysin titre, lysozyme activity and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to vaccinated and non-vaccinated animals were evaluated. The results showed that Lf supplements, particularly at 100mg level, significantly (P<0.05) enhanced serum lysozyme level, oxidative radical production and level of protection against A. hydrophila challenge in non-vaccinated animals irrespective of length of exposure. The specific immunity was not influenced by Lf feeding as evidenced from the bacterial agglutination titre and level of protection in vaccinated animals. As Lf feeding at 100mg/kg for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, these results support the possible use of Lf as an immunostimulant for farmed catfish.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Antibodies; Bacterial Vaccines; Catfishes; Cattle; Diet; Disease Susceptibility; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Hemolysin Proteins; Lactoferrin; Muramidase

Yellow drum (Nibea albiflora), a commercially important fish species in the coastal regions of southeast China, is highly susceptible to red-head disease caused by Vibrio harveyi B0003. Probiotics have been shown to enhance disease resistance in fish, but whether commensal probiotics could improve of the resistance to red-head disease in yellow drum and possible mechanisms has yet not been reported. A six-week feeding trial was conducted to investigate the red-head disease resistance potentials of five probiotic candidates (Bacillus megaterium B1M2, B. subtilis B0E9, Enterococcus faecalis AT5, B. velezensis DM5 and B. siamensis B0E14), and the liver health, serum and skin immunities, gut and skin mucosal microbiota of yellow drum were determined to illustrate the possible mechanisms. The results showed that autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5, P < 0.05) effectively improved red-head disease resistance in yellow drum. Furthermore, B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) efficiently improve liver health by improving liver morphology and decreasing serum glutamic oxaloacetic transaminase and glutamic propylic transaminase activities pre and post challenged with V. harveyi B0003 (P < 0.05). B. subtilis B0E9 and E. faecalis AT5 led to significant improvement (P < 0.05) in the serum complement 3 content (un-detected after challenged with V. harveyi B0003), lysozyme activity and skin mucosal immunity (such as IL-6, IL-10 and lysozyme expression) pre and post challenged with V. harveyi B0003, which was generally consistent with the cumulative mortality after challenged with V. harveyi B0003. This induced activations of serum and skin mucosal immunities were consistent with the microbiota data showing that B. subtilis B0E9 and E. faecalis AT5 modulated the overall structure of intestinal and skin mucosal microbiota, and in particular, the relative abundance of potentially pathogenic Achromobacter decreased while beneficial Streptococcus, Rothia, and Lactobacillus increased in fish fed with B. subtilis B0E9 and E. faecalis AT5. Overall, autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) can improve liver health, serum and skin immunities (especially up-regulated lysozyme activity and inflammation-related genes expression), positively shape gut and skin mucosal microbiota, and enhance red-head disease resistance of yellow drum.

Topics: Animals; Bacillus subtilis; Disease Resistance; Enterococcus faecalis; Fish Diseases; Fishes; Immunity, Mucosal; Liver; Microbiota; Muramidase; Perciformes; Probiotics

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.

Topics: Animals; Antibodies, Bacterial; Fish Diseases; Gram-Positive Bacterial Infections; Immunoglobulin M; Lactococcus; Muramidase; Oncorhynchus mykiss; Peroxidases

Chlorella is one of the most widely accepted Chlorophyta used by many as livestock and aquaculture feed. Nonetheless, different studies on the overall performances of fish reported the unfavourable effect of high-level supplementations of Chlorella vulgaris. The current study determined the impact of low-level dietary supplementation of C. vulgaris alongside the different feeding durations and their interactions on the growth hormone (GH), growth performances, serum-biochemical indices, hepatic function and some immunological parameters of red hybrid tilapia. The fingerlings (mean weight: 14.25 ± 0.01 g, length: 13.5 ± 0.49 cm) were fed diets containing 0, 0.99%, 2.91% and 4.76% of C. vulgaris powder per kilogram dry diet for 90 days. GH, growth performance, serum-biochemical indices (total serum protein, albumin, globulin, glucose, aspartate aminotransferase and alanine aminotransferase) and some immunological (respiratory burst and lysozyme activities) parameters of the fish were examined after 30, 60 and 90 days of feeding. The results demonstrated that tilapia fed C. vulgaris-supplemented diets showed increased levels of respiratory burst, lysozyme, albumin and total protein, GH and growth performances (P < 0.05), and the effects were duration dependent. After the 90 days of feeding, there was no adverse effect on the hepatic function of the fish. Besides, low survivability was observed in the control group than in the group fed the experimental diets. The group fed the diet supplemented with 4.76% C. vulgaris had significantly higher (P < 0.05) lysozyme activity throughout the duration of the feeding experiment. These results indicate that C. vulgaris enhanced growth performances, GH concentration, serum-biochemistry and some immunological parameters of red hybrid tilapia.

Topics: Animal Feed; Animals; Chlorella vulgaris; Cichlids; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Growth Hormone; Muramidase; Tilapia

Common carp (Cyprinus carpio), a valuable aquaculture species susceptible to various infections, requires effective immune enhancement strategies. This study investigates the immunomodulatory effects of orally administered terpenoids and phenol fraction (TPF) from Padina gymnospora in C. carpio, focusing on stimulation of nonspecific immune response, immune gene expression, and protection against experimental infection. P. gymnospora is a brown seaweed species known for its bioactive compounds and medicinal properties. TPF was extracted using the Harborne fractionation method, and the presence of terpenoids and phenol compounds was confirmed by qualitative analysis and high-performance thin layer chromatography (HPTLC). TPF was administered orally in different doses to carp. Nonspecific immune responses were evaluated by measuring cellular ROS, RNI, and peroxidase production. The expression of immune genes (lysozyme and interleukin-1β) was assessed by reverse transcriptase PCR. Furthermore, the protective efficacy of TPF was determined by infecting carp with a virulent pathogen, Aeromonas hydrophila, and monitoring mortality rates and disease symptoms. The results demonstrate that oral TPF administration significantly enhances nonspecific immune responses, with increased ROS, RNI, and peroxidase production, indicating improved immune function. Expression levels of lysozyme and interleukin-1β were upregulated, suggesting immune system activation. Moreover, TPF exhibited significant protection against experimental infection, with lower mortality rates compared to the control group. These findings highlight TPF's potential as an effective immunostimulatory agent, enhancing immune responses and providing infection protection in carp. In conclusion, oral TPF administration stimulates nonspecific immune responses, modulates immune gene expression, and confers protection against experimental infection in carp, displaying its potential for enhancing immune responses and disease resistance in aquaculture species, and contributing to sustainable fish health management.

Topics: Administration, Oral; Aeromonas hydrophila; Animals; Carps; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Muramidase; Peroxidases; Phenol; Reactive Oxygen Species; Terpenes

This study evaluates the effects of longan seed powder (LS) on the growth performance, immunological response, and immune-antioxidant related gene expression of Nile tilapia (Oreochromis niloticus). Three hundred fish (13.82 ± 0.06 g) were divided into five experiments and fed 5 diets, including the basal diet (control without LS) and basal diet containing 10 (LS10), 20 (LS20), 40 (LS40), and 80 (LS80) g kg

Topics: Animal Feed; Animals; Antioxidants; Aquaculture; Cichlids; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression; Muramidase; Peroxidase; Powders; Sapindaceae; Seeds; Streptococcal Infections

This study aimed to evaluate the effects of rambutan peel powder (RP) on growth, skin mucosal and serum immunities, and immune-related gene expression of striped catfish (Pangasianodon hypophthalmus) reared in a biofloc system. Three hundred fingerlings (17.14 ± 0.12 g fish

Topics: Animal Feed; Animals; Aquaculture; Catfishes; Diet; Dietary Supplements; Fish Diseases; Gene Expression; Immunity; Muramidase; Peroxidases; Powders; Sapindaceae

Aquatic food is becoming an important food source that provides micronutrients to human beings. The decline of wild aquatic animals makes aquaculture become increasingly important to play this role. However, infectious diseases, especially bacterial infection, represent severe threat to aquaculture, which causes huge economic loss. Meanwhile, strategies in managing bacterial infection in an antibiotic-independent way are still lacking. In this study, we monitor the metabolomic shift of crucian carp upon

Topics: Aeromonas hydrophila; Animals; Carps; Fish Diseases; Fructose; Gram-Negative Bacterial Infections; Muramidase

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Topics: Animals; Aquaculture; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Perciformes; Photobacterium

Aeromonas veronii AvX005 is a pathogenic bacterium with high toxicity to grass carp (Ctenopharyngodon idellus). The expression levels of g-type (goose-type lysozyme, Lys-g) and c-type lysozyme (chicken-type lysozyme, Lys-c) in the spleen of grass carp infected with AvX005 were significantly increased by approximately 4.5 times and 27 times, respectively. The recombinant proteins rLys-g and rLys-c produced in a recombinant expression system of Escherichia coli showed significant antibacterial activity against the pathogenic bacteria AvX005. A challenge test was conducted after rLys-g and rLys-c were expressed in grass carp L8824 liver cells, and compared with the survival rate of the control cells (46.3%), the survival rate of the experimental cells (77.6% for rLys-g and 68.6% for rLys-c) was significantly increased. Grass carp were infected with AvX005 on the second day after delivering pcDNA3.1-lys-g and pcDNA-lys-c with the Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant, and both pcDNA3.1-lys-g and pcDNA-lys-c provided 70% relative protection for grass carp. The activity of lysozyme and alkaline phosphatase in the serum of grass carp was significantly increased after injection of DNA. The expression of the immune factors IgM, C3 and IL8 in the kidney was upregulated to varying degrees for pcDNA3.1-lys-g and immune factors C3 and IgM was upregulated for pcDNA-lys-c. The results indicated that pcDNA3.1-lys-g and pcDNA-lys-c may be used as immunostimulants to protect grass carp from the pathogenic bacterium AvX005.

Topics: Acrylic Resins; Adjuvants, Immunologic; Aeromonas hydrophila; Aeromonas veronii; Animals; Carps; Cholesterol; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulin M; Muramidase; Quillaja Saponins

Sustainable methods that increase farmed fish yield while controlling infections are required to prevent economic losses in aquaculture farms. In this study, we evaluated the effects of betaine-supplemented (0%, 0.1%, 0.5%, and 1.0%) feed on the growth and immunity of the olive flounder Paralichthys olivaceus. Feed conversion ratios, post-infection cumulative mortality rates and innate immune responses were monitored. Weight gain was significantly higher with 0.5% and 1.0% than with 0% and 0.1% betaine-supplemented feed. Lysozyme activity was highest with 1.0% betaine. Respiratory burst activity was highest with 0.5% and 1.0% betaine. Serum bactericidal activity against Edwardsiella tarda was highest with 1.0% betaine (40% increase in survival rates compared with those in the control). Furthermore, serum virucidal activity against the viral haemorrhagic septicaemia virus (VHSV) was higher with 1.0% betaine than with other concentrations. With 0.5% and 1.0% betaine, the survival rates against VHSV were higher than those in the control until day 11, after which they declined. Our study suggests that betaine is a promising agent for promoting the growth of and enhancing immunity against E. tarda in olive flounders. Our findings may further contribute to developing necessary alternatives to conventional antibiotics in fish farming.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; Betaine; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Flounder; Immunity, Innate; Muramidase

Natural products have gained considerable attention for improving fish growth performance and immunity to enhance disease resistance. This study evaluated the effect of dandelion polysaccharides (DP) on skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge in the Common carp Cyprinus carpio. Diets containing four different concentrations of DP (g Kg

Topics: Alkaline Phosphatase; Animal Feed; Animals; Antioxidants; Biological Products; Carps; Catalase; Claudin-3; Cytokines; Diet; Dietary Supplements; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Interleukin-10; Malondialdehyde; Muramidase; Occludin; Polysaccharides; Superoxide Dismutase; Taraxacum; Tumor Necrosis Factor-alpha

Individual and combined efficacy of chitooligosaccharides (COS) and alginic acid (AA) at 1 g, 2 g, and 3 g per kg diet was assessed on growth and disease resistance in silver carp (Hypophthalmichthys molitrix) against Edwardsiella ictaluri. Growth parameters including specific growth rate (SGR), weight gain (WG), and feed conversion rate (FCR) were significant in fish fed 2 g and 3 g kg

Topics: Abortifacient Agents; Alginic Acid; Amylases; Animal Feed; Animals; Antioxidants; Carps; Chitosan; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Glutathione Peroxidase; Interleukin-10; Interleukin-8; Lipase; Malondialdehyde; Muramidase; Nitric Acid; Oligosaccharides; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of Bacillus subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of Aeromonas veronii were tested in vitro, and the results showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 were evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. One of its effect mechanisms is the combination could inhibit the growth of A. veronii in vitro; the other is direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the simple combination group could significantly reduce the expression of nuclear factor kappa-B (NF-κB) and proinflammatory cytokine interleukin-1β (IL-1β), increase the expression of lysozyme gene; and the third is intestinal microbiota also plays a regulatory role: the gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of transforming growth factor-β (TGF-β) and lysozyme. Given the effectiveness of this simple combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

Topics: Aeromonas veronii; Alkaline Phosphatase; Animals; Bacillus subtilis; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Muramidase; NF-kappa B; Transforming Growth Factor beta; Transforming Growth Factors; Zebrafish

This study aimed to assess the effects of dietary addition with Chlorella sorokiniana on fish growth, gut histology, antioxidant capacity, immune response, and disease resistance in rainbow trout. Three diets with similar proximate composition and different Chlorella meal levels were formulated. The control diet, 5% Chlorella diet, and 10% Chlorella diet contained 0%, 5% Chlorella meal, and 10% Chlorella meal, respectively. Each diet was assigned to triplicate tanks containing 30 fish (165.3 ± 0.6 g) in each tank. Fish were fed experimental diets for ninety days. The results showed that the addition of 5% Chlorella in the diet significantly increased feed intake by 19.3% and weight gain rate by 17.3% (P < 0.05) without affecting feed efficiency and gut histology. Diets containing Chlorella meal significantly decreased malonaldehyde contents in the plasma after the lipopolysaccharide (LPS) challenge (P < 0.05). Dietary supplementation with Chlorella meal significantly increased lysozyme (LZM) activity levels (in the head kidney) and immunoglobulin M (IgM) (in the head kidney) and complement component 3 (C3) (in the spleen) contents before the LPS challenge, and simultaneously increased LZM activity levels (in the plasma) and C3 contents (in the plasma and head kidney) after the LPS challenge (P < 0.05). Furthermore, dietary administration of Chlorella meal significantly increased the survival rate of fish infected with Aeromonas salmonicida (P < 0.05). In conclusion, C. sorokiniana can be used to improve fish growth, antioxidant capacity, and immunity.

Topics: Aeromonas salmonicida; Animal Feed; Animals; Antioxidants; Chlorella; Complement C3; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Immunoglobulin M; Lipopolysaccharides; Malondialdehyde; Muramidase; Oncorhynchus mykiss; Oxidative Stress

Rainbow trout (

Topics: Acid Phosphatase; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Catalase; Fish Diseases; Infectious hematopoietic necrosis virus; Interferon-Induced Helicase, IFIH1; Interleukin-8; Liver; Malondialdehyde; MicroRNAs; Muramidase; Myeloid Differentiation Factor 88; NLR Proteins; Oncorhynchus mykiss; Receptors, Cytokine; RNA, Messenger; Superoxide Dismutase; TNF Receptor-Associated Factor 3; Toll-Like Receptor 3; Toll-Like Receptor 8; Water

Deltamethrin (DM) is one of the most toxic but widely used pyrethroid insecticides. Even though a non-target animal, fish are at high risk as they are deficient in the enzyme system that hydrolyses pyrethroids. Enhancing the immune system is a potential method in preventing fish diseases. The present investigation aims to study the modulations in the immune response-related parameters in Oreochromis niloticus that were exposed to DM, by dietary supplementation of aqueous root extract of Asparagus racemosus (ARE). The experiment compared fish in control, DM (1 μg/L) exposed (added to water), ARE (10 g, 20 g, and 30 g ARE/kg of feed) supplemented, and DM-ARE cotreated groups. After 21 days of experimental period, serological, histopathological, and immune response related-gene and protein analysis were carried out. The DM-ARE cotreated group showed significant increase in weight gain, specific growth rate, and decreased feed conversion ratio compared to the DM exposed group. The ARE cotreatment could significantly revert the alteration induced by DM in lysozyme, respiratory burst, myeloperoxidase, C-reactive protein, glucose, cortisol, total protein, albumin, and triglyceride levels. The liver histopathology showed membrane breakage, severe necrosis, infiltration of inflammatory cells, melano-macrophages, and nuclear atrophy, and the kidney showed tubular necrosis, hematopoietic necrosis, Bowman's capsule edema, and glomerulus degeneration in DM exposed group. In ARE cotreated group, the liver showed regenerative cellular changes and only mild to moderate cellular damages, and the kidney tubules and glomerulus had intact structure. ARE discernibly regulated the expression of immune-related genes and proteins (IgM, TNFα, IFN-γ, IL-1β, and IL-8) in fish. The DM-ARE cotreated groups showed reduced cumulative mortality and higher relative percent survival on experimental challenge with Aeromonas hydrophila compared to the DM group. Thus, ARE possess protective potential against DM-induced toxicity, and can be used as a cost-effective technique in aquafarming.

Topics: Animal Feed; Animals; C-Reactive Protein; Cichlids; Diet; Dietary Supplements; Fish Diseases; Glucose; Hydrocortisone; Immunoglobulin M; Insecticides; Interleukin-8; Muramidase; Necrosis; Nitriles; Peroxidase; Plant Extracts; Pyrethrins; Triglycerides; Tumor Necrosis Factor-alpha; Water

This study aimed to investigate the effects of mango peel powder (MGPP) on growth, innate immunity, and immune-antioxidant related gene expression of Nile tilapia reared under biofloc system. Three hundred Nile tilapia (average weight 14.78 ± 0.05 g) were distributed into 15 fiber tanks (300 L per tank) assigned to five treatments in triplication. Fish were fed basal diet containing different levels MGPP as follows: 0 (MGPP0: control), 6.25 (MGPP 6.25), 12.5 (MGPP 12.25), 25 (MGPP 25), and 50 (MGPP 50) g kg

Topics: Animal Feed; Animals; Antioxidants; Aquaculture; Cichlids; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression; Mangifera; Muramidase; Peroxidases; Powders

The present study was conducted to evaluate the immunostimulatory effect of tea leaf extract (Camellia sinensis) on Labeo rohita and its resistance against Aeromonas hydrophila infection. The ethanolic extract of green tea (GTEE) was found to be the most potent as compared to other solvent extract which was used for further study. It was used to evaluate immune-biochemical response of L. rohita fingerlings, fed with tea leaf extract (control- 0.0%, 0.2% (T1), 0.4% (T2), 0.8% (T3) and 1% (T4) of GTEE kg

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Antioxidants; Blood Proteins; Cyprinidae; Fish Diseases; Gene Expression Profiling; Glucose; Gram-Negative Bacterial Infections; Immunity; Interleukin-10; Muramidase; Peroxidase; Plant Extracts; Solvents; Tea

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of β-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of β-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of β-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1β were significantly upregulated, while ifn-γ and il-10 were unaffected by β-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, β-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of β-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CT

Topics: Aeromonas salmonicida; Animal Feed; Animals; Antioxidants; beta-Glucans; Cyprinidae; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Muramidase; Protease Inhibitors; RNA, Messenger

Seven immune factors, lysozyme, hepcidin, heat-shock protein (HSP) 70, HSP90, immunoglobulin M, C-type lectin, and Lily-type lectin, were measured by PCR in the livers, spleens, and head kidneys of turbot infected with Vibrio anguillarum. Additive main effects and multiplicative interaction (AMMI) and genotype main effects and genotype × environment interaction (GGE) biplot analysis were used to analyze genotype × tissue interactions for immunological factors. The AMMI analysis revealed that immune factor expression was significantly affected by genotype, tissue, and genotype × tissue interactions. Genotype (65.85%) was the major contributor to the total variation in immune factor expression in comparison to tissue effects (7.54%) and genotype × tissue interactions (12.52%). GGE biplot analysis revealed differences in the ranking of the seven immune factors in the three tissues; head kidney possessed the strongest ability to distinguish the seven immune factors. The test tissue locations were divided into liver-spleen and head kidneys regions; HSP70 was expressed the highest in the liver-spleen regions, and lysozyme had the highest expression in the head kidney region. Overall, HSP70 and HSP90 had the best expression and stability in the three tissues.

Topics: Animals; Dissection; Fish Diseases; Flatfishes; Genotype; Immunologic Factors; Muramidase; Vibrio; Vibrio Infections

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1β, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Topics: Administration, Oral; Animals; Antigens, Bacterial; Bacterial Vaccines; Dihydrolipoamide Dehydrogenase; Drug Carriers; Fish Diseases; Fish Proteins; Gene Expression; Interferon-gamma; Interleukin-10; Interleukin-13; Interleukin-1beta; Interleukin-2; Interleukin-4; Intestines; Kidney; Muramidase; Nanoparticles; Perciformes; Silicon Dioxide; Spleen; Tumor Necrosis Factor-alpha; Vaccination; Vibrio alginolyticus; Vibrio Infections

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

Topics: Animals; Antibodies, Bacterial; Aquaculture; Bacterial Load; Bacterial Proteins; Bacterial Vaccines; Fish Diseases; Fructose-Bisphosphate Aldolase; Gram-Negative Bacterial Infections; Muramidase; Perciformes; Photobacterium; Recombinant Proteins; Spleen; Vaccination; Vaccine Efficacy; Vaccines, Subunit

The effect of cinnamaldehyde (CM) enriched diet on immunity and cytokine gene expression in Channa striatus against Aphanomyces invadans is reported. C. striatus was uniformly divided into eight groups (n = 25 fish each) and fed with formulated diets with 0, 5, 10, and 15 mg kg

Topics: Acrolein; Animals; Aphanomyces; Complement Activation; Diet; Fish Diseases; Fishes; Gene Expression; Immunity, Innate; Immunoglobulin M; Infections; Leukocyte Count; Muramidase; Phagocytosis; Reactive Oxygen Species

In this study, we sequenced and characterized the goose-type lysozyme gene, termed as BsLysG, from the Chinese black sleeper (Bostrychus sinensis). The BsLysG encodes 196 amino acids and contains a soluble bacterial lytic transglycosylases domain, three catalytic residues (Glu

Topics: Amino Acid Sequence; Animals; Base Sequence; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacteria; Gram-Positive Bacteria; Immunity, Innate; Muramidase; Phylogeny; Poly I-C; Sequence Alignment

Aloe vera is a traditional medicinal plant; however, its use in fish is fairly recent. We evaluated the effects of dietary A. vera on stress, innate immunity, and energy metabolism in pacu inoculated with Aeromonas hydrophila. For 7 days, 192 fish were fed with diets supplemented with 0% (control), 0.5%, 1.0%, and 2.0% of the plant extract and then inoculated with bacteria and sampled 3, 6, and 24 h later. All concentrations of A. vera reduced basal levels of cortisol, and 1.0% reduced cortisol levels more intensely 3 h after inoculation. A. vera increased the basal respiratory activity of leukocytes/RAL (0.5 and 1.0%), increased the serum levels of lysozyme (1.0 and 2.0%) 6 h after inoculation, and increased the activity of the complement system after 3 h. Spleen somatic index/SSI increased with 1.0 and 2.0% A. vera. A. vera also promoted metabolic effects. It increased basal levels of lipids in the liver and muscle, as well as hepatosomatic index (1.0%) and, 3 h after inoculation, prevented the reduction of serum triglyceride (1.0%) and reduced the mesenteric fat (1.0%). Bacterial inoculation increased RAL from 3 to 24 h and lysozyme levels at 24 h, increased serum cholesterol at 24 h, and decreased serum triglyceride from 3 to 24 h, regardless of A. vera. We concluded that A. vera offered for only 7 days had stress-reducing effects, stimulated innate immunity, protected triglyceride levels in blood, lipid depots in the liver and muscle, and directed the energy mobilization to visceral depots.

Topics: Aeromonas hydrophila; Aloe; Animals; Blood Glucose; Characiformes; Cholesterol; Energy Metabolism; Fish Diseases; Glycogen; Gram-Negative Bacterial Infections; Hydrocortisone; Immunity, Innate; Leukocytes; Lipids; Liver; Muramidase; Muscles; Plant Extracts; Random Allocation; Reactive Oxygen Species; Stress, Physiological; Triglycerides

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Topics: Alginates; Animal Feed; Animals; Body Composition; Catalase; Cell Encapsulation; Cells, Immobilized; Chitosan; Cholesterol; Complement Pathway, Alternative; Diet; Disease Resistance; Fish Diseases; Fish Proteins; Gene Expression Regulation; Interleukin-1; Lacticaseibacillus rhamnosus; Muramidase; Oncorhynchus mykiss; Probiotics; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Weight Gain; Yersinia Infections; Yersinia ruckeri

This study evaluated the effectiveness of dietary Ziziphus mauritiana leaf powder (ZLP) to control Aeromonas hydrophila infection in Nile tilapia and reduce damage to vital immune organs. Four experimental groups were fed a diet supplemented with ZLP at concentrations of 0, 5, 10, and 20 g/kg (w/w) for 6 weeks. At the end of the feeding trial, all groups were intraperitoneally injected with pathogenic A. hydrophila. It was found that Z. mauritiana significantly (P < 0.05) upregulated (lysozyme, interleukin 1 beta) and superoxide dismutase gene expressions as well as improved the activity of serum lysozyme and liver antioxidant enzymes. The fish that were fed a ZLP-supplemented diet also exhibited significantly higher survival rates after A. hydrophila challenge than those that were fed a ZLP-free diet (P < 0.05). Supplementation of 10 g/kg ZLP most effectively reduced the histopathological alterations caused by A. hydrophila challenge in the liver, spleen, kidney, and muscle of the fish. In conclusion, ZLP can be effective in controlling A. hydrophila infection in Nile tilapia (particularly at a concentration of 10 g/kg) through enhancement of its immune and antioxidant status.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Aquaculture; Cichlids; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Kidney; Liver; Muramidase; Muscles; Plant Extracts; Plant Leaves; Random Allocation; Spleen; Superoxide Dismutase; Up-Regulation; Ziziphus

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bacterial Vaccines; Fish Diseases; Flatfishes; Immunity, Humoral; Immunity, Innate; Immunoglobulin M; Kidney; Muramidase; Spleen; Vaccines, Inactivated; Vibrio; Vibrio Infections

Topics: Aeromonas hydrophila; Animals; Arguloida; Carps; Cloning, Molecular; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Immunization; Liver; Muramidase; Phylogeny; Poly I-C; RNA, Double-Stranded

Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg

Topics: Alkaline Phosphatase; Animals; Cathepsin B; Fish Diseases; Gram-Negative Bacteria; Gram-Positive Bacteria; Hydrogen-Ion Concentration; Mucus; Muramidase; Perciformes; Peroxidase; Protein Stability; Skin

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

Yellow catfish (Pelteobagrus fulvidraco) is an important economic cultured fish in China. Here we report antioxidative activity and immune regulation in head kidney using a central composite design based on water temperature (20-34 °C) and dietary lipid (2-17%). Response values were optimized using response surface methodology to maximize the immune response and relieve oxidative stress. The experiment was conducted under laboratory conditions and lasted for seven weeks. The results showed that the linear effects of lipid level on superoxide dismutase (SOD, and lysozyme (LYZ) activity, and malondialdehyde (MDA) content in head kidney, respiratory burst activity (RBA) of head kidney macrophages, and cumulative mortality of fish infected by Streptococcus iniae (S. iniae) were significant (P < 0.05). Similarly, the linear effects of water temperature on SOD activity, MDA content, and cumulative mortality were significant (P < 0.05). In addition, the quadratic effects of water temperature and lipid level on all experimental response values were significant (P < 0.05), and no interactive effect was found between water temperature and lipid level (P > 0.05). High water temperature and high lipid diet significantly reduced the antioxidative activity and immune response in head kidney, and increased MDA content, which caused increased mortality of the S. iniae-infected fish. The adjusted R

Topics: Animals; Antioxidants; Aquaculture; Catfishes; Fish Diseases; Fish Proteins; Head Kidney; Immunity, Innate; Lipid Metabolism; Macrophages; Malondialdehyde; Models, Statistical; Muramidase; Streptococcal Infections; Streptococcus iniae; Superoxide Dismutase; Temperature

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Sequence Alignment

The present study was designed to assess the effect of sublethal concentrations of electoplating industry effluent (EIE) on the non-specific and specific immune responses in the freshwater fish, Cyprinus carpio. Sublethal concentrations of electroplating industry effluent such as 0.004, 0.007, 0.010 and 0.013% were chosen based on the LC

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Antibody Formation; Carps; Electroplating; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Industrial Waste; Muramidase; Peroxidase; Protease Inhibitors; Water Pollution, Chemical

The dietary supplementation of synbiotic in Cirrhinus mrigala juvenile (with initial body weight ranging from 2.87 ± 0.01 g to 3.26 ± 0.05 g) was evaluated in terms of changes in innate immunity, antioxidant activity and disease resistance against Aeromonas hydrophilla infection. One hundred eighty acclimatized juveniles of mrigal were randomly distributed in the three replicates of each of four experimental groups i.e. control (without Probiotic and Prebiotic), T

Topics: Aeromonas hydrophila; Animals; Bacillus subtilis; Cyprinidae; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Probiotics; Synbiotics

The present study investigates the effects of orange peels derived pectin (OPDP) on skin mucus and serum immune parameters, disease resistance and growth performance of O. niloticus cultured under indoor biofloc system. Six hundred Nile tilapia (average weight 9.09 ± 0.05 g) were distributed into 15 fiber tanks (300 L per tank) assigned to five treatments repeated in triplicate. Fish were fed experimental diets contain different levels OPDP as follows: 0 (control in clear water), 0 (control in biofloc system), 5, 10, and 20 g kg

Topics: Animals; Cichlids; Citrus sinensis; Complement Pathway, Alternative; Disease Resistance; Fish Diseases; Fruit; Immunity, Innate; Mucus; Muramidase; Pectins; Peroxidases; Phagocytosis; Respiratory Burst; Skin; Streptococcal Infections; Streptococcus agalactiae

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Cytokines; Fish Diseases; Kidney; Muramidase; Perciformes; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphoric Monoester Hydrolases; Phosphotransferases (Nitrogenous Group Acceptor); RNA, Messenger; Streptococcal Infections; Streptococcus; Vaccination

In the present study, effect of dietary Withania sominefera (W. sominefera) root powder was evaluated to modulate immune and antioxidant response against Aeromonas hydrophila (A. hydrophila) infection in Nile tilapia (Oreochromis niloticus). W. sominefera root powder supplemented diets at two concentrations 2.5% (W 2.5%) and 5% (W 5%); fed for 6 weeks prior to the A. hydrophila challenge and continued the same respective diets during the post challenge period (2 weeks). Results showed that fish fed W. sominefera at 5% enhanced immune response in both pre and post-challenge period. NBT level exhibited only significant increase (P < 0.05) in the pre-challenge period compared to control. Malondialdehyde (MDA) levels in liver and muscle revealed significant decrease in both Withania supplemented groups compared to the control in post challenge period. Antioxidant enzymes activities (catalase/CAT/, glutathione S-transferase/GST/, glutathione/GSH; and superoxide dismutase/SOD) were improved in liver and muscle in post challenge period. Glutathione peroxidase (GPx) level in muscle and serum total antioxidant capacity (TAC) showed a significant increase in both Withania supplemented groups compared to the control post challenge. Withania supplementation enhanced disease resistance against A. hydrophila and reduced mortalities (20%), especially at supplemented concentration of 5%. Our findings suggest that W. sominefera root powder may have protective and immunotherapeutic roles in Nile tilapia against A. hydrophila infection which may be useful in controlling important fish bacterial diseases.

Topics: Aeromonas hydrophila; Animals; Blood Cells; Catalase; Cichlids; Diet; Fish Diseases; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Gram-Negative Bacterial Infections; Liver; Malondialdehyde; Muramidase; Muscles; Nitric Oxide; Phagocytosis; Plant Roots; Protective Agents; Respiratory Burst; Superoxide Dismutase; Withania

The present study investigated the effects of dietary supplementation of yeast nucleotides on the hematology, antioxidant activity, non-specific immunity, expression of intestinal cytokines, and disease resistance in Nile Tilapia (Oreochromis niloticus). Fish weighing 42.90 ± 0.14 g were randomly divided into four groups. Each group was set in triplicate (15 fish per replicate). Fish were fed on four dietary levels of yeast nucleotides (NTs) supplemented with the basal diet 0% (control), 0.05%, 0.15%, and 0.25% NTs. Significantly higher total serum protein, albumin, total serum globulin, total WBC counts, and lymphocyte and granulocyte contents were recorded in 0.25% NT group as compared to the control. The albumin/globulin ratio (A:G) showed a considerable decrease in the 0.25% NT group. The non-specific immune parameters; serum killing percentage, lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity, were significantly higher in the 0.25% NT group as compared to the control. Moreover, a 15-day feeding trial demonstrated improved results in terms of serum lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity than a 30-day feeding trial. A significant increase in the anti-oxidant status of O. niloticus was noticed, as reflected by increased superoxide dismutase and malondialdehyde activity in the serum of 0.25% NT group compared to the control, while glutathione peroxidase displayed a significant increase in all groups as compared to the control. The intestinal cytokines TGF-β, IL-1β, IL-10β, and TNF-α mRNA levels showed a pattern of 0.25% NT > 0.15% NT > 0.05% NT > 0% NT, as relative to the control Ef-1α levels. The relative survival percentages of fish fed on yeast nucleotide-supplemented diets, as analyzed by exposure to Aeromonas sobria, were significantly better than the control group. In conclusion, dietary yeast nucleotide administration at 0.25% improved blood proteins, leukocytes, antioxidant activity, non-specific immunity, cytokine gene expression, and disease resistance of Nile Tilapia.

Topics: Aeromonas; Animals; Cichlids; Cytokines; Diet; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Immunoglobulin M; Intestines; Leukocyte Count; Malondialdehyde; Muramidase; Nitric Oxide; Nucleotides; RNA, Messenger; Saccharomyces cerevisiae; Superoxide Dismutase

Orange-spotted grouper Epinephelus coioides reared at 34‰ and 27 °C were abruptly transferred to 6‰, 20‰ and 34‰ (control) and examined for innate cellular and humoral parameters after 3-96 h. Total leucocyte count (TLC), respiratory burst (RB), phagocytic activity (PA), alternative complement pathway (ACP) and lysozyme activity were significantly decreased 3-6 h, 3-6 h, 3-96 h, 3-96 h and 3-96 h, respectively after transferal into 6‰ salinity. TLC, RB and PA significantly increased after 3-48 h, 3-96 h and 3-24 h, respectively, with recovery of TLC and PA after 96 h and 48-96 h, whereas ACP and lysozyme activity significantly decreased 3-96 h after being transferred to 20‰. In another experiment, grouper reared at 34‰ and 27 °C were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at 2.3 × 10

Topics: Animals; Complement Pathway, Alternative; Disease Susceptibility; Fish Diseases; Immunity, Cellular; Immunity, Humoral; Leukocyte Count; Muramidase; Perciformes; Phagocytosis; Respiratory Burst; Salinity; Stress, Physiological; Vibrio alginolyticus; Vibrio Infections

Bacterial ghosts (BGs) can be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria. They are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. Here we prepared Edwardsiella tarda ghost (ETG) and tested different concentrations in vaccination trials. The results showed that serum IgM antibody titers were significantly higher in three different concentration immunization groups than control group (P < 0.05), However, there was no significant (P > 0.05) difference between the immunized groups. The phagocytic percentage (PP) was significantly higher (P < 0.05) in ETG immunized groups than in the control group from 3 days post-treatment. The PP continued to rise with time until day 21, when the values of three ETG immunized groups were 45.7%,51.2% and 50.7%, respectively. In addition, phagocytic index (PI) was significantly higher (P < 0.05) in ETG immunized groups than in the control group after 7 days post-treatment. However, there was no significant (P > 0.05) difference of PP or PI between immunized groups. In addition, non-specific immune immunity, such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activities displayed a similar pattern in all immunized groups, all immunized fish showed significantly higher activities than control group fish (P < 0.05). Most importantly three ETG immunized groups were all significantly more protected against the E. tarda challenge (19/25, 76% survival), (21/25, 84% survival) and (20/25, 80% survival) respectively, compared to (9/25, 36% survival) survival in the control group, but there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between immunized groups. All these results suggest that an ETG could stimulate cellular and humoral immunity, and could be used as a vaccine candidate in S.m. In summary, ETG can protect fish from Edwardsiellosis, and there is no significant difference in SR and RPS when three different concentrations of ETG are used, so it can easily be developed as a vaccine for mechanical and artificial operations.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bacterial Vaccines; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Immunization; Leukocytes; Muramidase; Perciformes; Phagocytosis; Staphylococcus aureus; Superoxide Dismutase

The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.

Topics: Aeromonas hydrophila; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Ceruloplasmin; Cyprinidae; Fish Diseases; Gram-Negative Bacterial Infections; Hemolysis; Muramidase; Peroxidase; Porins; Recombinant Proteins

The large yellow croakers (Larimichthys crocea) are mainly present in the Chinese coast and near seas with high economic importance, but vulnerable to many diseases, especially in the breeding and aquaculture. The purpose of this research was to boost the innate immune system of the large yellow croaker by administering bitter peptides into their peritoneal cavity. Total 120 Juvenile of large yellow croakers in very even weight of 60 g were divided into 4 different groups in 200/300 L of water tank, respectively. Fish growth were observed for 3 months before and after different treatments. The bitter peptides from pepsin hydrolysis were applied because they possess the highest bitter sensory scores. The blood of fish from the different groups was collected and tested for different immune parameters to evaluate the effectiveness of bitter peptides as immune stimulants after administration for 8 weeks. The average ratio of leukocytes/total blood cells (%) for control was found at 14.6%, for the low dose of bitter peptides 0.6 mg/fish was at 29.3%, for middle dose of 1.2 mg/fish was at 35%, and high dose of 2.4 mg/fish was at 30%. The lysozyme assay showed that the OD (optical density) units of relative progress lysis activity at 60 min were 0.17, 0.101, 0.307 and 0.198, respectively. Similarly in the same order as in phagocyte assay, most importantly the middle dose (1.2mg/fish) gave the highest survival rate throughout the assay. The results showed that bitter peptides can be used as immune boosters for the yellow croakers and the optimum dose was 1.2 mg/fish due to both leukocytes and lysozyme activity in the treated samples increased significantly compared with the control group. According to the results obtained, we suggest that the incorporation of middle dose of bitter peptides into fish feeds may reduce the fish diseases in aquaculture, at least for large yellow croakers.

Topics: Adjuvants, Immunologic; Animals; Fish Diseases; Hydrolysis; Immunity, Innate; Leukocytes; Muramidase; Pepsin A; Peptides; Perciformes; Phagocytosis

The aim of the present study was to investigate beneficial effects of dietary 1,8-cineole (cineole) supplementation on physiological, immunological and antioxidant responses of rainbow trout (Oncorhynchus mykiss) to crowding stress. The fish were fed for 50 days with diets containing 0 (control), 0.05, 0.1, 0.25, 0.5 and 1% cineole prior to exposure to a 14-day crowding stress. Serum stress markers (cortisol, glucose, lactate, T

Topics: Animals; Blood Glucose; Catalase; Crowding; Diet; Eucalyptol; Fish Diseases; Hydrocortisone; Lactic Acid; Leukocyte Count; Muramidase; Oncorhynchus mykiss; Stress, Psychological; Superoxide Dismutase; Triiodothyronine

Topics: Aeromonas veronii; Animals; Antigens, Bacterial; Bacterial Vaccines; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunoglobulin M; Interleukin-1beta; Muramidase; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

A 120-day feeding trial was conducted to evaluate the effect of different stocking densities on growth, the non-specific immunities, antioxidant status and digestive enzyme activities of Oreochromis niloticus fingerlings under a zero-water exchange biofloc system. Tilapias (0.51 ± 0.05 g) were randomly distributed in twelve tanks, each with 300 L water. The experimental design was completely randomized using three replications with four treatments 166 orgs m

Topics: Amylases; Animals; Aquaculture; Cichlids; Complement C3; Crowding; Fish Diseases; Glucose; Glutathione; Hydrocortisone; Lipase; Liver; Malondialdehyde; Muramidase; Serotonin; Stress, Psychological; Trypsin; Vibrio Infections

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

This study evaluated the effect of sublethal doses of antifungal drug miconazole nitrate (MCZ) on immunological responses and its role as a prophylactic drug against S. parasitica in Labeo rohita fingerlings. Fish were fed with sublethal doses of MCZ, that is, T1-6.30 mgMCZ kgBW

Topics: Age Factors; Animal Feed; Animals; Antiparasitic Agents; Cyprinidae; Dietary Supplements; Fish Diseases; Infections; Miconazole; Muramidase; Protease Inhibitors; Reactive Oxygen Species; Saprolegnia

This study investigated the effects of tuna hydrolysate (TH) inclusion in fishmeal (FM) based diets on the growth performance, innate immune response, intestinal health and resistance to Streptococcus iniae infection in juvenile barramundi, Lates calcarifer. Five isonitrogenous and isoenergetic experimental diets were prepared with TH, replacing FM at levels of 0% (control) 5%, 10%, 15% and 20%, and fed fish to apparent satiation three times daily for 8 weeks. The results showed that fish fed diets containing 5% and 10% TH had significantly higher final body weight and specific growth rate than the control. A significant reduction in blood glucose was found in fish fed 10%, 15% and 20% TH compared to those in the control whereas none of the other measured blood and serum indices were influenced by TH inclusion. Histological observation revealed a significant enhancement in goblet cell numbers in distal intestine of fish fed 5 to 10% TH in the diet. Moreover, fish fed 10% TH exhibited the highest resistance against Streptococcus iniae infection during a bacterial challenge trial. These findings therefore demonstrate that the replacement of 5 to 10% FM with TH improves growth, immune response, intestinal health and disease resistance in juvenile barramundi.

Topics: Animal Feed; Animals; Complement System Proteins; Diet; Disease Resistance; Fish Diseases; Intestines; Kaplan-Meier Estimate; Liver; Muramidase; Perciformes; Streptococcus iniae; Tuna

β-glucan has been shown to increase non-specific immunity and resistance against infections or pathogenic bacteria in several fish species, but information regarding its trans-generational immune-enhancing effects is still rather limited. Lysozyme is a maternal immune factor playing an important role in the developing embryos of zebrafish. Here we clearly showe that β-glucan enhanced the level of C-type lysozyme in eggs of zebrafish, and the embryos derived from β-glucan-treated zebrafish were more resistant to bacterial challenge than control embryos. Moreover, the transferred lysozyme was apparently linked with the antimicrobial defense of early embryos. In addition, we also showed that β-glucan induced a significant increase in the synthesis of C-type lysozyme in previtellogenetic oocytes. Therefore, we show for the first time that β-glucan can enhance the lysozyme level in offspring via both inducing the transfer of the molecule from mothers to eggs and stimulating its endogenous production in oocytes.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; beta-Glucans; Cells, Cultured; Diet; Eggs; Female; Fish Diseases; Gene Expression Regulation, Developmental; Immunity, Innate; Muramidase; Oocytes; Up-Regulation; Zebrafish; Zebrafish Proteins

Black rockfish (Sebastes schlegelii), an important aquaculture species in Korea, has been affected by bacterial diseases leading to a drastic decline in production. Goose-type lysozyme (LysG) is a key enzyme of the innate immune system to eradicate bacterial infections. In this study, two isoforms of LysG from black rockfish, designated as RfLysG1 and RfLysG2, have been identified and characterized at the molecular, transcriptional, and functional levels. The deduced amino acid sequences had the LysG family characteristics and exhibited conserved properties, including active residues and domains. The cDNA sequences of RfLysG1 and RfLysG2 were 1514 bp and 900 bp in length, respectively. The 567-bp open reading frame (ORF) of RfLysG1 encoded a protein of 188 amino acids with molecular mass 20.11 kDa, and the 600-bp ORF of RfLysG2 encoded a polypeptide with 199 amino acids and molecular mass of 22.19 kDa. Homology studies indicated that RfLysG1 showed the highest identity (84.6%) with LysG-B of Oplegnathus fasciatus, while RfLysG2 showed the highest identity (74.4%) with LysG of Siniperca chuatsi. Both sequences possessed a soluble lytic trans-glycosylase domain. Both lacked signal peptide and they were not identified as proteins secreted by non-classical pathway by the SecretomeP server. Transcriptional analysis of the two genes showed constitutive expression, where both genes were highly expressed in blood under normal physiological conditions. In response to the immune challenges lipopolysaccharide (LPS), Streptococcus iniae, and poly I:C injection, the expression of RfLysG1 and RfLysG2 was significantly upregulated in blood and spleen tissues in a time-dependent manner. Turbidimetric assays indicated that both recombinant proteins tagged with maltose-binding protein (MBP) were reactive against several Gram-positive and Gram-negative bacteria, but MBP was inactive. Optimum temperatures for the recombinant RfLysG1 and RfLysG2 were 40 °C and 50 °C, respectively, and both were highly active at pH 3.0. The results provide evidence for the vital immunological role and bacteriolytic potential of RfLysG1 and RfLysG2.

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

The marbled spinefoot rabbitfish (Siganus rivulatus) is an economically valuable fish species that has potential for commercial production in aquaculture. To overcome challenges in its sustainable production, a formulated diet is required for imparting health and robustness. This study evaluates the effect of dietary supplementation with arachidonic acid (ARA; 20:4n-6) on growth, survival, immune function and fatty acid composition of red blood cells (RBCs) in rabbitfish. We conducted two feeding trials using juvenile fish (to evaluate growth and survival) and adults (to evaluate immune function and fatty acid incorporation). Fish were fed diets supplemented with three different levels of ARA (in % of total fatty acids): 0.6 (unsupplemented control), 2.6 (moderate) and 4.7 (high). The fish fed with moderate ARA levels exhibited improved (p < 0.05) growth over the control and the high ARA level groups. During an outbreak of Streptococcus iniae, fish fed with moderate ARA survived significantly (p < 0.05) better (89%) than the control and the high ARA groups (59% and 48%, respectively). Moderate ARA supplementation resulted in elevated lysozyme and complement levels in the plasma of rabbitfish. A significant increase in the total serum immunoglobulin levels was observed in both the medium and the high ARA level groups; however, a decrease in antiprotease activity was recorded in the supplemented groups as compared to the control. Fatty acid analysis in fish red blood cells revealed a significant (p < 0.05) increase in the proportion of ARA of total fatty acids in the groups fed with the medium and the high ARA level diets (9.5% and 11.2%, respectively, compared to 7.1% in the control). Concomitantly, there was a decrease in the proportion of eicosapentaenoic acid (EPA; 20:5n-3), dihomo-γ linolenic acid (DGLA; 20:3n-6) and several 18-carbon unsaturated fatty acids in these groups. In conclusion, ARA in rabbitfish feeds improved growth, survival as well as innate and acquired humoral immune functions. Thus ARA supplementation in the diet of this species could be a valuable step towards establishing the commercial culture of rabbitfish.

Topics: Animal Feed; Animals; Arachidonic Acid; Diet; Dietary Supplements; Erythrocytes; Fatty Acids; Fish Diseases; Fish Proteins; Immunity, Innate; Immunoglobulins; Muramidase; Perciformes; Streptococcal Infections; Streptococcus iniae

The effects of oral vaccination by two immune routes (gavage and mixed fodder administration) using a recombinant DNA vaccine SL7207-pVAX1-sip against Streptococcus agalactiae were assessed. SL7207-pVAX1-sip significantly enhanced various innate immune responses of tilapia, such as total serum protein, superoxide dismutase activity, lysozyme activity, complement C3 concentration in serum, serum antibacterial activity, and interleukin 1β and tumor necrosis factor-α. Vaccinated fish had higher relative percent survival values (57% and 63% for gavage group and mix fodder administration, respectively) at 10 weeks after S. agalactiae infection. When administered mixed fodder, the DNA vaccine SL7207-pVAX1-sip against S. agalactiae may produce more effective protection. These findings can promote the application and development of DNA vaccines in aquaculture.

Topics: Administration, Oral; Animals; Bacterial Vaccines; Complement C3; Drug Administration Routes; Fish Diseases; Fish Proteins; Immunity, Innate; Interleukin-1beta; Muramidase; Streptococcal Infections; Streptococcus agalactiae; Superoxide Dismutase; Tilapia; Tumor Necrosis Factor-alpha; Up-Regulation; Vaccination; Vaccines, DNA

This study aimed to investigate the impacts of dietary threonine on intestinal immunity and inflammation in juvenile grass carp. Six iso-nitrogenous semi-purified diets containing graded levels of threonine (3·99-21·66 g threonine/kg) were formulated and fed to fishes for 8 weeks, and then challenged with Aeromonas hydrophila for 14 d. Results showed that, compared with optimum threonine supplementation, threonine deficiency (1) decreased the ability of fish against enteritis, intestinal lysozyme activities (except in the distal intestine), acid phosphatase activities, complement 3 (C3) and C4 contents and IgM contents (except in the proximal intestine (PI)), and it down-regulated the transcript abundances of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, IgZ, IgM and β-defensin1 (except in the PI) (P<0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-17D mRNA levels partly related to NF-κB signalling; (3) could down-regulate intestinal anti-inflammatory cytokine transforming growth factor (TGF)-β1, TGF-β2, IL-4/13A (not IL-4/13B) and IL-10 mRNA levels partly by target of rapamycin signalling. Finally, on the basis of the specific growth rate, against the enteritis morbidity and IgM contents, the optimum threonine requirements were estimated to be 14·53 g threonine/kg diet (4·48 g threonine/100 g protein), 15.05 g threonine/kg diet (4·64 g threonine/100 g protein) and 15·17 g threonine/kg diet (4·68 g threonine/100 g protein), respectively.

Topics: Aeromonas hydrophila; Animals; Antimicrobial Cationic Peptides; Blood Proteins; Carps; Cytokines; Diet; Down-Regulation; Enteritis; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Hepcidins; Immunoglobulin M; Intestines; Muramidase; NF-kappa B; Signal Transduction; Threonine; TOR Serine-Threonine Kinases; Up-Regulation

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Aquaculture; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunization; Lipoproteins; Muramidase; Peptides; Peroxidase; Protease Inhibitors; Staphylococcus hominis; Surface-Active Agents; Tilapia

Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55 ± 5% DO) or normoxia (85 ± 5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V. anguillarum were significantly higher in normoxic fish. Serum killing of V. anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 β, TCR-β, MHC-II β), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.

Topics: Animals; Antibodies, Bacterial; Cichlids; Fish Diseases; Lymphocyte Count; Muramidase; Oxygen; Serum Bactericidal Test; Spleen; Vaccination; Vibrio; Vibrio Infections

Olive flounder (Paralichthys olivaceus), also known as the Japanese flounder in Japan, is one of the most important commercial marine finfish species cultured in Korea and Japan. The purpose of this study was to evaluate how a species of brown algae (Ecklonia cava, E. cava) affects the growth rate of olive flounder and its immune response to pathogenic bacteria. First, the experimental fish were divided into four groups: the control group was fed the diet containing only 1.0% Lactobacillus plantarum (L. plantarum), group I was fed 1.0% L. plantarum and 1.0% E. cava (EC), group II was fed 1.0% L. plantarum and 0.1% ethanol extract of EC (EE), and group III was fed 1.0% L. plantarum and 0.5% EE. The diets fed to the fish twice a day for 16 weeks. The results indicated that supplementation with 1.0% EC and 0.1% EE improved the growth and body weight of olive flounder, and decreased its mortality. This diet, however, did not significantly affect the biochemical profiles of the experimental flounder. The supplementation of 1.0% EC also enhanced the innate immune response of the fish, as evidenced by the high respiratory burst, and increased serum lysozyme and myeloperoxidase activity. The addition of 1.0% EC and either 0.1% or 0.5% EE also decreased the accumulative mortality of olive flounder infected by pathogenic bacteria (Edwardsiella tarda, Streptococcus iniae, and Vibrio harveyi). Overall, these results suggest that E. cava can act as a prebiotic by improving the innate immune response in fish infected with pathogenic bacteria as increased the growth of the probiotic.

Topics: Animals; Body Weight; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Flounder; Lactobacillus plantarum; Muramidase; Peroxidase; Phaeophyceae; Prebiotics; Respiratory Burst; Streptococcal Infections; Streptococcus iniae; Vibrio; Vibrio Infections

Lysozyme as an important nonspecific immune factor, can kill bacteria by hydrolyzing β-1,4-glycosidic linkages of peptidoglycan layer, and plays an important role in innate immune response against pathogen infection. In the present study, we report molecular cloning, tissue distribution and functional characterization of the c-type and g-type lysozymes in Qihe crucian carp Carassius auratus (designated as Ca-clys and Ca-glys, respectively). The full-length of Ca-clys and Ca-glys cDNA were cloned using RT-PCR and RACE methods. Catalytic and other conserved residues, required for functionality, were identified by multiple sequence alignment and structure predicted. The findings indicating the Ca-clys with signal peptide sequence, while the Ca-glys without, imply that the two isozymes function in different sites of cell. Phylogenetic analysis revealed that Ca-clys and Ca-glys genes evolve at different rates. Moreover, spatial expression analysis showed that Ca-clys transcript was most abundant in kidney and least in gill. However, the expression level of Ca-glys was significantly lower compared with Ca-clys in various tissues, which was the most abundant in spleen and least in brain. After intraperitoneal injection with A. hydrophila and lipopolysaccharide (LPS), the mRNA levels of Ca-clys and Ca-glys were generally up-regulated in liver and gill, but indicated the different expression changes in spleen, kidney and head kidney. With regard to the lysozyme activity, it was showed that the total enzyme activities generally increased in liver, gill, spleen, and head kidney after stimulation. These results confirmed that both Ca-clys and Ca-glys play an important role in non-specific immunity after A. hydrophila invasion. In this study, it was speculated that expressions of Ca-clys and Ca-glys were regulated in different patterns against pathogens.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; Fish Diseases; Fish Proteins; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Lipopolysaccharides; Molecular Conformation; Muramidase; Phylogeny; Sequence Alignment; Transcriptome

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 c-type lysozyme gene (To-lysC) was cloned from golden pompano, Trachinotus ovatus. The To-lysC cDNA is composed of 743 bp with a 36 bp of 5'-UTR, 432 bp open reading frame (ORF) and 275 bp 3'-UTR, encoding a polypeptide of 144 amino acids (GenBank accession no: KT935522). Phylogenetic analysis revealed that To-lysC showed highest similarity to Perca flavescens lysC. Quantitative real-time PCR (qRT-PCR) analysis showed that To-lysC had relatively high expression level in the head kidney, gill and brain. After Vibrio harveyi infection, transcripts of To-lysC increased and reached its peak at 12 h p.i. These results indicated that To-lysC may play an important role in innate immune response to bacteria.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Gene Expression Regulation, Enzymologic; Muramidase; Perciformes; Phylogeny; RNA, Messenger; Sequence Alignment; Vibrio; Vibrio Infections

The aim of this study was to investigate the protective effects of dietary administration of commercial prebiotic, Immunogen, on immunological indicators, enzymatic responses and stress tolerance in juvenile (81.65 ± 1.49) rainbow trout (Oncorhynchus mykiss) following Aeromonas hydrophila infection. The first group of fish was fed with the diet containing 2 g kg(-1) Immunogen whilst the control group received the diet free of Immunogen. There were three replicates per group. After 6 weeks feeding, the control group were divided into two treatments injected with saline buffer (control), and 1.5 × 10(8) CFU A. hydrophila respectively. The fish fed with the Immunogen supplemented diet were also injected with 1.5 × 10(8) CFU A. hydrophila. Our results revealed that dietary Immunogen increased the level of white blood cell (WBC) and percentage of lymphocyte (P < 0.05), however, the level of red blood cell (RBC), Hematocrit (Hct), hemoglobin (Hb) and percent of monocyte decreased in Untreated-Challenged group but unaffected in the group fed with Immunogen (P < 0.05). The level of lysozyme, alternative complement, antiprotease activity, total protein, albumin and globulin decreased in Untreated- Challenged group compared to control group. However, there was an increase in the level of lysozyme, alternative complement, antiprotease activity, bactericidal activity, in the Treated- Challenged group compared to other groups (P < 0.05). Serum alkali phosphatase (ALT) and aspartate aminotransferase, significantly increased fallowing challenge with A. hydrophila but in the Treated-Challenged group, there was no significant difference compared to the control group (P < 0.05). Lactate dehydrogenase (LDH) level was not different between groups (P > 0.05). Serum cortisol and glucose levels were higher in the challenge group, but these levels were lower in fish under challenge that were fed Immunogen-supplemented diet in contrast to the group fed control diet. The stress responses affected by A. hydrophila challenge (P < 0.05). Serum sodium, potassium and calcium concentration decreased by A. hydrophila exposure (P < 0.05), and Immunogen showed protection effect against this change.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Diet; Disease Resistance; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Head Kidney; HSP70 Heat-Shock Proteins; Immunity, Humoral; Muramidase; Oncorhynchus mykiss; Prebiotics; Tumor Necrosis Factor-alpha

The mucosal surfaces constitute the first line of host defense against infection, and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes, while in constantly contact with a wide range of pathogens. The lysozymes are considered as key components for innate immune response to pathogen infection with their strong antibacterial activities. But their activities in mucosal immune responses were always overlooked, especially for g-type lysozymes, whose expression patterns in mucosal tissues following bacterial challenge are still limited. Towards to this end, here, we characterized the g-type lysozymes, Lyg1 and Lyg2 in turbot, and determined their expression patterns in mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot g-type lysozyme genes showed the closest relationship to Cynoglossus semilaevis. The two lysozyme genes showed different expression patterns following challenge. Lyg2 was significantly up-regulated in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge, while Lyg1 showed a general trend of down-regulation. The significant mucosal expression signatures of g-type lysozyme genes indicated their key roles to prevent pathogen attachment and entry in the first line of host defense system. Further functional studies should be carried out to better characterize the availability of utilization of g-type lysozyme to increase the disease resistance in the mucosal surfaces and facilitate the disease resistant breeding selection.

Topics: Amino Acid Sequence; Animals; Fish Diseases; Fish Proteins; Flatfishes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Immunity, Mucosal; Muramidase; Phylogeny; Sequence Alignment; Streptococcal Infections; Streptococcus iniae; Vibrio; Vibrio Infections

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

Topics: Amino Acid Sequence; Animals; Cells, Cultured; Chickens; Fish Diseases; Fish Proteins; Francisella; Gadus morhua; Geese; Gene Expression; Interferon-gamma; Lipopolysaccharides; Macrophages; Models, Molecular; Muramidase; Organ Specificity; Phylogeny

Topics: Animals; Bacteria; Bacterial Infections; Cloning, Molecular; DNA, Complementary; Fish Diseases; Fish Proteins; Flatfishes; Gene Expression; Muramidase; Phylogeny; Recombinant Proteins

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu

Topics: Amino Acid Sequence; Animals; Cloning, Molecular; DNA, Complementary; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Fish Proteins; Lipopolysaccharides; Muramidase; Pathogen-Associated Molecular Pattern Molecules; Phylogeny; Poly I-C; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Smegmamorpha; Streptococcal Infections; Streptococcus iniae

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non-stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI-, NI- and I-treated groups (94.5, 64 and 62.3 μg mg(-1), respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re-infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3-75%, higher than in the non-exposed control (40.6% mortality).

Topics: Animals; Ciliophora Infections; Cold Temperature; Fish Diseases; Immersion; Injections, Intraperitoneal; Lethal Dose 50; Muramidase; Poecilia; Tetrahymena

Edwardsiella tarda, a bacterial pathogen to farmed fish as well as humans, possesses the genes of two lysozyme inhibitors, ivy and mliC (ivy(Et) and mliC(Et)). We recently studied IvyEt and found it to be implicated in E. tarda virulence. In the present study, we characterized MliC(Et) in comparison with Ivy(Et) in a turbot model. MliC(Et) contains the FWSKG motif and two cysteines (C33 and C98) that are highly conserved in subgroup 1 MliCs but are of unknown functional importance. To examine the essentialness of these conserved structural features, recombinant MliC(Et) (rMliC) and its mutants bearing C33S and W79A (of the FWSKG motif) substitutions were prepared. Subsequent analysis showed that rMliC (i) inhibited lysozyme-induced lysis of a Gram-positive bacterium, (ii) reduced serum-facilitated lysozyme killing of E. tarda, and (iii) when introduced into turbot, promoted bacterial dissemination in fish tissues. The C33S mutation had no influence on the activity of rMliC, while the W79A mutation slightly but significantly enhanced the activity of rMliC. Knockout strains of either mliC(Et) or ivy(Et) were severely attenuated for the ability of tissue invasion, host lethality, serum survival, and intracellular replication. The lost virulence of the mliC transformant (TXΔmliC) was restored by complementation with an introduced mliC(Et) gene. Compared to the Δivy(Et) or ΔmliC(Et) single-knockout strains, the ΔmliC(Et) Δivy(Et) double-knockout strain was significantly impaired in most of the virulence features. Together, these results provide the first evidence that the conserved cysteine is functionally dispensable to a subgroup 1 MliC and that as a virulence factor, MliC(Et) most likely works in a concerted and parallel manner with Ivy.

Topics: Animals; Bacterial Proteins; Base Sequence; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Flatfishes; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Kidney; Monocytes; Muramidase; Recombinant Proteins; Sequence Analysis, DNA; Virulence Factors

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

This study was conducted to test the hypothesis that elevated dietary arginine enhances immunity of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed graded levels of dietary arginine for 9 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. Head kidney and spleen weights, as well as erythrocyte and leukocyte counts were significantly influenced by dietary arginine levels. A similar trend was also observed for hemagglutination titre, serum lysozyme activity, IgM concentration, C3 and C4 content. The highest survival rates following A. hydrophila infection were obtained in fish fed the diets containing arginine at 16.1-21.9 g/kg diet. Phagocytic activity of leukocytes was significantly enhanced by dietary arginine supplementation. In contrast, acid phosphatase activity significantly decreased with dietary arginine levels. Dietary arginine levels did not have a significant effect on the total iron-binding capacity. Gene expression of TNF-α and TGF-β in head kidney significantly increased with dietary arginine levels up to 21.9 g/kg diet, and decreased thereafter. Fish fed the basal diet exhibited the highest IL-10 mRNA expression level. Gene expression of IL-1β and TOR increased with dietary arginine addition, reaching a plateau at 18.5 and 21.9 g arginine/kg diet, respectively. In spleen, higher IL-1β and TNF-α gene expressions were obtained in fish fed the diets containing 24.5 g arginine/kg diet than in fish fed the other dietary treatments. TGF-β mRNA expression levels were significantly lower in fish fed the diets containing ≤21.9 g arginine/kg diet. IL-10 and TOR mRNA expression levels were lower in fish fed 16.1 g arginine/kg diet, while 4E-BP mRNA expression levels increased with dietary arginine levels up to 12.7 g/kg diet and decreased thereafter. Our results indicate that arginine has beneficial effects on regulating mRNA expression of inflammatory cytokines, as well as TOR and 4E-BP and improving humoral and cellular immunity, therefore enhancing disease resistance of fish.

Topics: Aeromonas hydrophila; Animals; Arginine; Carps; Complement C3; Complement C4; Cytokines; Diet; Disease Resistance; Erythrocyte Count; Fish Diseases; Fish Proteins; Gene Expression; Gram-Negative Bacterial Infections; Head Kidney; Immunoglobulin M; Leukocyte Count; Muramidase; Organ Size; Phagocytosis; RNA, Messenger; Spleen; TOR Serine-Threonine Kinases

Enzyme activities and gene expression of a number of innate immune parameters in the serum, mucus and skin of Atlantic salmon (Salmo salar) were investigated after challenge with a pathogenic strain of Aeromonas salmonicida (A. salmonicida). Fish were injected in the dorsal muscle with either 100 μl bacterium solution, about 3.05 × 10(7) CFU/ml A. salmonicida, or 100 μl 0.9% NaCl (as control group) and tissue samples were collected at days 0, 2, 4 and 6 post-injection. Lysozyme (LSZ) and alkaline phosphatase (AKP) activities in serum, mucus and skin, and LSZ and AKP mRNA expression in skin of the challenged fish were higher than those of the control at most of the experimental time, with significant differences at several time points (P < 0.05), indicating the involvement of LSZ and AKP in the innate immunity of Atlantic salmon to A. salmonicida. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in mucus and skin, along with the SOD, POD and CAT mRNA expression in skin significantly decreased at day 4 and 6, indicating the decreased antioxidant capacity of the challenged fish. Glutamate pyruvate transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activities in serum, mucus and skin of the challenged group were all higher than those of the control after the injection, and at several time points significant differences were found between the two groups, suggesting organs of fish were impaired after the pathogen infection. The changes of the GPT and GOT activities could be used as potential biomarkers for the impairment of physiological functions caused by the pathogen infection. Identified biomarkers of the immune responses will contribute to the early-warning system of the disease. So this study will not only provide a theoretical basis for vaccine development, but also provide basic data for the establishment of early warning systems for diseases caused by A. salmonicida in Atlantic salmon rearing.

Topics: Aeromonas salmonicida; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Catalase; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Mucus; Muramidase; Peroxidase; RNA, Messenger; Salmo salar; Skin; Superoxide Dismutase

To investigate the response of pompano fish (Trachinotus ovatus) to white spot disease, we used the protozoan Cryptocaryon irritans to infect live 450-g specimens at concentrations of 40,000 theronts/fish. We assessed the relative infection intensity (RII), serum immobilizing titer, and immunity-related enzyme activities (ACP, AKP, LZM), and assessed feeding, serum ion concentrations (Na(+), Cl(-), Ca(2+) and K(+)) and blood biochemistry (ALT, AST, LDH) of pompano. The fish were then treated with a lethal dose of C. irritans (70,000 theronts/fish) and the number of deaths was recorded. We found that the relative infection intensities of the control group, group I, and group II were 0, 0.630 ± 0.179, and 0.014 ± 0.006. Poly-infection induced a significant increase in the serum immobilizing titer (853.33 ± 295.60) of group II. In terms of the biochemical assessment, group II had significantly higher alkaline phosphatase and acid phosphatase activities than the other groups, and the lowest lysozyme activity (P < 0.05), compared to higher activity in the control group and the highest level in group I. Only the fishes of group I had stopped feeding after treatment. The concentrations of Na(+), Cl(-), and Ca(2+) in blood serum did not differ significantly among the three groups, but K(+) concentration increased with the increasing infection frequency. Alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities in fish of group II were significantly higher than those of the other groups. Survival of the fish subjected to the lethal dose of C. irritans was 0, 0, and 100 in groups control, I, and II, respectively. In conclusions, based on the food intake of group II, along with the results of relative infection intensity, serum immobilizing titer, and survival, we speculate that the fish in that group acquired high protective immunity following poly-infection by C. irritans, experiencing limited harm for pompano.

Topics: Acid Phosphatase; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Calcium; Chlorine; Ciliophora Infections; Fish Diseases; Fisheries; Fishes; L-Lactate Dehydrogenase; Muramidase; Potassium; Random Allocation; Seawater; Sodium

In the present study, KGM was degraded by H2O2 and HCl to obtain two products with different molecular weights: oxidized konjac glucomannan (OKGM, 4.7 × 10(5) Da) and low-molecular-weight oxidized konjac glucomannan (L-OKGM, 9.2 × 10(3) Da). The effects of the two OKGM products on IL-1β, TNF-α, and TLR22 gene expression, and immune parameters and the resistance to Aeromonas hydrophila of Schizothorax prenanti were determined. The results showed that the lysozyme activity was significantly enhanced by the L-OKGM diets. The SOD activity was significantly increased by both OKGM and L-OKGM diets. The MDA level of fish fed the OKGM and L-OKGM diets was significantly lower than the control group. IL-1β mRNA level in the spleen significantly increased in all L-OKGM fed groups. The 8.0 g kg(-1) L-OKGM diet also significantly up-regulated IL-1β gene expression in the head kidney. In the gut, IL-1β mRNA levels were significantly higher in fish fed with the 8.0 g kg(-1) OKGM and 16.0 g kg(-1) L-OKGM diets. The TNF-α mRNA level of L-OKGM group significantly increased in the spleen, head kidney and gut. High dosing of OKGM significantly up-regulated TNF-α transcription in the head kidney, while only the 8.0 g kg(-1) OKGM group showed significantly higher TNF-α mRNA expression in the mesonephros. Fish fed the L-OKGM diets showed significantly higher expression of TLR22 in the spleen, head kidney and mesonephros. After the injection of A. hydrophila, the 8.0 g kg(-1) L-OKGM group showed a significantly higher survival rate than did the control group. Present study suggests that OKGM and L-OKGM can up-regulate immune-related gene expression and enhance disease resistance in S. prenanti, and L-OKGM exhibits higher immunomodulatory activity.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Head Kidney; Interleukin-1beta; Intestinal Mucosa; Malondialdehyde; Mannans; Muramidase; RNA, Messenger; Spleen; Superoxide Dismutase; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund's Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.

Topics: Adhesins, Bacterial; Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Endopeptidases; Fish Diseases; Gene Expression; Ictaluridae; Immunity; Immunization; Muramidase; Streptococcus; Vaccines, Subunit

The present study aimed to investigate the effects of Chlorophytum borivilianum polysaccharide (CBP), as a dietary supplement administered at varying concentrations with feed (basal diet), on various cytokine-related responses in Labeo rohita fingerlings. Immune parameters and immune-related gene expressions were measured at 3rd, 4th, and 5th week after feeding. The results revealed that dietary administration of CBP at 0.2% and 0.4% for 4 weeks significantly upregulated serum lysozyme and phagocytic activity. Complement C3 and respiratory burst activity (RBA) were significantly higher after 4 weeks of CBP feeding. The immune related genes IL-8, IL-1β, TNF-α, and iNOS were downregulated (P < 0.05) in groups with 0.2% and 0.4% CBP supplemented diets at week 4. Expression of anti-inflammatory cytokines (IL-10 and TGF-β) was also downregulated (P < 0.5) after 4 weeks of feeding with 0.2% to 0.8% CBP. However, five weeks of CBP administration had no significant effect on immune gene expression, except TNF-α and IL-8. Fish fed with 0.4% CBP for 4 weeks showed maximum resistance against Aeromonas hydrophila (73.3% survival) compared to control. From these results, we recommend that CBP administration at 0.4% for 4 weeks could effectively improve immune response and disease resistance in L. rohita.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Complement C3; Cyprinidae; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Interleukin-10; Interleukin-1beta; Interleukin-8; Liliaceae; Muramidase; Nitric Oxide Synthase Type II; Phagocytosis; Polysaccharides; Respiratory Burst; Survival Analysis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The present study was designed to test the immunostimulatory efficacy of poly-β hydroxybutyrate-hydroxyvalerate (PHB-HV) extracted from Bacillus thuringiensis B.t.A102 on the immune system of Oreochromis mossambicus. Fish were fed with 0%, 1%, 3% or 5% PHB-HV supplemented feed and were bled at regular intervals of 5 days. The specific immune response was measured in terms of antibody response to sheep red blood cells, the nonspecific immune mechanisms were analysed in terms of serum lysozyme activity, total peroxidases activity and antiprotease activity. The overall functional immunity was tested by experimental challenge with live virulent Aeromonas hydrophila. The results revealed that all the doses of PHB-HV supplementation in feed were effective in stimulating both specific and nonspecific immune mechanisms. The bacterial challenge experiment showed that highest dose of 5% PHB-HV supplementation was more effective than 1% and 3% doses. The study concludes that PHB-HV can be used as a potential immunostimulant in finfish aquaculture.

Topics: Aeromonas hydrophila; Animals; Antibody Formation; Bacillus thuringiensis; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination Tests; Immunization; Male; Muramidase; Peroxidase; Polyesters; Protease Inhibitors; Spectroscopy, Fourier Transform Infrared; Tilapia

The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10(8) cell g(-1) of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Blood Proteins; Carps; Complement C3; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Immunity, Innate; Intestines; Microbiota; Muramidase; Phagocytosis; Random Allocation; Real-Time Polymerase Chain Reaction; Respiratory Burst; RNA

Lysozyme is an important component of the innate immune response against pathogen infection. The gene coding for c-type lysozyme in red-spotted grouper Epinephelus akaara was cloned and designated EaClys. The complete cDNA contains a 432 bp open reading frame encoding a protein of 144 amino acids displaying 65-91% similarity with the amino acid sequences of human, mouse, chicken, and fish counterparts. Recombinant EaClys (rEaClys) was expressed in Escherichia coli, displayed antibacterial activity against Gram-positive and Gram-negative bacteria, and possessed bactericidal activity against Vibrio alginolyticus. EaClys mRNA was constitutively expressed in all tested E. akaara tissues, and its expression increased after pathogen challenge. Most notably, challenges with LPS, SGIV or V. alginolyticus upregulated EaClys mRNA expression in the head, kidney, and blood. Its expression peaked between 16 and 24 h after challenge before dropping back to the baseline level. By using recombinant cytokines as signaling pathway mimetics and blocking antibodies and chemical inhibitors as pathway inhibitors, we show that LPS-induced lysozyme release from macrophages is promoted by cytokines TNF-α and IL-1β, and dependent on NF-κB pathway activation. These data suggest that EaClys is a constitutive and inducible acute-phase protein that is involved in the innate immune defense of E. akaara, and provide new clues about the molecular mechanisms that regulate innate immune responses in fish.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Fish Diseases; Iridovirus; Molecular Sequence Data; Muramidase; Perciformes; Phagocytosis; Phylogeny; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sequence Alignment; Sequence Analysis, DNA; Vibrio; Vibrio Infections

The gram-negative bacterium, Vibrio alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing brown-marbled grouper aquaculture, causing fish farmers globally to suffer substantial economic losses. The objective of this study was to investigate the proteins involved in the immune response of brown-marbled grouper fingerlings during their initial encounter with pathogenic organisms. To achieve this objective, a challenge experiment was performed, in which healthy brown-marbled grouper fingerlings were divided into two groups. Fish in the treated group were subjected to intraperitoneal injection with an infectious dose of V. alginolyticus suspended in phosphate-buffered saline (PBS), and those in the control group were injected with an equal volume of PBS. Blood samples were collected from a replicate number of fish from both groups at 4 h post-challenge and analysed for immune response-related serum proteins via two-dimensional gel electrophoresis. The results showed that 14 protein spots were altered between the treated and control groups; these protein spots were further analysed to determine the identity of each protein via MALDI-TOF/TOF. Among the altered proteins, three were clearly overexpressed in the treated group compared with the control; these were identified as putative apolipoprotein A-I, natural killer cell enhancement factor and lysozyme g. Based on these results, these three highly expressed proteins participate in immune response-related reactions during the initial exposure (4 h) of brown-marbled grouper fingerling to V. alginolyticus infection.

Topics: Animals; Apolipoprotein A-I; Fish Diseases; Gene Expression Regulation; Immunity, Innate; Muramidase; Perciformes; Vibrio alginolyticus; Vibrio Infections

Oreochromis niloticus bred in net cages were supplemented with cell wall of Saccharomyces cerevisiae (Sc) (0.3%) or chromium carbochelate (Cr) (18 mg/kg of feed) or in association (Sc + Cr), for 90 days. After this period, acute inflammation was induced in the swim bladder by inoculation of 3 × 10(8) CFU of inactivated Streptococcus agalactiae, and another group received 0.65% saline solution (control). Twelve, 24, and 48 h after stimulation, the inflammation was evaluated through total and differential counting of accumulated cells, and through leukocyte respiratory burst in the blood, cortisolemia, glycemia and serum lysozyme concentration. The results showed that there were greater total numbers of cells in the exudate of fish inoculated with inactivated bacterium than in those injected with saline solution, with predominance of lymphocytes, thrombocytes, macrophages and granulocytes. Tilapia supplemented with Cr presented increased total numbers of cells with significant accumulation of lymphocytes and reductions in cortisolemia and glycemia, but the different treatments did not have any influence on leukocyte respiratory burst or serum lysozyme concentration. Tilapia supplemented with Sc and the Cr + Sc association did not present significant changes to the variables evaluated, despite higher accumulation of lymphocytes in the inflammatory exudate from fish treated with Sc. The results indicate that tilapia bred in net cages and supplemented with Cr presented higher total accumulation of cells at the inflammatory focus, thus indicating an increase in the inflammatory response induced by the bacterium, probably due to the reduction in cortisolemia and higher glucose consumption. Thus, supplementation with Cr had beneficial action, which facilitated development of acute inflammation induced by the bacterium, but did not affect neither leukocyte respiratory burst in the blood nor serum lysozyme concentration.

Topics: Air Sacs; Animals; Blood Glucose; Chromium; Cichlids; Fish Diseases; Hydrocortisone; Muramidase; Probiotics; Random Allocation; Respiratory Burst; Respiratory Tract Infections; Saccharomyces cerevisiae; Streptococcal Infections; Streptococcus agalactiae

To explore the effect of low-dose Cryptocaryon irritans infection on growth, feeding and antiparasitic immunity of orange-spotted grouper (Epinephelus coioides), this study utilized C. irritans at concentrations of 5500 theronts/fish (Group I, 1/10 of 96 h LC50) or 11,000 theronts/fish (Group II) to infect E. coioides weighing 38 g on average at week 0, 2 and 4, respectively. Food consumption was recorded daily; the fish were weighed weekly; serum immobilizing titer (SIT), and acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM) activity were recorded every 2 weeks; the fish were treated with lethal dose (70,000 theronts/fish) of C. irritans in the 8th week and death number were recorded. The result shows that in the 1st week after the first infection, the fish's weight gain (WG), length gain (LG), and specific growth rate (SGR) dropped as parasite dose increased, and WG, SGR values were negative; while, after the 2nd and the 3rd infection, no significant differences were detected among the three groups. These results indicated that the 1st infection affected the fish most, while the following infections were protected by some immunity. In the 3rd, 7th, and 8th week, condition factor (CF) increased with the increased infectious dose, indicating that the parasite affected body length more than body weight. As the experiment went on, accumulated food consumption (AFC) of all three groups steadily grew (control > Group I > Group II). But on the 2nd day after the first infection, daily food consumption (DFC) of Group I and II significantly dropped, the decline of Group II was greater than that of Group I, DFC recovered in the following week, with Group I earlier than Group II. After the 2nd infection, DFC of Group I and II dropped again, Group II still dropped more than Group I, and both groups recovered on the 3rd day after infection. The 3rd infection caused no significant difference in week food consumption (WFC). These results indicated that a higher dose of infection causes a greater drop in FC and a slower recovery. Weekly feed conversion ratio (WFCR) values of Group I and II in the 1st week was negative; in the 2nd week, WFCR was lower in the group infected by a higher dose of parasite; while in the 3rd and following weeks, no significant pattern was observed. Accumulate feed conversion ratio (AFCR) dropped as the infectious dose increased (control > Group I > Group II), AFCR of Group I and II reached above 0 i

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Body Weight; Ciliophora Infections; Eating; Fish Diseases; Hymenostomatida; Muramidase; Perciformes; Superoxide Dismutase

This study investigated the effects of long-term heat exposure on Japanese flounder (Paralichthys olivaceus) and its hybrids (P. olivaceus ♀ × summer flounder Paralichthys dentatus ♂). From 24 ± 0.5°C, temperature was increased by 1 ± 0.5°C in a day and was kept at that temperature for 5 days before next rise. Cumulative survival rate (CSR), cumulative survival rate under different temperature (CSR-T), histological alteration, and related enzyme activities were investigated. In P. olivaceus, mass mortality occurred at 29 and 32 °C (the CSR-T dropped to 42.39%), and serious gill damages appeared at 30 and 32°C. Meanwhile, the activities of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), and pyruvate kinase (PK) declined around 29 and 32°C (except for CAT). In comparison with P. olivaceus, the CSR of the hybrids was higher, the gill kept a better structural integrity, and the activities of SOD, CAT, LZM, and PK showed tiny fluctuations. The results suggested that during the process of chronic heat stress, P. olivaceus seemed to be more sensitive to 29 and 32°C, and the manifestations in survival, histology, and enzyme activity were generally consistent. For the hybrids, the comparatively insensitivity to high temperature might imply its better heat tolerance.

Topics: Analysis of Variance; Animals; Catalase; Fish Diseases; Flounder; Gills; Heat Stress Disorders; Hybridization, Genetic; Muramidase; Pyruvate Kinase; Species Specificity; Superoxide Dismutase; Survival Analysis; Temperature

The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund's complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Cell Proliferation; Cyprinidae; Edwardsiella tarda; Enterobacteriaceae Infections; Enzymes; Fish Diseases; Immunity, Humoral; Immunity, Mucosal; Immunization; Leukocytes; Macrophage-Activating Factors; Mucus; Muramidase; Peroxidase; Survival Analysis

This study was carried out to investigate the effect of an acute stressor on the variation of some physiological and immunological parameters of Siberian sturgeon (Acipenser baerii) juveniles. Fish, reared in 3 tanks for 10 weeks, were used for this study. The acute stress of fish consisted of 2 min of air exposure stress. Plasma levels of cortisol, glucose, and lactate as well as lysozyme activity in plasma were measured before stress and 1 hr, 3 hr, 6 hr, 9 hr, 12 hr, and 24 hr after stress. The plasma cortisol significantly increased in the highest level 1 hr after stress, yet it gradually declined after 3 hr. The glucose significantly increased only 1 hr after stress. There was no significant difference between plasma lactate prestress and poststress. Moreover, lysozyme activity was enhanced by stress, thus reaching the highest level 9 hr after stress. The results of this study indicate that Siberian sturgeon not only have a rapid response to acute stress, but also a great capacity for recovery from stress, thus returning physiological parameters to prestress levels after 6 hr.

Topics: Animals; Blood Glucose; Female; Fish Diseases; Fishes; Hydrocortisone; Lactic Acid; Male; Muramidase; Stress, Physiological

This study investigates the effects of prebiotic Immunogen on lysozyme, TNFα and HSP70 gene expression in head kidney, humoral innate immune parameters and resistant to Aeromonas hydrophila of rainbow trout. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two groups fed control or diet supplemented with 2 g kg(-1) Immunogen for 45 days. The results revealed that administration of Immunogen significantly (P < 0.05) up regulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly (P < 0.05) lower in Immunogen fed fish at the end of trial. Humoral innate immune parameters (lysozyme activity, ACH50 and bactericidal activity) were significantly (P < 0.05) increased whether 15 or 45 days after feeding on Immunogen supplemented diet. However, significant (P < 0.05) increase in agglutination antibody titer observed just after 45 days feeding on Immunogen. Rainbow trout fed with 2 g kg(-1) Immunogen showed remarkably higher resistance against A. hydrophila (64.44% survival) compared to the control group (24.44% survival). These results confirm that Immunogen can up regulates immune related genes expression, stimulates immune response that per se enhances disease resistance in rainbow trout.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Hematologic Tests; HSP70 Heat-Shock Proteins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Prebiotics; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Evaluation of functional mechanism of immunostimulant in fish is essential for realizing their therapeutic potential. The impact of dietary supplementation of seeds of Achyranthes aspera on the immune system of Catla catla was studied. Catla (37.73 ± 6.7 g) were fed with test diets containing 0.5 (D1) and 1.0% (D2) seeds of A. aspera and control diet (D3) for 50 days. Then fish were immunized with heat-killed Aeromonas hydrophila. Blood and tissue samples were collected after 7 days of immunization. Three fish were sacrificed for each parameter. Respiratory burst activity, bactericidal activity and antibody response were significantly (P < 0.05) higher in test diets fed catla compared to the control one. Nitric oxide synthase and serum lysozyme were significantly (P < 0.05) higher in fish fed with diet D2 compared to the other treatments. There were up-regulations of TNFα, lysozyme c and g gene expressions in kidney and IL-10, lysozyme c and g expressions in hepatopancreas of test diets fed fish compared to the control one. The expression of IL-10 was down-regulated in the kidney of D2 diet fed fish compared to others. In the gill, significantly (P < 0.05) higher expressions of TNFα was found in D1 diet feed fish and lysozyme c and in D2 diet fed fish compared to the other groups. This study showed tissue-specific gene expression pattern in catla. An inverse relationship was found between the expression of TNFα and IL-10 in kidney of catla.

Topics: Achyranthes; Aeromonas hydrophila; Animals; Aquaculture; Carps; Fish Diseases; Gene Expression Regulation; Gills; Gram-Negative Bacterial Infections; Hepatopancreas; Immunomodulation; Interleukin-10; Kidney; Muramidase; Nitric Oxide Synthase; Random Allocation; Respiratory Burst; Tumor Necrosis Factor-alpha

The effect of diet enriched with 1% chitin or chitosan on innate immune response and disease resistance in Cirrhina mrigala against Aphanomyces invadans was investigated at weeks 1, 2, and 4. In the un-infected and infected groups the white blood cells (WBC), red blood cells (RBC), haematocrit (Ht), lympocytes, monocytes, and neutrophils significantly increased when fed with 1% chitin (CH) or chitosan (CT) enriched diet from weeks 1 to 4 when compared to control; however, the haemoglobin (Hb) and thrombocytes significantly increased only on weeks 2 and 4. The total protein and albumin levels also significantly increased with any enriched diet on weeks 2 and 4; but the globulin and albumin:globulin ratio increased on week 4 as compared to control; similarly the phagocytic activity significantly increased on weeks 2 and 4 while the lysozyme activity increased from weeks 1 to 4. The complement activity was significantly enhanced in CT and CTI fed groups on weeks 2 and 4. In un-infected fish fed with 1% CH and CT diets, the cumulative mortality was 10% and 5% whereas the infected fish suffered 20% and 25% mortality. The present results suggest that infected fish fed with 1% chitin or chitosan enriched diet modulates the immune system conferring disease resistance in C. mrigala against A. invadans.

Topics: Analysis of Variance; Animals; Aphanomyces; Blood Cell Count; Chitin; Chitosan; Complement System Proteins; Cyprinidae; Dietary Supplements; Disease Resistance; Fish Diseases; Hematocrit; Immunity, Innate; Infections; Muramidase; Nephelometry and Turbidimetry; Phagocytosis

The effects of a Chinese herbal mixture (CHM) composed of astragalus, angelica, hawthorn, Licorice root and honeysuckle on immune responses and disease resistant of Nile tilapia (Oreochromis niloticus GIFT strain) were investigated in present study. Fish were fed diets containing 0 (control), 0.5%, 1.0%, 1.5% or 2.0% CHM (w/w) for 4 weeks. And series of immune parameters including lysozyme, cytokine genes TNF-α and IL-1β, superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA) were measured during test period. After four weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Nile tilapia with CHM-supplementation diet stimulated lysozyme activity, SOD activity and POD activity in serum, induced TNF-α and IL-1β mRNA expression in head kidney and spleen, but decreased serum MDA content. All CHM-supplemental groups showed reduced mortalities following A. hydrophila infection compared with the group fed the control diet. These results suggested that this CHM can be applied as a tilapia feed supplement to elevate fish immunity and disease resistance against A. hydrophila.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Analysis of Variance; Animal Feed; Animals; Cichlids; Dietary Supplements; DNA Primers; Drugs, Chinese Herbal; Fish Diseases; Gram-Negative Bacterial Infections; Malondialdehyde; Muramidase; Peroxidase; Real-Time Polymerase Chain Reaction; Superoxide Dismutase

Despite the importance and success of developing a candidate vaccine against Aeromonas hydrophila infection in fish, little is known about the molecular mechanisms of the vaccine-induced immunoprotection in Indian major carp, Labeo rohita, primarily due to lack of information on most of the immune related genes of the species. In this study, a novel candidate antigen recombinant outer membrane protein R (rOmpR) of A. hydrophila was evaluated as a vaccine candidate along with a modified adjuvant formulation. Protective efficacy of the rOmpR immunization was assessed in terms of survival against A. hydrophila challenge as well as modulation of immune response in vaccinated fish after 1, 3, 6, 12, 24, 72 h and 10 days post-injection (using immune gene expression analysis) and 10, 28, 56 and 140 days post-injection (serum immune parameter analysis). The generated immune response was compared with a formalin-killed A. hydrophila antigen preparation using mineral oil only and modified adjuvant alone. We report a variable up-regulation of the immune-related genes viz., lysozyme G, complement factor 4, immunoglobulin M, β2-microglobulin, major histocompatibility complex I and II, and interleukin-1β in anterior kidney and spleen tissues at early time points post-immunization in all the groups, when compared to the control fish. The vaccinated fish also showed an increase in serum natural hemolysin titer, lysozyme and myeloperoxidase activities, and antibody titer irrespective of vaccine formulations as compared to control fish on days 10, 28 and 56. However, the increase in the serum parameters was more pronounced on day 140 in rOmpR-modified adjuvant injected group, indicating the modulatory role of this new vaccine formulation. Upon challenge with live A. hydrophila on days 56 and 140 post-immunization, significantly reduced percent mortality was noted in the group immunized with modified adjuvant based rOmpR vaccine formulation. Taken together, our results suggest that rOmpR along with modified adjuvant could potentially be used as a vaccine formulation to handle A. hydrophila infection on a long-term basis.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Carps; DNA Primers; DNA, Complementary; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Peroxidase

The fish pathogenic oomycete Aphanomyces invadans is the causative agent of epizootic ulcerative syndrome (EUS), a fish disease of international significance and reportable to the World Organisation for Animal Health. In spite of the current and potential impact of A. invadans infection on fisheries and aquaculture sectors of the world, very little is known about the host-A. invadans interactions. In the present study, following experimental infection with A. invadans in one of the Indian major carps, Labeo rohita, sequential changes in various innate immune parameters were monitored. The results indicated that at early stages of infection, no significant changes in any of the studied innate immune parameters were observed. However, at the advanced stages of infection from 6 to 12 days post infection (dpi), the respiratory burst and alternate complement activity were significantly higher whereas lysozyme, antiproteases and α-2 macroglobulin values were significantly lower than the control group and also from the infected group at earlier stages of infection. Since, the possibility of vaccination of fish against A. invadans appears remote due to difficulties in eliciting a specific antibody response, the information generated in the present study could be useful for developing strategies for improving resistance to A. invadans infection by stimulating the innate immunity through immunomodulation.

Topics: Analysis of Variance; Animals; Aphanomyces; Carps; Complement Pathway, Alternative; Fish Diseases; Immunity, Innate; Infections; Muramidase; Protease Inhibitors; Respiratory Burst; Serum Albumin; Serum Globulins

The present study was conducted to investigate the effects of dietary carbohydrate (CHO) levels on non-specific immune responses, hepatic antioxidative status and disease resistance of juvenile golden pompano. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO levels for 8 weeks. After the feeding trial, fish were challenged by Vibrio harveyi and survival rate was recorded for the next 12 days. Plasma total protein and albumin content, respiratory burst activity, alkaline phosphatase, slightly increased with dietary starch level from 0% to 16.8%, but significantly decreased at dietary starch levels of 16.8%-28%. Plasma lysozyme, complement 3 and complement 4 levels increased with increasing dietary carbohydrate up to 11.2% and then declined (P < 0.05). Contrary to glutamic-oxalacetic transaminase and triiodothyronine, plasma cortisol content increased with increasing dietary carbohydrate up to 22.4%, and then levelled off. The hepatic total antioxidative capacity, reduced glutathione and catalase levels reached the peak at the fish fed diet with 16.8% carbohydrate (P < 0.05). This also held true for hepatic superoxide dismutase activities, whereas the hepatic malondialdehyde content of fish fed dietary starch level of 16.8% was significantly lower than that of fish fed no CHO diet, but showed little difference (P > 0.05) with those of the other treatments. After challenge, fish fed 11.2% and 16.8% dietary CHO showed higher survival rate than that of fish in 0% CHO group (P < 0.05). However, survival rate showed little difference among 0%, 5.6%, 22.4% and 28% CHO groups (P > 0.05). The results of this study suggest that ingestion of 11.2-16.8% dietary CHO can enhance the non-specific immune responses, increase the hepatic antioxidant abilities, and improve resistance to V. harveyi infection of juvenile golden pompano.

Topics: Analysis of Variance; Animals; Catalase; Complement System Proteins; Dietary Carbohydrates; Disease Resistance; Fish Diseases; Glutathione; Hydrocortisone; Liver; Muramidase; Perciformes; Respiratory Burst; Superoxide Dismutase; Vibrio Infections

This trial was carried out to investigate the effects of dietary administration of Vitacel(®), a commercial fermentable fiber, on immune related genes (Lysozyme, TNFα and HSP70) expression, innate immune response and resistance of rainbow trout against Aeromonas hydrophila. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two treatments. The treatments were feeding rainbow trout with diets supplemented with 0 (control) or 10 g kg(-1) Vitacel(®) for 45 days. The results revealed that administration of fermentable fiber significantly (P < 0.05) upregulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly lower in Vitacel(®) fed fish at the end of trial (P < 0.05). Furthermore dietary administrations of Vitacel(®) remarkably elevated rainbow trout innate immune parameters include serum lysozyme, ACH50, bactericidal activity and agglutination antibody titer (P < 0.05). Administration of 10 g kg(-1) Vitacel(®) significantly increased rainbow trout resistance against A. hydrophila (P < 0.05). The results of present study revealed that dietary Vitacel(®) can upregulates immune related genes expression and elevates innate immune response and disease resistance of rainbow trout.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Dietary Fiber; Disease Resistance; DNA Primers; Fermentation; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; HSP70 Heat-Shock Proteins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5-8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach.

Topics: Alkaline Phosphatase; Animals; Antibodies, Viral; Aquaculture; Carps; China; Cloning, Molecular; Complement System Proteins; DNA Primers; Drug Delivery Systems; Fish Diseases; Immunization; Muramidase; Nanotubes, Carbon; Real-Time Polymerase Chain Reaction; Reoviridae; Reoviridae Infections; Respiratory Burst; Reverse Transcriptase Polymerase Chain Reaction; Superoxide Dismutase; Viral Vaccines

Fucoidan is sulfated polysaccharide extracted from seaweed brown algae. This study was designed to evaluate the immunomodulatory effects and disease resistance of dietary fucoidan on catfish, Clarias gariepinus, immunosuppressed by cadmium. Three hundred and sixty African catfish, C. gariepinus, was allocated into six equal groups. The first group served as a control. Groups (F1 and F2) were fed on fucoidan supplemented ration at concentrations of 4 and 6g/kg diet respectively for 21 days. Groups (Cd, CdF1 and CdF2) were subjected throughout the experiment to a sub-lethal concentration of 5ppm cadmium chloride solution and groups (CdF1 and CdF2) were fed on a ration supplemented with fucoidan. Macrophages oxidative burst, phagocytic activity percentages and lymphocytes transformation index were a significant increase in the fucoidan-treated groups (F1 and F2), while serum lysozyme, nitric oxide and bactericidal activity were enhanced only in group (F2) when compared with controls. These parameters as well as absolute lymphocyte count and survival rate were significantly increased in group (CdF2) when compared with cadmium chloride immunosuppressed group (Cd). It could be concluded that the fucoidan can be used as immunostimulant for the farmed African catfish, C. gariepinus as it can improve its resistance to immunosuppressive stressful conditions.

Topics: Aeromonas hydrophila; Animals; Aquaculture; Cadmium Chloride; Catfishes; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Immunocompromised Host; Immunosuppressive Agents; Kaplan-Meier Estimate; Leukocyte Count; Muramidase; Nitric Oxide; Phagocytosis; Polysaccharides; Random Allocation; Respiratory Burst

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

Lysozyme is an important component of the innate immune system. In this study, four lysozyme genes including one c-type lysozyme and three g-type lysozymes were identified from channel catfish (Ictalurus punctatus). The lysozyme genes are highly conserved in their structural features as compared to those from other species. Phylogenetic analyses were conducted allowing annotation of these genes. Additional analyses using conserved syntenies allowed determination of orthologies for the c-type lysozyme. Phylogenetic analysis indicated that the g-type lysozyme may have gone through species-specific gene duplications leading to multiple copies in some teleost species. Channel catfish possessed three copies of the g-type lysozyme genes. Expression analysis revealed that the catfish lysozyme genes were expressed in a broad range of tissues. The highest levels of expression were found in head kidney, liver, spleen, and trunk kidney, compatible with the immune functions of these tissues/organs. The c-type and g-type lysozymes were drastically induced after bacterial infection, but exhibited large differences in the extent of induction and the tissue with the highest level of induction, with the g-type lysozyme being most highly induced in the head kidney whereas the other three lysozymes being most highly induced in the liver, suggesting their cooperative actions in the immune responses but difference in their detailed functions.

Topics: Amino Acid Sequence; Animals; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Fish Proteins; Ictaluridae; Muramidase; Organ Specificity; Phylogeny; Polymerase Chain Reaction; Sequence Alignment

Streptococcus parauberis causing systemic infections has been recognized as a major bacterial disease in olive flounder Paralichthys olivaceus in South Korea. Although an emerging outbreak of S. parauberis has affected heavily farmed fish species starry flounder Platichthys stellatus, no study of the innate immune responses and pathogenic mechanisms in starry flounder is available. In the present study, starry flounder were intraperitoneally challenged with four S. parauberis strains to investigate changes in innate immune responses. Significant increases in serum lysozyme activities, superoxide production of kidney leucocytes, and serum superoxide dismutase activities were observed following experimental injection of S. parauberis. All these data suggested that the innate immune parameters were highly modulated during the S. parauberis infection process to render protection to the starry flounder. However, S. parauberis also exhibited the mechanisms to complete disease establishment by avoiding host immune responses. S. parauberis could survive and proliferate in the mucus, serum and kidney leucocytes of starry flounder. In particular, the strain isolated from the starry flounder showed the higher survival ability than other originated strains in the tested host fish.

Topics: Animals; Fish Diseases; Flounder; Immunity, Innate; Injections, Intraperitoneal; Kidney; Leukocytes; Muramidase; Nitroblue Tetrazolium; Organ Specificity; Streptococcal Infections; Streptococcus; Superoxide Dismutase

To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Ictaluridae; Muramidase; Nitric Oxide; Phylogeny; Reactive Oxygen Species

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenic E. tarda strain TX01. IvyEt possesses the Ivy signature motif CKPHDC in the form of (82)CQPHNC(87) and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivyEt gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEt is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Cysteine; Edwardsiella tarda; Fish Diseases; Flatfishes; Gene Expression Regulation, Bacterial; Muramidase

The freshwater bacterial pathogen, Flavobacterium columnare, infects a variety of ornamental and farmed fish species worldwide through mucosal attachment points on the gill and skin. While previous studies have demonstrated a chemotactic response of F. columnare to fish mucus, little is known about how host gill mucosal molecular and cellular constituents may impact rates of adhesion, tissue invasion, and ultimately, mortality. Here, we describe the use of RNA-seq to profile gill expression differences between channel catfish (Ictalurus punctatus) differing in their susceptibility to F. columnare both basally (before infection) and at three early timepoints post-infection (1 h, 2 h, and 8 h). After sequencing and de novo assembly of over 350 million 100 base-pair transcript reads, between group comparisons revealed 1714 unique genes differentially expressed greater than 1.5-fold at one or more timepoints. In the large dataset, we focused our analysis on basal differential expression between resistant and susceptible catfish as these genes could potentially reveal genetic and/or environmental factors linked with differential rates of infection. A number of critical innate immune components including iNOS2b, lysozyme C, IL-8, and TNF-alpha were constitutively higher in resistant catfish gill, while susceptible fish showed high expression levels of secreted mucin forms, a rhamnose-binding lectin previously linked to susceptibility, and mucosal immune factors such as CD103 and IL-17. Taken together, the immune and mucin profiles obtained by RNA-seq suggest a basal polarization in the gill mucosa, with susceptible fish possessing a putative mucosecretory, toleragenic phenotype which may predispose them to F. columnare infection.

Topics: Animals; Disease Resistance; Fish Diseases; Fish Proteins; Flavobacterium; Gills; Host-Pathogen Interactions; Ictaluridae; Interleukin-8; Mucus; Muramidase; Nitric Oxide Synthase Type II; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, RNA; Time Factors; Transcriptome; Tumor Necrosis Factor-alpha

The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Aquaculture; Cyprinidae; Dietary Supplements; Disease Resistance; Diterpenes; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Nitroblue Tetrazolium; Peroxidase; Phagocytosis; Protease Inhibitors

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

Topics: Actinomycetales Infections; Adjuvants, Immunologic; Aeromonas hydrophila; Amino Acid Sequence; Animals; Cloning, Molecular; DNA, Complementary; Escherichia coli; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Ictaluridae; Micrococcus; Molecular Sequence Data; Muramidase; Phylogeny; Plasmids; Real-Time Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Sequence Alignment

To determine whether transcriptional levels of channel catfish (Ictalurus punctatus) genes are differentially regulated between a first infection with Aeromonas hydrophila and a re-infection, suppression subtractive hybridization (SSH) was performed in this study using anterior kidney cDNA after the re-infection as tester. Of the 96 clones isolated from the SSH library, 28 unique expressed sequence tags (ESTs) were obtained, of which eight were confirmed to be slightly but significantly (P < 0.05) more up-regulated by the re-infection at 6 h post infection (hpi). Expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the eight ESTs were significantly (P = 0.016) more up-regulated by the first infection, with a major peak at 3 hpi. A total of 96 genes reported in literature to be up-regulated by bacterial infections were selected and subjected to expression analysis at 3 hpi. Of the 96 selected genes, 19 were found to be significantly (P < 0.05) induced by A. hydrophila after the first infection and the re-infection. The 19 genes belonged to the following five main categories: 1) toll-like receptor (TLR2, TLR3, TLR5, TLR21); 2) antimicrobial peptide (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, cathepsin D, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1β, interleukin-10, tumor necrosis factor α, chemokine CXCL-10); 4) signaling proteins (cadherin EGF LAG seven-pass G-type receptor 1, very large inducible GTPase 1, arginine deiminase type 2, lymphokine-activated killer T-cell originated protein kinase); 5) lysozyme (lysozyme c). Overall, the total 27 genes (8 ESTs plus the 19 selected genes) were significantly (P < 0.001) more induced by the first infection. Peaked expression of lysozyme c and serum lysozyme activity after the first infection were seen at 24 hpi, whereas that after the re-infection were seen at 12 hpi, suggesting that both innate and adaptive immunity were involved in the defense against the re-infection of A. hydrophila.

Topics: Aeromonas hydrophila; Animals; Antimicrobial Cationic Peptides; Base Sequence; Cytokines; DNA Primers; Expressed Sequence Tags; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gene Library; Gram-Negative Bacterial Infections; Ictaluridae; Molecular Sequence Data; Muramidase; Sequence Analysis, DNA; Statistics, Nonparametric; Toll-Like Receptors

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

A bacterial strain isolated from intestines of hybrid catfish (Clarias gariepinus Male × Clarias macrocephalus Female) exhibited an in vitro inhibitory effect on a fish pathogen, Aeromonas hydrophila TISTR 1321. By using the 16S rDNA sequence analysis, it was identified as Lactobacillus plantarum C014. To examine whether L. plantarum C014 had potential for use as an immunostimulant and biocontrol agent in hybrid catfish, the fish diet supplemented with L. plantarum C014 (10(7) CFU/g diet) was prepared and used for the in vivo investigation of its effect on innate immune response and disease resistance of hybrid catfish. Two innate immune response parameters, phagocytic activity of blood leukocytes and plasma lysozyme activity, were significantly enhanced in the treated fish after 45 days of feeding. Feeding the fish with the L. plantarum C014 supplemented diet for 45 days before challenging them with A. hydrophila at the dose of LD50 could reduce the mortality rate of the fish from 50% (in control group) to 0% (in treated group). Based on its origin and beneficial effect on innate immune response and disease resistance, L. plantarum C014 may be a potential candidate for use as a natural and safe immunostimulant and biocontrol agent in hybrid catfish.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Base Sequence; Catfishes; Dietary Supplements; Disease Resistance; DNA Primers; Fish Diseases; Gram-Negative Bacterial Infections; Hybridization, Genetic; Immunity, Innate; Lactobacillus plantarum; Lethal Dose 50; Molecular Sequence Data; Muramidase; Phagocytosis; Probiotics; RNA, Ribosomal, 16S; Sequence Analysis, DNA

In this study, the antibacterial properties and active ingredient of plant extracts and its effect on the performance of crucian carp (Carassius auratus gibelio var. E'erqisi, Bloch) were assessed.. The transmission electron microscopy and flow cytometric analysis showed that the antibacterial activity of plant extracts is due to the disruption of the cell membrane and the leakage of cytoplasmic contents. The UPLC-MS/MS analysis showed that the contents of gallic acid, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin gallate, aloe-emodin, rhein, emodin, chrysophanol, and physcion, were 5.27%, 3.30%, 1.08%, 19.32%, 5.46%, 0.23%, 0.56%, 1.28%, 0.75% and 0.39% in plant extracts, respectively. Results of feeding experiment showed that feeding crucian carp with 1.0% and 2.0% plant extracts significantly enhanced specific growth rate, serum total protein, lysozyme, catalase and superoxide dismutase activities, and decreased the feed conversion rate, malondialdehyde contents and the mortality rate (P < 0.05).. It can be concluded that plant extracts added to fish feed can act as natural antimicrobial and immunostimulants to prevent pathogenic infection, enhance immune response, and promote growth of the fish.

Topics: Adjuvants, Immunologic; Aeromonas; Animal Feed; Animals; Anti-Bacterial Agents; Blood Proteins; Carps; Catalase; Cell Membrane; Cytoplasm; Energy Metabolism; Fish Diseases; Growth; Immunity; Infections; Malondialdehyde; Muramidase; Plant Extracts; Plant Tumors; Rheum; Rhus; Superoxide Dismutase

Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Fish Proteins; Flatfishes; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Immunoblotting; Muramidase; Organ Specificity; Plasmids; Polymerase Chain Reaction; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Species Specificity

This study evaluated the effects of dietary cholesterol on antioxidant capacity, non-specific immune response and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean meal-based diets. Fish were fed diets supplemented with graded cholesterol levels (0 [control], 0.3, 0.6, 0.9, 1.2, and 1.5%) for nine weeks. The fish were then challenged by A. hydrophila and their survival rate recorded for the next week. Dietary cholesterol supplementation generally increased the serum and hepatic superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (TAC) activities, but decreased the serum and hepatic malondialdehyde (MDA) contents. Further, the hepatic CAT and serum SOD, CAT, and TAC activities were significantly higher in fish fed diets supplemented with 0.9 or 1.2% cholesterol compared to those fed the control diet, whereas the serum and hepatic MDA contents were significantly lower. The respiratory burst activity, alternative complement activity, and hepatic lysozyme activity increased steadily when the supplemental cholesterol was increased by up to 1.2% and then declined with further addition. The serum lysozyme activity and phagocytic activity increased steadily with increasing dietary supplemental cholesterol level up to 0.9% and then declined with further addition. Dietary cholesterol supplementation generally enhanced the protection against A. hydrophila infection, and fish fed diets supplemented with 0.9 or 1.2% cholesterol exhibited the highest post-challenge survival rate. The results indicated that cholesterol may be under-supplied in rainbow trout fed soybean meal-based diets, and dietary cholesterol supplementation (0.9-1.2%) contributed to improved immune response and disease resistance of rainbow trout against A. hydrophila.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Antioxidants; Aquaculture; Cholesterol; Complement Pathway, Alternative; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Respiratory Burst

Topics: Animals; China; Cichlids; Fish Diseases; Immunity, Innate; Muramidase; Nitroblue Tetrazolium; Phagocytosis; Species Specificity; Streptococcal Infections; Streptococcus agalactiae

Lesions ranging from surface wounds to deep tissue wounds caused by cormorant predation were observed on several species of the farmed fish in Pohořelice, Czech Republic. Two-year-old stocked common carp Cyprinus carpio harvested in late March were examined for ectoparasites and endoparasites, injuries extent, and lysozyme concentration in skin mucus. Additionally, three body condition indices were measured. Endoparasite infection occurred only scarcely. Wounded fish were more susceptible to the ectoparasites Gyrodactylus spp. and Dactylogyrus spp. (Monogenea), and Ichthyophthirius multifiliis (Ciliophora). The intensity of infection of other ectoparasites Eudiplozoon nipponicum (Monogenea), Argulus spp. (Branchiura) and trichodinids (Ciliophora) did not significantly differ between wounded and control groups of fish. Lysozyme concentration in fish mucus was significantly higher in wounded fish and was positively associated with both the extent of damaged epithelium and Gyrodactylus spp. abundance. There were no differences in Fulton's condition factor and lipid content in muscle and liver tissues between wounded and non-wounded fish. Higher values of spleen-somatic index in wounded fish corresponded to increased intensity of parasite infection, most likely reflecting changes in immune system of infected fish. Although our results did not show any significant effect of cormorant attacks on fish condition, the wounded fish had significantly higher parasite numbers which could impact the growth or survival of the fish throughout the production season.

Topics: Animals; Aquaculture; Birds; Carps; Czech Republic; Disease Susceptibility; Ectoparasitic Infestations; Fish Diseases; Fresh Water; Linear Models; Muramidase; Predatory Behavior; Seasons; Wounds and Injuries

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

Three different antigenic preparations from the epizootic ulcerative syndrome (EUS) pathogen Aphanomyces invadans were evaluated as vaccine candidate in catla (Catla catla). Anti-catla enzyme immunoconjugate was prepared after isolating catla immunoglobulin and raising hyperimmune sera against it, in rabbit. Three antigens namely, fungal extract (FE), fungal extract mixed with Freund's incomplete adjuvant (FIA) in a 1:1 (v/v) ratio (FE + A) and extra cellular product (ECP) were prepared and three groups of catla were vaccinated intramuscularly with all these antigens (200 μg/fish). Different cellular and humoral immune responses were measured for the entire vaccinated and control group on 0th, 5th, 15th and 25th day post vaccination. Thirty days after the vaccination, the fish were challenged with an A. invadans zoospore dose of 1 × 10(5) ml(-1) and mortality and relative percent of survival (RPS) were recorded. Study of cellular immunological parameters including antigen-specific leukocyte proliferation, antigen-specific nitric oxide production and superoxide anion production showed significantly higher (p < 0.05) values, in general, on 5th and 15th day post vaccination than the 0th day. Among all the antigenic groups, FE + A showed most significant response compared to the other groups. Among the humoral immune responses, lysozyme activity showed almost similar trend like cellular parameters. Anti-Aphanomyces antibody production was measured by enzyme-linked immunosorbent assay (ELISA) and it was increased with increasing days of vaccination in all the vaccinated groups with the highest observed on 25th day. Among the antigens, FE + A showed the highest antibody production following vaccination. The result of the homologous pathogen challenge study showed reduction in mortality in all the vaccinated groups. However, this reduction was not statistically significant (p > 0.05). Increased immune responses and protection have important implications with regard to the control of EUS by vaccination.

Topics: Animals; Antibodies; Aphanomyces; Cell Proliferation; Cyprinidae; Fish Diseases; Leukocytes; Muramidase; Nitric Oxide; Superoxides; Vaccination; Vaccines

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Ciliophora Infections; Disease Susceptibility; Energy Metabolism; Fish Diseases; Gene Expression Regulation; Hymenostomatida; Mucus; Muramidase; Oncorhynchus mykiss; Skin; Time Factors

In this study, we investigated the immune enhancing effects of different adjuvants used in a pentavalent vaccine for turbots. The pentavalent vaccine consisted of inactive bacterial cells from five common pathogenic strains (Vibrio anguillarum, Vibrio scophtalmi, Edwardsiella tarda, Vibrio harveyi and Vibrio alginolyticus) and the adjuvants were astragalus polysaccharides (APS), propolis, and the Freund's complete adjuvant (FCA). Turbots were immunized with the pentavalent vaccine alone or with one of the adjuvants, and the immune efficiency was evaluated by measuring the activities of lysozyme (LSZ) and superoxide dismutase (SOD), and serum antibody titers. Fish were also challenged with the pathogens after immunization and the relative percent survival (RPS) was assessed. Our results showed that APS, propolis, and FCA had significant immune-enhancing effects on turbots as shown by the higher titers of antibodies against the pathogens, increased LSZ and SOD activities, and enhanced RPS after challenge with pathogens. Among the three adjuvants, FCA had the most significant immune synergistic effects with the vaccine, and APS and propolis had lower and similar immune synergies.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bacterial Infections; Bacterial Vaccines; Enterobacteriaceae; Enterobacteriaceae Infections; Fish Diseases; Flatfishes; Immunity; Immunization; Muramidase; Superoxide Dismutase; Survival Analysis

The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study.. Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR.. The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, β-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

Topics: Animals; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Flounder; Hemoglobins; Immunoglobulin M; Muramidase; Petroleum; Real-Time Polymerase Chain Reaction; Respiration; Water Pollutants, Chemical

The present study was conducted to investigate the effects of dietary chitooligosaccharides (COS) supplementation on the innate immune response and protection against Vibrio harveyi infection in Trachinotus ovatus. A basal diet was supplemented with 0.0 (control), 2.0, 4.0 and 6.0 g COS kg(-1) to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 × 1.0 × 2.0 m), and each cage was stocked with 80 fish (initial average weight 10.8 ± 0.05 g). After 8 weeks of feeding trial, Both the final weight and specific growth rate (SGR) significantly increased with increasing dietary COS levels up to 4.0 g kg(-1), whereas there were no significant differences for COS levels from 4.0 to 6.0 g kg(-1). A decreased feed conversion ratio (FCR) was observed with increasing dietary COS levels. The total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme and superoxide dismutase (SOD) activity were significantly increased with the increased levels of dietary COS (P < 0.05), and reached a maximum at level of 4.0 g kg(-1) COS. There were no significant differences in those immunological parameters between 4.0 and 6.0 g kg(-1) COS. Moreover, the dietary COS supplementation groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with V. harveyi. These results indicated that dietary intake containing COS could enhance the immune responses of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 4.0 g kg(-1) COS to the fish for 56 days showed considerable improvement in the growth, survival and immune response of the fish.

Topics: Animal Feed; Animals; Aquaculture; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Fish Diseases; Immunity, Innate; Leukocyte Count; Muramidase; Oligosaccharides; Perciformes; Random Allocation; Respiratory Burst; Superoxide Dismutase; Vibrio; Vibrio Infections

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 present study describes the effect of diet supplementation with Chaga mushroom, Inonotus obliquus extract at 0%, 0.01%, 0.1%, and 1.0% levels on the innate humoral (lysozyme, antiprotease, and complement), cellular responses (production of reactive oxygen and nitrogen species and myeloperoxidase), and disease resistance in olive flounder, Paralichythys olivaceus against Uronema marinum. The lysozyme activity and complement activity significantly increased in each diet on weeks 2 and 4 against pathogen. The serum antiprotease activity and reactive nitrogen intermediates production significantly increased in fish fed with 0.1% and 1.0% diets from weeks 1-4. However, reactive oxygen species production and myeloperoxidase activity significantly increased in 1.0% and 2.0% diets on weeks 2 and 4. In fish fed with 0.1% and 1.0% diets and challenged with U. marinum the cumulative mortality was 50% and 40% while in 0% and 0.01% diets the mortality was 85% and 55%. The results clearly indicate that supplementation diet with I. obliquus at 0.1% and 1.0% level positively enhance the immune system and confer disease resistance which may be potentially used as an immunoprophylactic in finfish culture.

Topics: Animals; Basidiomycota; Ciliophora Infections; Complement System Proteins; Dietary Supplements; Disease Resistance; Fish Diseases; Flounder; Immunity, Innate; Muramidase; Oligohymenophorea; Protease Inhibitors; Reactive Oxygen Species

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.

Topics: Animals; Blood Cell Count; Blood Chemical Analysis; Blood Physiological Phenomena; Chenopodiaceae; Ciliophora Infections; Diet; Dietary Supplements; Disease Resistance; Erythrocyte Indices; Fish Diseases; Flounder; Hematocrit; Hemoglobins; Immunity, Innate; Muramidase; Oligohymenophorea; Plant Extracts; Random Allocation; Superoxides

Potential immunostimulatory effects of orally administered β-glucan were investigated in combination with immersion vaccination against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). A linear, unbranched and pure (purity ≥98%) β-1,3-glucan (syn. paramylon) from the alga Euglena gracilis was applied at an inclusion level of 1% β-glucan in feed administered at a rate of 1% biomass day(-1) for 84 consecutive days. Fish were vaccinated after two weeks of experimental feeding and bath challenged with live Y. ruckeri six weeks post-vaccination. Blood and head kidney were sampled at day 0, 13 (1 day pre-vaccination), 15, 55, 59 (day 3 post-challenge (p.c.)), 70 and 84. Vaccination induced significantly increased survival p.c., whereas the β-glucan had no effect on survival in either unvaccinated or vaccinated fish. Expression in head kidney of genes related to the acute phase response, i.e. interleukin-1β (IL-1β), serum amyloid A (SAA), precerebellin, and hepcidin, was significantly different in vaccinated fish receiving β-glucan compared to vaccinated controls at day 3 p.c., while no effect of β-glucan was observed among unvaccinated fish. Significant interaction between β-glucan and vaccination was found for the regulation of IL-1β, tumour necrosis factor-α, interferon-γ, SAA, precerebellin and hepcidin p.c. For SAA, the significant effect of β-glucan in vaccinated fish persisted at day 14 p.c. and 28 p.c. The difference in gene expression among vaccinated fish was mainly observed as down-regulations in vaccinated, β-glucan fed fish compared to up-regulations or no regulation in vaccinated controls. Slightly increased levels of plasma lysozyme activity were found in fish (both unvaccinated and vaccinated) receiving β-glucan at day 3 p.c. compared to control fed groups. This was associated with a faster clearance of Y. ruckeri in unvaccinated fish receiving β-glucan. In contrast to the trend towards a beneficial effect of β-glucan on plasma lysozyme activity, a trend towards suppression of plasma antibodies was seen in both unvaccinated and vaccinated fish receiving β-glucan. However, the effects of β-glucan were not reflected in the survival curves, and the differences seen in plasma lysozyme activity and antibody levels may have counteracted and set off each other as well as counteracted any potential effect represented by the differences in gene expression found.

Topics: Acute-Phase Proteins; Animals; Antibodies, Bacterial; Bacterial Vaccines; beta-Glucans; Cytokines; Euglena gracilis; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Head Kidney; Immersion; Immunologic Factors; Muramidase; Oncorhynchus mykiss; Survival Analysis; Vaccination; Yersinia Infections; Yersinia ruckeri

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

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

Nocardiosis caused by Nocardia seriolae has been causing severe loss of fish production, so that an effective vaccine is urgently needed. Mycobacterium bovis BCG (BCG) is a live attenuated vaccine for tuberculosis, which is effective against various infectious diseases including nocardiosis in mammals. In this study, the protective efficacy of BCG against N. seriolae was evaluated in Japanese flounder Paralichthys olivaceus and antigen-specific immune responses induced in BCG vaccinated fish were investigated. Cumulative mortality of BCG-vaccinated fish was 21.4% whereas that of PBS-injected fish was 56.7% in N. seriolae challenge. However, gene expression level of IFN-γ was only slightly up-regulated in BCG-vaccinated fish after injection of N. seriolae antigen. In order to reveal non-specific immune responses induced by BCG vaccination, transcriptome of the kidney after BCG vaccination was investigated using oligo DNA microarray. Gene expression levels of antimicrobial peptides such as C-type and G-type lysozyme were significantly up-regulated after BCG vaccination. Consistently, BCG vaccination appeared to increase the bacteriolysis activity of the serum against Micrococcus luteus and N. seriolae. These results suggest that BCG-vaccinated Japanese flounder fight N. seriolae infection mainly by non-specific immune responses such as by the production of bacteriolytic lysozymes.

Topics: Animals; BCG Vaccine; Colony Count, Microbial; Fish Diseases; Fish Proteins; Flounder; Gene Expression Profiling; Gene Expression Regulation; Immunization; Muramidase; Mycobacterium bovis; Nocardia; Nocardia Infections; Polymerase Chain Reaction; Reproducibility of Results

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

The impact of agrichemicals on aquatic vertebrate species has been a matter of increasing concern to researchers and environmentalist. In the present study, we evaluated the effects of a sublethal concentration of atrazine (10% of the LC(50-96 h)), a world-wide used herbicide, on the innate immune system of silver catfish (Rhamdia quelen). A significant reduction on phagocytic index, bacteria agglutination and bactericidal activity of the serum, serum lysozyme and total serum peroxidase activity was observed in fish exposed to atrazine for 24 h. After 10 days exposure to atrazine, only bactericidal activity of the serum, bacteria agglutination and total serum peroxidase activity were significantly reduced. Atrazine had no effect on the natural complement hemolytic activity. Our results demonstrate that atrazine decreases the innate immune response of fingerlings, which might increase its susceptibility to opportunistic pathogens.

Topics: Actinomycetales Infections; Aeromonas hydrophila; Animals; Aquaculture; Atrazine; Catfishes; Complement Hemolytic Activity Assay; Dose-Response Relationship, Drug; Female; Fish Diseases; Gram-Negative Bacterial Infections; Herbicides; Immunity, Innate; Male; Micrococcus luteus; Muramidase; Peroxidase; Water Pollutants, Chemical

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.

Topics: Acclimatization; Animals; Animals, Domestic; Electrophoresis, Gel, Two-Dimensional; Fish Diseases; Gene Expression Regulation; Hypoxia; Immunity, Humoral; Muramidase; Perches; Proteomics; Transferrin

This study was investigated to examine the effects of dietary inosine monophosphate (IMP) supplementation on growth performance, feed utilization, innate immunity, hematological parameters and disease resistance of juvenile olive flounder. Five experimental diets were formulated to contain IMP at levels of 0, 0.1, 0.2, 0.4 and 1.0%. All diets were maintained isonitrogenous (48% crude protein) and isocaloric (20.7 MJ/kg diet). Triplicate groups of olive flounder (initial body weight, 7.5 ± 0.02 g) were fed one of the experimental diets to apparent satiation (twice a day) for 14 weeks. Final body weight of fish fed 0.1-0.2% IMP were significantly higher than that of fish fed the 1.0% IMP. Groups of fish fed 0.2 or 0.4% IMP diet had significantly higher myeloperoxidase and lysozyme activities than fish fed the control diet. However, nitro-blue-tetrazolium and superoxide dismutase activities were not significantly different among all treatments. In the challenge test against Streptococcus iniae, cumulative mortality of fish fed IMP supplemented diets was significantly lower (15%, 4%, 4% and 9% for 0.1%, 0.2%, 0.4% and 1.0% IMP, respectively) than that of fish fed the control group (87%). The results suggest that IMP supplementation of 0.46-1.84 g into a kg of fish meal based diet (0.1-0.4% IMP product) can enhance innate immunity and disease resistance of olive flounder.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Aquaculture; Blood Chemical Analysis; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Fish Diseases; Flounder; Hematocrit; Immunity, Innate; Inosine Monophosphate; Muramidase; Random Allocation; Respiratory Burst; Streptococcal Infections; Streptococcus; Superoxide Dismutase

Combined vaccines are immunological products intended for immunization against multifactorial infectious diseases caused by different types or variants of pathogens. In this study, the effectiveness of Vibrio alginolyticus (Va), Vibrio harveyi (Vh), Vibrio vulnificus (Vv) and infectious spleen and kidney necrosis virus (ISKNV), an iridovirus, combined-vaccine (Vibrio and ISKNV combined vaccines, VICV), Va+Vh+Vv inactive vaccine (VIV) and ISKNV whole cell inactive vaccine (IWCIV) in Epinephelus coioides were evaluated using various immunological parameters including antibody titer, serum lysozyme activity (LA), respiratory burst (RB) activity, bactericidal activity (BA) and relative percentage survival (RPS). E. coioides immunized with VICV and challenged with Va+Vh+Vv+ISKNV had an RPS of 80%. The RPS was 73.3% in E. coioides immunized with VIV and challenged with Va+Vh+Vv. E. coioides immunized with IWCIV and challenged with ISKNV had an RPS of 69.6%. Serum LA in the vaccinated group was significantly higher than the control group on days 21 and 28 post-vaccination (P<0.01). The RB activity of head kidney cells in the vaccinated group was significantly higher (P<0.01) compared to that in the control group. However, RB activity of spleen cells in the vaccinated group and the control group were not significantly different (P>0.05). After immunization with VICV, BA values of blood leucocytes and head kidney cells increased significantly more than spleen cells. BA value of blood leucocytes was higher than that in head kidney cells. There were distinct difference between BA values in head kidney cells and in spleen cells (P<0.05) as well as between BA value of blood leucocytes and head kidney cells (P<0.01). E. coioides vaccinated with VICV have significantly higher antibody levels than control groupers (P<0.01). Our study suggests that the VICV candidate can effectively protect groupers against multiple bacterial and viral pathogens.

Topics: Animals; Antibodies, Bacterial; Antibodies, Viral; Aquaculture; Bacterial Vaccines; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immunization; Muramidase; Perciformes; Random Allocation; Respiratory Burst; Survival Analysis; Trager duck spleen necrosis virus; Vaccines, Combined; Vibrio; Vibrio Infections; Viral Vaccines

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.

Topics: Aeromonas; Animals; Catfishes; Complement Hemolytic Activity Assay; Dose-Response Relationship, Drug; Female; Fish Diseases; Glycine; Glyphosate; Herbicides; Male; Muramidase; Peroxidase; Time Factors; Water Pollutants, Chemical

The effect of diet supplemented with Phellinus linteus fed for 30 days was investigated in grouper Epinephelus bruneus challenged with Vibrio anguillarum, Vibrio harveyi, Vibrio alginolyticus, and Vibrio carchariae; infected and treated fish had a significantly higher percent weight gain and feed efficiency. In groups fed with enriched diet and challenged with V. anguillarum and V. harveyi the mortality rate declined with a consequent rise in survival rate than with other pathogens. On the other hand, in groups fed with P. linteus enriched diet and challenged with V. anguillarum, V. harveyi, and V. alginolyticus the cellular and humoral immune responses, such as the alternative complement activity (ACH(50)), serum lysozyme activity, phagocytic activity (PA), phagocytic index (PI) significantly higher than in the control group. The respiratory bursts (RB), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were found significantly enhanced when the groups fed with enriched diet against V. anguillarum and V. harveyi. The results reveal that kelp grouper fed for 30 days with P. linteus enriched diet had higher cellular and humoral immune response and disease protection from vibriosis than the group fed on basal diet with the protection linked to stimulation of immune system.

Topics: Agaricales; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Diet; Dietary Supplements; Fish Diseases; Glutathione Peroxidase; Immunity, Innate; Muramidase; Perciformes; Phagocytes; Plant Extracts; Respiratory Burst; Superoxide Dismutase; Vibrio Infections

The effect of diets containing sodium alginate at 0, 0.5, 1.0, and 2.0 g kg⁻¹ following challenge with Streptococcus iniae in kelp grouper Epinephelus bruneus were assessed with reference to survival rate and innate immune parameters such as alternative complement, lysozyme, natural haemagglutination, respiratory burst, superoxide dismutase, and phagocytic activities on week 1, 2, and 4. Fish fed with sodium alginate containing diet at 1.0 and 2.0 g kg⁻¹ after being challenged with S. iniae had higher survival rates of 75% and 60%, respectively than those fed with control diet (0 g kg⁻¹). With any enriched diet the percentage of macrophages significantly decreased from week 1-4, while the percentage of neutrophils and lymphocytes significantly increased. The alternate complement activity, natural haemagglutination, and phagocytic activities of infected fish fed with sodium alginate containing diet at 1.0 g kg⁻¹ on week 2 and 1.0 and 2.0 g kg⁻¹ diets on week 4 were significantly higher when compared to the control. The lysozyme, respiratory bursts, and superoxide dismutase activities of fish fed with enriched diets at 1.0 and 2.0 g kg⁻¹ were significantly increased on week 2 and 4. We therefore recommend that at 1.0 or 2.0 g kg⁻¹ dietary administration of sodium alginate can enhance innate immunity and disease resistance in kelp grouper against S. iniae.

Topics: Alginates; Animals; Complement Pathway, Alternative; Diet; Fish Diseases; Glucuronic Acid; Hemagglutination; Hexuronic Acids; Immune System; Immunologic Factors; Leukocytes; Muramidase; Phagocytosis; Respiratory Burst; Streptococcal Infections; Streptococcus; Superoxide Dismutase

Lysozyme is a muramidase that inflicts damage on bacterial cell wall by catalyzing the cleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan. Lysozymes are classified into several types, one of which is the goose-type (g-type). In this study, we identified and analyzed a g-type lysozyme (SmLysG) from turbot Scophthalmus maximus. The deduced amino acid sequence of SmLysG contains 193 residues and is most closely related to that of the g-type lysozyme of Scophthalmus rhombus (94% overall identity). SmLysG possesses a Goose Egg White Lysozyme (GEWL) domain with conserved residues essential for catalytic activity. Recombinant SmLysG (rSmLysG) purified from yeast exhibits strong lysozyme activity against Micrococcus luteus. Enzyme assays showed that the optimal temperature and pH of rSmLysG are 30°C and pH 7.0, respectively. Substrate spectrum analysis indicated that rSmLysG inhibited the growth of a number of important fish pathogens of both Gram-negative and Gram-positive natures. SmLysG transcription was detected in multiple tissues and was upregulated in kidney and spleen by experimental challenges with lipopolysaccharide and bacterial pathogens that are, respectively, sensitive to and resistant against the lytic effect of rSmLysG. Comparative analysis showed that although bacterial infection also induced the expression of c-type lysozyme, the induction levels were much lower than those of SmLysG. Taken together, these results indicate that SmLysG is a functional g-type lysozyme with a wide working range and is involved in innate immune defense against general bacterial infection.

Topics: Animals; Bacteria; Bacterial Infections; Fish Diseases; Flatfishes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Immune System; Molecular Sequence Data; Muramidase; Recombinant Proteins; Sequence Alignment; Substrate Specificity; Temperature

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

The development and duration of immune protection against Aeromonas hydrophila infections with garlic as an immunostimulant in rainbow trout Oncorhynchus mykiss was studied. Rainbow trout fingerlings of 14 g average weight were fed with 0 g (=Control), 0.5 g and 1.0 g of garlic 100 g(-1) of feed for 14 days. Physiological factors, biochemical, immunological, haematological parameters and electrolyte indices were evaluated after a further 14, 21 and 28 days before challenge with Aeromonas hydrophila. Fourteen days after the cessation of feeding with garlic, mortality rates of 12% (relative percent survival [RPS] = 86%) and 16% (RPS = 80%) were recorded in groups which received 0.5 g and 1.0 g of garlic 100 g(-1) of feed, respectively, compared to 84% mortalities in the controls. The corresponding RPS 21 days after ending the feeding regime was 75% and 68, respectively. One week later, the RPS had dropped to 55% and 46% in the groups fed with 0.5 g and 1.0 g garlic 100 g(-1) of feed, respectively.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Cell Count; Diet; Fish Diseases; Garlic; Gram-Negative Bacterial Infections; Muramidase; Oncorhynchus mykiss; Peroxidase; Respiratory Burst

This study determines the effect of diet enriched with the herb Baical skullcap Scutellaria baicalensis, and/or probiotics Lactobacillus sakei BK19 in rock bream, Oplegnathus fasciatus (32 ± 3 g) against Edwardsiella tarda. The changes in haematological parameters, innate immune response, and disease resistance were investigated after 1, 3, and 6 weeks. The white blood cell count (WBC: 10(4) mm(-3)), red blood cell count (RBC: 10(6) mm(-3)), and haemoglobin (Hb: g dl(-1)) levels significant increased (P < 0.05) with mixed diet on 3rd and 6th week and probiotics enriched diet on 6th week. The haematocrit (Ht: %) level significantly increased (P < 0.05) when fed with mixed diet on weeks 1-6. Interestingly, in mixed diet group the lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) significantly increased from week 1-6. The eosinophils (EOS) significantly increased in all the treated groups. In the probiotics or mixed diet groups the total protein (TP: g dl(-1)) increased significantly on weeks 3 and 6. The serum lysozyme activity significantly was enhanced in all the treated groups indicating an increase in the innate immunity level. Serum complement, antiprotease activities, reactive oxygen species (ROS) and reactive nitrogen species (RNS) production significantly increased from week 1-6 with mixed diet. The maximum protection against E. tarda was recorded in mixed diet group with a minimum cumulative mortality of 20% and a high relative percent survival (RPS) of 72.84. In the probiotics and herbal diet groups the cumulative mortality was 25% and 35% and RPS was 68.63 and 59.42, respectively. This study indicates that administration of probiotics or mixed diets can effectively minimize the mortality and restore the altered hematological parameters and enhancing the innate immunity in O. fasciatus against E. tarda.

Topics: Animals; Complement System Proteins; Diet; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Muramidase; Perciformes; Probiotics; Protease Inhibitors; Reactive Nitrogen Species; Reactive Oxygen Species; Scutellaria baicalensis

The effect of five probiotics, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus sakei, Bacillus subtilis, and Saccharomyces cerevisiae as individual and mixed enriched diet on the seasonal prevalence, activity and intensity of Uronema marinum infection in olive flounder Paralichthys olivaceus is reported. The growth performance, feed efficiency, blood biochemistry, survival rate, and non-specific immune response of U. marinum infected olive flounder on week 0, 1, 2, 4, 6, and 8 were quantified. The prevalence and infection intensity reached a peak from June to December and then it declined from December to March. The scuticocidal activity in the serum was significantly higher when fed with L. plantarum, L. acidophilus, and S. cerevisiae diets on weeks 2-8. All enriched diets significantly enhanced the weight gain significantly between week 6 and 8; the feed efficiency registered a significantly increase from week 4 to 8 when compared to infected fish fed with control diet. Infected fish fed with L. plantarum-supplemented diet had higher survival rate than with other enriched diets. The serum aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) levels significantly increased when fed with L. plantarum, L. acidophilus or S. cerevisiae-supplemented diet. Total protein (TP) and glucose (GLU) level significantly increased with any enriched diet from week 4 to 8. The superoxide anion production and serum lysozyme activity registered a significant increase when fed with L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diet from week 4-8. The present study concludes that L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diets act as immunostimulants enhancing the growth, feed efficiency, blood biochemistry, survival rate, and non-specific immune response in U. marinum infected olive flounder.

Topics: Animals; Blood; Blood Glucose; Blood Proteins; Ciliophora Infections; Diet; Enzymes; Fish Diseases; Flounder; Immunomodulation; Muramidase; Oligohymenophorea; Prevalence; Probiotics; Superoxides; Survival Analysis

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 μl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 10⁷ CFU ml⁻¹). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis.

Topics: Alanine Transaminase; Animal Feed; Animals; Aspartate Aminotransferases; Bacillus subtilis; Complement System Proteins; Diet; Dietary Supplements; Eleutherococcus; Fish Diseases; Flounder; Glycyrrhiza uralensis; Immunity, Innate; Lactobacillus; Muramidase; Panax; Phagocytosis; Plant Extracts; Probiotics; Respiratory Burst; Saccharomyces; Streptococcal Infections; Streptococcus

Lysozymes are the key molecules in innate immune system and possess high bactericidal properties. In the present study, a full-length c-type lysozyme cDNA has been cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques from kelp grouper, Epinephelus bruneus (Eb-CTL). The cDNA consists of 753 base pairs (bp) with a 432 bp open reading frame (ORF) that encodes 144 amino acids (aa) residues and polyadenylation signal sequence AATAAA. The deduced aa sequence polypeptide had a predicted molecular weight of 16 kDa and theoretical isoelectric point of 7.3. The deduced aa sequence have a long lysozyme domain which contains all catalytic sites and other conserved residues required for lysozyme activity. Pair-wise alignments showed a higher identity (76.4%) with Psetta maxima lysozyme and low identity (38.9%) with lysozymes of insect Bombyx mori. Interestingly, phylogenetic analysis revealed that the kelp grouper lysozyme was more closely related to other fish and vertebrate lysozymes. Quantitative real-time reverse transcriptase-PCR analysis showed that Eb-CTL transcripts are constitutively expressed in hematopoietic organs such as heart, spleen, and kidney after stimulation with LPS or infection with Vibrio anguillarum, indicating the involvement of Eb-CTL in the innate immunity of kelp grouper. This study is a first step on the genetics and functions of the c-type lysozyme in kelp grouper, and their role in anti-bacterial activity with reference to immunological properties which might have biotechnological applications.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Complementary; Fish Diseases; Gene Expression Regulation, Enzymologic; Immunity, Innate; Molecular Sequence Data; Muramidase; Open Reading Frames; Perciformes; Phylogeny; Random Allocation; Real-Time Polymerase Chain Reaction; Republic of Korea; Reverse Transcriptase Polymerase Chain Reaction; Vibrio; Vibrio Infections

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

The present study investigated the effect of Eriobotrya japonica extracts at 0%, 0.1%, 1.0%, and 2.0% doses supplementation with feed on non-specific immune response, hematological and biochemical profile, and disease resistance against Vibrio carchariae in kelp grouper Epinephelus bruneus at weeks 1, 2, and 4. The white blood cell (WBC) significantly increased in fish fed with 0.1%, 1.0%, and 2.0% diets on weeks 1 and 2 when compared to the control. However, the glucose always decreased from the control except on week 2 against pathogen. The serum total protein, albumin, and globulin significantly increased at week 2 but they did not changed significantly at weeks 1 and 4. The superoxide anion, lymphokines production index, and phogocytosis did not significantly increased in any diet on the first week whereas it was significantly enhanced in 1.0% and 2.0% supplementation diets on weeks 2 and 4 against V. carchariae when compared to control. All diets significantly enhanced the serum lysozyme activity, bactericidal activity, and haemolytic complement activity from weeks 1-4 as compared to control. The serum agglutinating antibody titre did not significantly enhance on the first week whereas it was significantly enhanced on weeks 2 and 4. Fish fed with 1.0% and 2.0% doses diets was found lower mortality than 0.1% diet. Thus, this study suggested that 1.0% and 2.0% doses supplementation diets could be advocated to enhance the immune response and production disease from V. carchariae in E. bruneus.

Topics: Animals; Aquaculture; Blood Glucose; Blood Proteins; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Eriobotrya; Fish Diseases; Immunity, Innate; Leukocyte Count; Muramidase; Perciformes; Plant Extracts; Superoxides; Vibrio Infections

To determine the effect of lipopolysaccharide (LPS) for the prevention of infection by Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss Walbaum) fingerlings.. Rainbow trout fingerlings were fed with 0 mg (= controls), 1.875 mg, 3.75 mg, 7.5 mg and 15 mg of LPS per 100 g of commercial feed for 14 days before experimental challenge with A. hydrophila. The results revealed a reduction in mortalities to 5% in the two lowest doses and 15% in the group, which received 15 mg LPS per 100 g of feed, compared with 45% mortalities in the control. LPS exerted a powerful oxidative burst effect and was a potent mediator of phagocytic, lysozyme, bactericidal and antiprotease activities and total protein. However, whereas there were increases in specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) in LPS-treated fish, the data were not significantly (P > 0.05) different.. LPS was effective at preventing disease caused by A. hydrophila and in stimulating the innate immune response of rainbow trout.. The results of this study highlight the role of LPS in fish disease control.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Lipopolysaccharides; Muramidase; Oncorhynchus mykiss; Respiratory Burst

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

Goldfish, Carassius auratus (47 + or - 3 g, n = 300) were inoculated intramuscularly (50 microl) with Aeromonas hydrophila (1.8 x 10(6) cells ml(-1)). On the 6th day of post-infection the fishes were divided into i) control, without infection fed with normal diet (C), ii) infected fish, fed with normal diet (IU), and infected fishes treated with different doses of iii) 100 mg kg(-1), iv) 200 mg kg(-1), iv) 400 mg kg(-1) and vi) 800 mg kg(-1) mixed herbal extracts supplementation diets. Hematological and immunological parameters were determined on week 1, 2 and 4. In infected goldfish were fed diets containing 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds, the white blood cell (WBC) levels significantly increased (P < 0.05) throughout the experimental trial compared to the control. During the experimental period, the red blood cell (RBC) and haemoglobin (Hb) level in goldfish significantly decreased (P < 0.05) when fish fed with 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds while it was restored near control when infected fish fed with 400 or 800 mg kg(-1) of herbal extracts supplementation feeds. On the other hand, the haematocrit (Ht) values decline significantly (P < 0.05) in 100, 200 and 400 mg kg(-1) of mixed herbal supplementation feeding groups on weeks 2 and 4 when compared to control group. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) values almost significantly differ from the control values. The infected goldfish and treated with 100 or 200 mg kg(-1) of herbal supplementation feeds exhibited significantly decline (P < 0.05) in total protein (TP), glucose (GLU) and cholesterol (CHO) levels on week 1-4 whereas it was restored when infected fish fed with 400 or 800 mg kg(-1) of herbal supplementation feeds on week 4. In comparison to untreated control goldfish, the respiratory burst activity and phagocytic activity of blood cells was significantly enhanced in infected fish feeding with 200, 400 and 800 mg kg(-1) of herbal supplementation feeds compared to the control. On the other hand, infected fish fed with all the doses of mixed herbal supplementation feeds, the lysozyme activity was significantly enhanced throughout the experimental period. This study shows that the infected goldfish treated with 400 and 800 mg kg(-1) of herbal supplementation feeds preceding the challenge with live A. hydrophila had 30% and 25% mortality

Topics: Aeromonas hydrophila; Animal Feed; Animals; Blood Cell Count; Diet; Dietary Supplements; Fish Diseases; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Plants, Medicinal; Respiratory Burst; Time Factors

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40+/-3.1% to 79.4+/-1.7%) and it decreased from August to November (63+/-2.3% to 30+/-2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49+/-7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100ppm for 30min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.

Topics: Animals; Anti-Infective Agents, Local; Bacterial Infections; Ciliophora Infections; Complement System Proteins; Disinfectants; Fish Diseases; Fisheries; Flounder; Formaldehyde; Hydrogen Peroxide; Immunomodulation; Movement; Muramidase; Oligohymenophorea; Phagocytosis; Prevalence; Republic of Korea; Respiratory Burst; Seasons; Seawater; Zeolites

Allicin was fed at 0 (= control), 0.5 and 1.0 mL of Allimed liquid 100 g(-1) of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), fingerlings before infection with Aeromonas hydrophila with a resultant reduction in mortalities from 80% in the controls to 8% [relative percentage survival (RPS) = 90%] and 0% (RPS = 100%) among the treated fish. Allicin was strongly antibacterial compared to the control, with a minimum inhibitory concentration (MIC) of >400 microL mL(-1) of Allimed liquid. Use of allicin led to a lower number of white blood cells (132.0 +/- 0.4 x 10(3)) compared to 175.0 +/- 0.1 x 10(3) in the controls, but elicited increased phagocytic activity, i.e. a phagocytic value of 39.2% compared to 13.6% in the controls, and serum lysozyme activity, which showed significant (P > 0.05) differences compared to the control at 15 and 30 min after the first reading at 0 min of incubation.

Topics: Aeromonas hydrophila; Animals; Anti-Infective Agents; Disulfides; Enzyme Activation; Fish Diseases; Gram-Negative Bacterial Infections; Leukocyte Count; Microbial Sensitivity Tests; Muramidase; Oncorhynchus mykiss; Phagocytosis; Sulfinic Acids; Superoxides; Time Factors

Feeding rainbow trout, Oncorhynchus mykiss (Walbaum), with 1% lupin, Lupinus perennis, mango, Mangifera indica, or stinging nettle, Urtica dioica, for 14 days led to reductions in mortality after challenge with Aeromonas hydrophila. In addition, there was significant enhancement in serum bactericidal activity, respiratory burst and lysozyme activity in the treatment groups compared to the controls. Use of lupin and mango led to the highest number of red blood and white blood cells in recipient fish, with use of stinging nettle leading to the highest haematocrit and haemoglobin values; the highest value of mean corpuscular volume and haemoglobin was in the control groups and those fed with stinging nettle.

Topics: Aeromonas hydrophila; Animals; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Lupinus; Magnoliopsida; Mangifera; Muramidase; Oncorhynchus mykiss; Respiratory Burst; Survival Analysis; Urtica dioica

We report the growth, innate immune response, and disease resistance in olive flounder (Paralichthys olivaceus) challenged with Streptococcus iniae after feeding with diet enriched with Zooshikella sp. strain JE-34 three different concentration i.e. Low (3.4 x 10(4), n = 50), medium (3.5 x 10(6), n = 50), and high (3.4 x 10(8), n = 50) cfu ml(-1) supplemented diets, the changes were monitored on weeks 1, 2, 4, 8, 12, and 16. With all diets the innate immune parameters, such as superoxide anion production, phagocytic and lysozyme activity were not enhanced on week 1 and 4. On the other hand, all tested immune parameters in the treated groups significantly enhanced after 8th week; the weight gain significantly increased after 4th week in fish fed with enriched diets. The mortality in olive flounder after administration with high concentration diet showed 25%. With low and medium enriched diets the mortality was 40% and 35%, respectively. In the infected untreated group mortality was 85% while there was no mortality in the control group. The results suggested that Zooshikella sp. strain JE-34 enriched diets could be used to enhance the innate immune response and disease resistance of P. olivaceus against S. iniae.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Blood Glucose; Cell Survival; Cholesterol; Fish Diseases; Flounder; Gammaproteobacteria; Hemoglobins; Leukocytes, Mononuclear; Muramidase; Phagocytosis; Phosphorus; Probiotics; Streptococcal Infections; Streptococcus; Triglycerides

We report the effect of aqueous-, ethanol- and methanol-solvent-derived extracts of three traditional Korean herbs, Punica granatum, Chrysanthemum cinerariaefolium and Zanthoxylum schinifolium, by monitoring the innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and the functional immunity in terms of percentage mortality and relative percent survival (RPS) in olive flounder (Paralichthys olivaceus) against Uronema marinum (1 x 10(5)ciliates ml(-1)) for 30 days. Fish were intraperitoneally administered with 5, 50 and 100 mg kg(-1) body weight of each traditional Korean medicinal (TKM) solvent extract except the control and infected untreated groups. In all the treated groups at concentrations of 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters were found significantly enhanced when compared to 0 mg kg(-1) dose. However, at 5 mg kg(-1) the tested immune parameters did not vary. Administration of TKM solvent extracts preceding the challenge with U. marinum for 30 days significantly reduced the percentage mortality with the consequent increase in RPS. Administration of 50 and 100 mg kg(-1) TKM solvent extracts clearly enhanced the innate immune responses and disease resistance in P. olivaceus against U. marinum.

Topics: Animals; Chrysanthemum cinerariifolium; Ciliophora; Ciliophora Infections; Complement Pathway, Alternative; Fish Diseases; Flounder; Immunity, Innate; Lythraceae; Medicine, Korean Traditional; Muramidase; Phagocytosis; Plant Extracts; Respiratory Burst; Zanthoxylum

Parameters of non-specific immune response and level of specific and natural antibodies were investigated after an experimental challenge with Aeromonas hydrophila in genetically different common carp (Cyprinus carpio) families. Ten resistant and ten sensitive families were used for the experiment, which had been selected out of 96 families, based on the results of a preliminary challenge test. Blood samples were collected 12 h, one week and 21 days following the challenge. Phagocytic and respiratory burst activities of phagocytic cells, lysozyme activity of the blood plasma were determined. Level of specific antibodies against A. hydrophila and level of natural antibodies were measured in the samples taken on the 28th day. Non-infected fish from resistant and sensitive families were used as controls. Significant differences of phagocytic and lysozyme activities were measured between the challenged resistant and sensitive families. The level of specific antibodies between the same families was also found to be significantly different. There were no significant differences of the studied parameters between the control groups. Based on our results, phagocytic activity of leukocytes, plasma lysozyme activity and specific antibody titre were found to be higher in the resistant families than in the sensitive ones following infection with A. hydrophila.

Topics: Aeromonas hydrophila; Animals; Antibodies, Bacterial; Carps; Female; Fish Diseases; Genetic Predisposition to Disease; Gram-Negative Bacterial Infections; Leukocytes, Mononuclear; Male; Muramidase; Phagocytosis; Respiratory Burst; Selection, Genetic

Expression of four reference genes of grass carp, including beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1alpha), was studied in tissues of normal individuals and bacteria-infected individuals. EF1alpha had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1alpha in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1alpha is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.

Topics: Animals; Bacterial Infections; Bacterial Toxins; Carps; Escherichia coli Proteins; Fish Diseases; Fish Proteins; Gene Expression Profiling; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Muramidase; Reference Standards; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction

The present study evaluated the efficacy of dietary doses of Withania somnifera (Ashwagandha) root powder on immunological parameters and disease resistance against the bacterial pathogen Aeromonas hydrophila infections in Indian major carp, Labeo rohita fingerlings. Fishes were fed with dry diet containing 0 gkg(-1) (control), 1 gkg(-1) (T(1)), 2 gkg(-1) (T(2)) and 3 gkg(-1) (T(3)) W. somnifera root powder for 42 days. Immunological (NBT level, Phagocytic activity, total immunoglobulin and lysozyme activity) parameters of fishes were examined at 0 days, 14 days, 28 days and 42 days of feeding. Fishes were challenged with A. hydrophila 42 days post feeding and mortalities (%) were recorded over 14 days post-infection. The results demonstrate that fishes fed with W. somnifera root showed enhanced NBT level, Phagocytic activity, total Immunoglobulin level and lysozyme activity (p<0.05) compared with the control group. The survivability was higher in experimental diets than the control group. Dietary W. somnifera at the level of 2 gkg(-1) showed significantly (P<0.05) higher protection (RPS 42.85+/-0.65%) against A. hydrophila infection than control. The results suggest that the W. somnifera root powder have a stimulatory effect on immunological parameters and increases disease resistance in L. rohita fingerlings against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Diet; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulins; Muramidase; Nitroblue Tetrazolium; Phagocytosis; Plant Roots; Survival Analysis; Withania

We report the effect of aqueous, ethanol, and methanol solvent leaf extracts of Punica granatum on innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity and functional immunity in terms of percentage cumulative mortality and Relative Percent Survival (RPS) in olive flounder Paralichthys olivaceus naturally infected with lymphocystis disease virus (LDV) after 8 weeks. Infected fish were intraperitoneally administered with 0, 5, 50, and 100 mg kg(-1) body weight of solvent extracts. In groups treated with 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters significantly increased after 8 weeks when compared to 0 mg kg(-1) dose, but not with 5 mg kg(-1). Administration of P. granatum solvent extracts for 8 weeks significantly reduced the percentage mortality with the consequent increase in RPS. The results suggest that intraperitoneal administration of the leaf extracts of P. granatum at 50 or 100 mg kg(-1) dose clearly enhance the innate immune responses and disease resistance after 8 weeks in P. olivaceus against natural LDV infection.

Topics: Animals; Complement System Proteins; DNA Virus Infections; Fish Diseases; Flounder; Immunity, Innate; Iridovirus; Lythraceae; Muramidase; Phagocytosis; Plant Extracts; Respiratory Burst

We report the effect of two probiotics, Lactobacil and Sporolac as separate or mixed diets on innate immune mechanisms, such as phagocytosis activity, superoxide anion production of blood leukocytes, complement activity and plasma lysozyme activity, and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder, Paralichthys olivaceus (523.5 +/- 18.3 g) on week 1, 2, and 4. In infected fish, administration of diet supplemented with Lactobacil individually or mixed with Sporolac significantly enhanced the immune parameters like phagocytic activity superoxide anion production, complement activity, and plasma lysozyme. However administration of supplemented diet with Sporolac alone, all the chosen immune parameters did not enhance when compared to control group; this diets resulted in lower mortality (30% and 25%) than Sporolac diet group (45%) in 30 days. We conclude that Lactobacil individually or mixed with Sporolac act as immunostimulants that enhance the innate immune response and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder.

Topics: Analysis of Variance; Animals; Aquaculture; Dietary Supplements; DNA Virus Infections; Fish Diseases; Flounder; Immunity, Innate; Iridoviridae; Muramidase; Phagocytosis; Probiotics; Superoxides

In olive flounder, Paralichthys olivaceus infected with lymphocystis disease virus (LDV) bath treatment with formalin, hydrogen peroxide or Jenoclean at 50, 100, and 200 ppm daily 10 min for 60 days enhanced the innate immune response and disease resistance. Jenoclean enhanced the immune parameters at the lowest concentration of 50 ppm; on the other hand, hydrogen peroxide bath treatment enhanced the immunity level at 100 ppm, while formalin was effective only at 200 ppm. A low cumulative mortality and high relative percent survival was noted in Jenoclean treated group followed by formalin and hydrogen peroxide treated groups. In olive flounder at low concentrations of 50 ppm or 100 ppm hydrogen peroxide and Jenoclean effectively prevent LDV infection.

Topics: Analysis of Variance; Animals; Complement Pathway, Alternative; DNA Virus Infections; Dose-Response Relationship, Drug; Fish Diseases; Flounder; Formaldehyde; Hydrogen Peroxide; Immunity, Innate; Iridoviridae; Muramidase; Phagocytosis; Polymerase Chain Reaction; Republic of Korea; Respiratory Burst; Survival Analysis

The percent weight gain (PWG) and feeding efficiency (FE) of Epinephelus coioides were calculated. The survival of Saccharomyces cerevisiae P13 in the posterior intestines using a specific primer pair of YMR245w-F/YMR245w-R, non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing S. cerevisiae at 0 (control), 10(3), 10(5), or 10(7) colony-forming units (cfu) kg(-1) for 4 weeks. Results showed that grouper fed a diet containing S. cerevisiae at the levels of 10(3), 10(5), and 10(7) cfu kg(-1) had significantly increased PGW and FE especially in the 10(7) cfu kg(-1) group which were 211.6% and 1.2, respectively. S. cerevisiae was able to survive in the fish posterior intestines during the S. cerevisiae feeding period. Fish fed a diet containing S. cerevisiae at 10(7) cfu kg(-1) had significantly higher survival rates than those fed the 10(3) cfu kg(-1)S. cerevisiae diet and the control diet after challenge with Streptococcus sp. and an iridovirus, with increased survival rates of 26.6% and 36.6%, respectively, compared to the challenge control group. The phagocytic activity, respiratory burst and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH(50)) of fish fed diets containing S. cerevisiae at 10(5) and 10(7) cfu kg(-1) were significantly higher than those of fish fed the 10(3) cfu kg(-1)S. cerevisiae-contained diet and the control diets after 4 weeks of feeding, and had increased by 20% and 20%, 27.6% and 19.7%, 30.5% and 36.2%, 205.8% and 169.6%, and 90.8% and 80.3%, respectively, compared to the control group. We therefore recommend dietary S. cerevisiae administration of 10(5) and 10(7) cfu kg(-1) to E. coioides to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus especially in the 10(7) cfu kg(-1) group.

Topics: Animals; Body Weight; Complement Pathway, Alternative; Fish Diseases; Immunity, Innate; Muramidase; Perciformes; Phagocytosis; Probiotics; Respiratory Burst; Saccharomyces cerevisiae; Streptococcal Infections; Streptococcus; Superoxide Dismutase

The effect of Lactobacillus sakei BK19 (10(8) cells g(-1)) supplemented diet fed to kelp grouper, Epinephelus bruneus against streptococcosis caused by Streptococcus iniae and Streptococcus parauberis with reference to the innate immune response and disease resistance was evaluated at 1, 2, and 4 weeks. Maximum reduction in mortalities was observed in kelper feeding the probiotic diet for two weeks after challenged with the pathogens when compared to the infected group fed with basal diet; similarly the cellular and humoral immune responses such as head kidney macrophage phagocytic and peroxidase activities, serum lysozyme activity, and total protein levels increased significantly. The results reveal that, in streptococcosis infected kelp grouper feeding L. sakei BK19 enriched diet affords a higher level of disease protection due to stimulation of immune system.

Topics: Animals; Dietary Supplements; Fish Diseases; Lactobacillus; Macrophages; Muramidase; Perciformes; Peroxidase; Phagocytosis; Probiotics; Respiratory Burst; Streptococcal Infections; Streptococcus; Survival Analysis

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)-induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham-injected phosphate-buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E₂ (PGE₂) in the Vo-fed and LPS-injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX-2) mRNA expression in bone was higher in fish fed Fo compared to Vo-fed fish. Gene expression of immunoglobulin M (IgM) was up-regulated in bone of all LPS-injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation-related spinal deformities. At the same time, we found evidence that localized injection-related processes could trigger the development of vertebral body malformations.

Topics: Animal Feed; Animals; Bone and Bones; Diet; Dietary Fats; Fatty Acids; Fish Diseases; Inflammation; Irritants; Lipopolysaccharides; Muramidase; Radiography; Salmo salar; Spinal Diseases; Spine

The effect of traditional Chinese medicine (TCM) formulated from propolis and Herba Epimedii extracts at the ratio of 3:1 (w/w) on non-specific immune response of Chinese sucker (Myxocyprinus asiaticus) was investigated. Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% TCM extracts for five weeks. The respiratory burst and phagocytic activities of blood leukocytes, lysozyme and natural haemolytic complement activities in plasma were measured weekly. After five weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Chinese sucker with different dosage of TCM extracts stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme activity in plasma. They had no effect on plasma natural haemolytic complement activity. All dosage of treated groups showed reduced mortality following A. hydrophila infection. Feed containing 0.5% TCM extracts was the most effective with the mortality of the fish significantly reduced by 35% compared to the control. The results indicate that propolis and Herba Epimedii extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Complement System Proteins; Cypriniformes; Drugs, Chinese Herbal; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Propolis; Respiratory Burst

Orange-spotted grouper Epinephelus coioides held at 27 degrees C were then further cultured at 19, 27 (control), and 35 degrees C, and were examined for innate cellular and humoral responses after 3-96 h. The total leucocyte count, respiratory burst, and phagocytic activity significantly decreased 3, 48, and 96 h after fish were transferred to 19 and 35 degrees C. Both the alternative complement pathway (ACH(50)) and the lysozyme activity significantly decreased at 3-96 h after fish were transferred to 19 and 35 degrees C. In another experiment, groupers reared at 27 degrees C at 34 per thousand salinity were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at a dose of 2.3 x 10(9) colony-forming units (cfu) fish(-1), and then further reared in water temperatures of 19, 27 (control), and 35 degrees C. The cumulative mortalities of V. alginolyticus-injected fish held in 19 and 35 degrees C were significantly higher than that of injected fish held in 27 degrees C. Resistance had decreased after 12 h for the challenged grouper held at 35 degrees C. All injected fish held in 19 degrees C had died after 72 h. It was concluded that at 12 h after transfer of grouper from 27 to 19 and 35 degrees C, immunity was suppressed and resistance against V. alginolyticus had decreased.

Topics: Animals; Antibody Formation; Complement Pathway, Alternative; Disease Susceptibility; Fish Diseases; Immunity, Innate; Leukocyte Count; Muramidase; Perciformes; Phagocytosis; Respiratory Burst; Survival Analysis; Temperature; Time Factors; Vibrio alginolyticus; Vibrio Infections

Bacterium Flavobacterium columnare is the causative agent of columnaris disease in many wild and farmed fish species. Immunostimulants are used with success in aquaculture against many pathogens, but the ability to improve innate resistance to columnaris disease has not been studied. Fingerling rainbow trout were treated with two immunostimulants, yeast beta-glucan and beta-hydroxy-beta-methylbutyrate (HMB). Selected innate immune function parameters, the production of reactive oxygen species (ROS) by whole blood and by isolated head kidney leukocytes, plasma lysozyme activity and complement bacteriolytic activity, were determined to assess the immune status of fish. The fish were then bath challenged with virulent F. columnare bacteria, and the mortality of fish was recorded. Given orally both stimulants raised the levels of immune function parameters, but did not improve survival in challenge at any concentration of the stimulants used. Intra peritoneal injection of beta-glucan increased parameter values several fold, but no beneficial effect of injected glucan on survival was noted. As a control, antibiotic medication administered prior to and during the challenge infection prevented the mortality. Innate immune mechanisms, even when induced to high levels with immunostimulants, as evidenced here, were not able to increase resistance against F. columnare. This may be connected to the external character of the infection. The results from the treatments with beta-glucan and HMB suggest that there is little prospect of preventing columnaris disease by means of immunostimulants in early life stage of rainbow trout. However, the efficacy of other immune stimulants remains open.

Topics: Adjuvants, Immunologic; Animals; Aquaculture; beta-Glucans; Complement System Proteins; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Kaplan-Meier Estimate; Muramidase; Oncorhynchus mykiss; Reactive Oxygen Species; Valerates

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg(-1) dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg(-1) dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Proteins; Cyprinidae; Erythrocyte Count; Euglena; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Leukocyte Count; Muramidase; Superoxides; Survival Analysis

The brown bullhead Ameiurus nebulosus is a species of the family Ictaluridae commonly used as a sentinel of environmental contamination. While these fish have been utilized for this purpose in areas contaminated with polychlorinated biphenyls (PCBs), few controlled, laboratory-based studies have been designed to document the effects of PCB mixtures in this species. Here, brown bullhead were exposed to the PCB mixture, Aroclor 1248, via intraperitoneal injection and the effects on immune function, plasma hormones and disease resistance were evaluated. Exposure to this mixture led to a decrease in bactericidal activity and circulating antibodies to Edwardsiella ictaluri present from a previous exposure to this pathogen. A subsequent E. ictaluri disease challenge led to significantly higher mortality in A1248 treated fish compared to vehicle-control fish. The mitogenic response to the T-cell mitogen, phytohemaglutinin-P, was increased compared to vehicle-control fish. The steroid hormone, cortisol, and the thyroid hormone, T3, were also significantly lower in A1248 exposed fish. In summary, we have validated a number of functional immune assays for application in brown bullhead immunotoxicity studies. Additionally, we have demonstrated that the PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead. Such data may compliment the interpretation of data yielded from applied field studies conducted in PCB contaminated aquatic ecosystems.

Topics: Animals; Antibodies, Bacterial; Aroclors; Cytochrome P-450 CYP1A1; Edwardsiella ictaluri; Enterobacteriaceae Infections; Enzyme-Linked Immunosorbent Assay; Estradiol; Fish Diseases; Ictaluridae; Kidney; Leukocytes, Mononuclear; Mitogens; Muramidase; Random Allocation; Respiratory Burst; Testosterone; Thyroxine; Triiodothyronine; Water Pollutants, Chemical

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 microg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 +/- 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T(1) were fed on basal diet (control). The fish of T(2) were given the basal diet, containing propolis-ethanolic-extract. The fish of T(3) were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 x 10(7) cells ml(-1)) at the end of the feeding period and kept for 15 more days. The best growth rate and feed conversion ratio were obtained with T(2.) The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T(2) followed by T(3) when compared with the control group. The HCT-level and monocyte-counts were increased (T(2)). No significant change, in the large lymphocytic-count was found among the three treatments (28-27-28%), while the neutrophil-count was significantly decreased (7%) with T(2) and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T(2). The RLP against A. hydrophila was high with T(2) and T(3). The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Anti-Infective Agents; Cichlids; Fish Diseases; Gram-Negative Bacterial Infections; Hematocrit; Immunity, Innate; Leukocyte Count; Microbial Sensitivity Tests; Muramidase; Propolis; Time Factors

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg(-1) body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 x 10(7) cells ml(-1)). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Azadirachta; Complement System Proteins; Curcuma; Fish Diseases; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Magnoliopsida; Muramidase; Ocimum; Phagocytosis; Plant Extracts; Respiratory Burst

The present experiment was carried out to study the effect of different dosages of beta-glucan suspension derived from barley on the innate immune response and disease resistance of Anabas testudineus spawns against infection caused by Aeromonas hydrophila. Four different dosages of beta-glucan suspension in phosphate buffered saline at the rate of 0, 5, 10, 15 mg l(-1) were taken and 8 days old spawn were exposed for 2 h and 3 h. The cell suspension of spawn was assayed for total protein, acid phosphatase activity, lysozyme activity, bactericidal and NBT. Further, the spawns were challenged with 3 x 10(5) cells ml(-1) of A. hydrophila and survivability percentage and immunological parameters were assayed upto day 7. On day 7, most of the immunological parameters such as lysozyme activity, bactericidal activity and NBT activity were significantly enhanced after exposing the fish to all the concentrations of beta-glucan. Challenge study indicated least mortality in the group of spawns immersed in 15 mg l(-1) beta-glucan suspension for 3 h. Thus, 3 h exposure to beta-glucan suspension could reduce the mortality and increase the immunity of A. testudineus spawns.

Topics: Acid Phosphatase; Aeromonas hydrophila; Animals; beta-Glucans; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Perches; Superoxides; Survival Analysis

Garlic, Allium sativum, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At doses of 0.5 and 1.0 g garlic per 100 g of feed, there was a reduction in mortalities to 4% compared with the controls (88%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was stimulation of the number of erythrocytes and leucocytes, a significantly higher haematocrit, enhancement of phagocytic activity, respiratory burst, lysozyme, anti-protease and bactericidal activities following feeding with garlic.

Topics: Aeromonas hydrophila; Animals; Blood Cell Count; Diet; Dietary Supplements; Fish Diseases; Garlic; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Random Allocation; Superoxides

Ginger, Zingiber officinale, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At 0.5 g ginger per 100 g of feed, there was a reduction in mortalities to 0% compared with the controls (64%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was proliferation in the number of neutrophils, macrophages and lymphocytes, and enhanced phagocytic, respiratory burst, lysozyme, bactericidal and anti-protease activities compared with the controls.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Cell Count; Colony Count, Microbial; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Superoxides; Zingiber officinale

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

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.

Topics: Animals; Colony Count, Microbial; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Gene Expression Regulation; Ictaluridae; Kidney; Muramidase; Survival Analysis; Time Factors; Toll-Like Receptors; Virulence

The present study was performed to investigate the immunostimulatory effects of Korean mistletoe extract (KM-110; Viscum album Coloratum) on the non-specific immune response and protection against Aeromonas hydrophila infection in Japanese eel (Anguilla japonica). Eels were fed under 4 regimes, 0%, 0.1%, 0.5% and 1.0% KM-110 mixed diet. On day 14 after feeding, 15 fish from each group were injected i.p. with live A. hydrophila (3 x 10(6)CFU) and the remaining unchallenged fish from each group were used to study the innate immune response. On 14 days post-infection, the total survival rates were 26.6% in control, and 33.3%, 66.6% and 80% in 0.1%, 0.5% and 1% KM-110-treated groups, respectively. The maximum lysozyme activity was observed in the 1% KM-110-treated group. There was no significant difference of lysozyme activity between 0.1% and 0.5% KM-110 group. Superoxide anion (O(2)(-)) production was significantly (p<0.05) augmented in the 0.5% and 1% KM-110 groups compared to the control and 0.1% KM-110 group. No significant difference of (O(2)(-) production was found between 0.5% and 1% KM-110 group. Likewise, there was a significant increase in phagocytic activity in the 0.5% KM-110 group compared with the 0.1% group (p<0.05), but no significant difference between the 0.5% and the 1% KM-110 group indicating that 0.5% KM-110 concentration is suitable for stimulating maximum phagocytic activity resulting in a high amount of ROI production. Considering the present results, KM-110 could be utilized as a promising immunostimulating substance for a diet in aquaculture.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Anguilla; Animals; Diet; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Cellular; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Reactive Oxygen Species; Survival Analysis; Viscum album

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.

Topics: Amino Acid Sequence; Animals; Fish Diseases; Flatfishes; Gene Expression Regulation, Enzymologic; Gram-Negative Bacterial Infections; Lipopolysaccharides; Molecular Sequence Data; Muramidase; Photobacterium; Phylogeny; Sequence Alignment

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (approximately 93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi. Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of approximately 93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Bacteriocins; Cross Reactions; Fish Diseases; Muramidase; Oncorhynchus mykiss; Probiotics; Subcellular Fractions; Time Factors; Vibrio; Vibrio Infections

The lysozyme activity, alternative complement activity (ACH50), respiratory burst, SOD (superoxide dismutase) activity and phagocytic activity of orange-spotted grouper Epinephelus coicoides were examined when the fish were injected intraperitoneally with sodium alginate at 10, 20, 30mgkg-1 and iota-carrageenan at 10, 20, 30mgkg-1, respectively after 24, 72 and 120 h. Serum ACH50 increased directly with dose after 24 and 72 h for both sodium alginate and iota-carrageenan treatments. The fish that received sodium alginate at 20mgkg-1 after 24 and 72 h, and the fish that received iota-carrageenan after 72 and 120 h showed significantly increased respiratory burst, SOD activity and phagocytic activity, respectively. In another experiment, E. coicoides which had been injected individually with sodium alginate and iota-carrageenan at 10, 20, 30mgkg-1, were challenged with Vibrio alginolyticus at 1.8x10(9) colony-forming units (cfu)fish-1 and then placed in seawater of 33 per thousand. The survival of fish that received sodium alginate at 20mgkg-1, and the fish that received iota-carrageenan at 30mgkg-1 was significantly higher than that of fish which received saline and the control fish after 48 h as well as at the termination of the experiment (120 h after the challenge). It is therefore concluded that E. coicoides which received sodium alginate at 20mgkg-1 or iota-carrageenan at 30mgkg-1 increased the non-specific immune response and resistance from V. alginolyticus infection.

Topics: Adjuvants, Immunologic; Alginates; Animals; Biocompatible Materials; Carrageenan; Complement Pathway, Alternative; Fish Diseases; Glucuronic Acid; Hexuronic Acids; Immunity, Innate; Muramidase; Perciformes; Phagocytosis; Respiratory Burst; Superoxide Dismutase; Time Factors; Vibrio alginolyticus; Vibrio Infections

The effects of GH on immune and endocrine responses to channel catfish challenged with the bacterium Edwardsiella ictaluri were examined. Catfish (11.7+/-1.0 g) treated with recombinant bovine growth hormone (rbGH) and challenged with E. ictaluri experienced similar mortality as control-exposed fish. Plasma activity of lysozyme was higher (P<0.01) in rbGH-exposed fish. Compared to day 0 controls (non-exposed fish), IGF-I levels decreased (P<0.05) in challenged fish while levels were similar (P>0.10) between treatments. Abundance of GH receptor (GHR) mRNA tended to decrease (P=0.055) in liver of challenged fish while toll like receptor 5 (TLR5) mRNA increased (P<0.05) in liver compared to d 0 controls. An increase in lysozyme may suggest GH enhances a nonspecific immune response. A decrease in GHR mRNA and plasma IGF-I suggests a downregulation of the somatotropic axis in response to disease. The increase in TLR5 mRNA suggests that TLR5 may play a role in host response to bacterial challenge. While exogenous rbGH may play a stimulatory role to increase lysozyme levels, there was no apparent effect of rbGH on mortality to E. ictaluri.

Topics: Animals; Cattle; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Growth Hormone; Ictaluridae; Insulin-Like Growth Factor I; Muramidase; Receptors, Somatotropin; Recombinant Proteins; RNA, Messenger; Toll-Like Receptor 5

Mozambique tilapia Oreochromis mossambicus acclimated to 27 degrees C were then held at 19, 23, 27 (control), 31 and 35 degrees C, and were examined for non-specific cellular and humoral responses after 12-96 h. Total leucocyte count decreased significantly when fish were transferred to 19 and 23 degrees C after 48 and 96 h, and when transferred to 35 degrees C over 12-96 h, respectively. Respiratory burst decreased significantly when fish were transferred to 19, 31 and 35 degrees C over 24-96 h, whereas phagocytic activity and phagocytic index decreased significantly when fish were transferred to low temperatures (19 and 23 degrees C) and high temperatures (31 and 35 degrees C) over 12-96 h. Lysozyme activity decreased significantly when fish were transferred to 19 degrees C after 12-96 h, but increased significantly when transferred to 31 and 35 degrees C over 48-96 h. Alternative complement pathway (ACH(50)) also decreased significantly when transferred to 19 and 23 degrees C after 12h, but increased significantly when transferred to 31 and 35 degrees C after 24h. In another experiment, tilapia reared at 27 degrees C were injected intraperitoneally with Streptococcus iniae at a dose of 1 x 10(7)colony-forming units (cfu)fish(-1), and then reared onward at water temperatures of 19, 23, 27 (control), 31 and 35 degrees C. Over 48-168 h, the cumulative mortality of S. iniae-injected fish held in 19 and 35 degrees C was significantly higher than that of injected-fish held in 23, 27 and 31 degrees C. It is concluded that transfer of tilapia O. mossambicus from 27 degrees C to low temperatures (19 and 23 degrees C) after 12h, and transfer of fish from 27 degrees C to high temperatures (31 and 35 degrees C) reduced their immune capability. Moreover, tilapia under temperature stress at 19 and 35 degrees C from 27 degrees C decreased its resistance against S. iniae.

Topics: Animals; Disease Susceptibility; Fish Diseases; Leukocyte Count; Leukocytes; Muramidase; Phagocytosis; Respiratory Burst; Streptococcal Infections; Streptococcus; Stress, Physiological; Temperature; Tilapia; Time Factors

A preliminary study with a 75days feeding trial was conducted to study the immunomodulatory effect of microbial levan on Cyprinus carpio juveniles. Five purified isonitrogenous and isocaloric diets with graded levels of levan, namely (T(1)) 0.1% levan, (T(2)) 0.2% levan, (T(3)) 0.5% levan, (T(4)) 1.0% levan, and a control group without levan were fed to five groups of fishes in triplicate. The total erythrocyte count and haemoglobin content was significantly (p<0.05) enhanced in the T(3) group, though the total leucocyte counts, total protein and A/G ratio did not show any significant difference (p>0.05) when compared with the control group. The respiratory burst activity (NBT) of blood phagocytes and lysozyme activity was also highest in T(3) group. The relative survival percentage after challenge with Aeromonas hydrophila was highest (100%) in the T(3) group followed by 83.33% and 66.67% in the T(2) and T(4) groups, respectively. This suggests that microbial levan at 0.5% can be used as a dietary immunostimulant for C. carpio juveniles. This is the first report on microbial levan having an immunomodulatory effect on C. carpio.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; Carps; Diet; Erythrocytes; Fish Diseases; Fructans; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Muramidase; Phagocytes; Random Allocation; Respiratory Burst; Survival Analysis; Time Factors

The study evaluated the efficacy of dietary doses of Mangifera indica (mango) kernel on the immune response and disease resistance of Labeo rohita fingerlings against the bacterial pathogen Aeromonas hydrophila infections in. L. rohita fingerlings fed diet containing 0 (Control), 1g, 5 g, 10 g mango kernel kg(-1) dry diet for 60 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio, blood glucose), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined at 20, 40 and 60 days of feeding. Fish were challenged with A. hydrophila 60 days post feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with mango kernel showed enhanced superoxide anion production, lysozyme, serum bactericidal, serum protein, albumin (P<0.05) compared with the control group. The mortality (%) was recorded up to 10 th day post-challenge. Less survivability was observed in control group (50%) up to day 10 after infection. The survivability was higher in experimental diets. The group fed 5 g kernel kg(-1) dry diet showed highest percentage survival (98%). These results indicate that mango kernel stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Blood Chemical Analysis; Blood Proteins; Cyprinidae; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Mangifera; Muramidase; Seeds; Serum Albumin; Superoxides; Survival Analysis; Time Factors

A 60 days experiment was conducted to study the effect of dietary gelatinized (G) and non-gelatinized (NG) starch on immunomodulation of Labeo rohita juveniles. Two hundred and thirty four juveniles (av. wt. 2.53+/-0.04) were randomly distributed in six treatment groups with each of three replicates. Six semi-purified diets containing NG and G corn starch, each at six levels of inclusion (0, 20, 40, 60, 80, 100) were prepared viz., T(1) (100% NG, 0% G starch), T(2) (80% NG, 20% G starch), T(3) (60% NG, 40% G starch), T(4) (40% NG, 60% G starch), T(5) (20% NG, 80% G starch) and T(6) (0% NG, 100% G starch). After a feeding period of 60 days, the juveniles were challenged with Aeromonas hydrophila to study their immunomodulation due to feeding of G and NG starch. RBC and haemoglobin content were significantly (P<0.05) reduced due to bacterial challenge, but dietary starch (G/NG starch) had no effect on it. G:NG starch ratio in the feed had significant effect on total leukocyte count during pre- and post-challenge periods. The leukocyte count concomitantly increased with the increased level of G starch in the diet. Highest albumin/globulin (A/G) ratio was recorded in T6 group (100% G starch) and lowest in T1 group (100% NG starch) group followed by T2 group both in pre- and post-challenge periods. NBT, lysozyme activity, total protein and globulin content were highest in T2 group (80% NG, 20% G starch) both in pre- and post-challenge periods. After challenge with A. hydrophila, the highest survival was recorded in T2 group, whereas lowest survival was recorded in T6 group. Conclusively high level of G starch was found to be immunosuppressive in Labeo rohita juveniles and NG:G starch ratio of 80:20 seems to be optimum for promoting growth and protecting immunity in L. rohita juveniles.

Topics: Aeromonas hydrophila; Animals; Blood Cell Count; Blood Proteins; Body Weight; Carps; Cholesterol; Dietary Carbohydrates; Fish Diseases; Gelatin; Gram-Negative Bacterial Infections; Hemoglobins; Immunologic Factors; Muramidase; Random Allocation; Respiratory Burst; Starch; Survival Analysis; Triglycerides

The purpose of this study was to determine the effect of water and hexane soluble fractions of the Indian medicinal plant, Solanum trilobatum on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish were intraperitoneally injected with different doses of 0, 4, 40 or 400 mg kg(-1) body weight of water or hexane soluble fraction. The nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, reactive oxygen species production and reactive nitrogen species production by peripheral blood leucocytes. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) was assessed by a challenge with live Aeromonas hydrophila. Almost all the doses of both water and hexane soluble fractions enhanced the serum lysozyme activity. All the doses of water soluble fraction significantly enhanced the ROS production on most of the days tested. In hexane soluble fraction treated groups, the enhancement in the ROS production was observed at least on 2 days. All the doses of water soluble fraction significantly enhanced the production of RNS only on one day. The RNS production was enhanced significantly only in the group treated with 40 mg kg(-1) of hexane fraction. The leaf fraction administration preceding the challenge with live A. hydrophila, decreased the percentage mortality in the experimental group with the consequent increase in RPS values. This preliminary study indicates that S. trilobatum could be used to promote the health status of fish in intensive finfish aquaculture.

Topics: Aeromonas hydrophila; Animals; Aquaculture; Fish Diseases; Gram-Negative Bacterial Infections; Male; Muramidase; Plant Extracts; Plant Leaves; Plants, Medicinal; Reactive Nitrogen Species; Reactive Oxygen Species; Solanum; Tilapia

The effects of unmethylated CpG oligodeoxynucleotides (ODN) on the mammalian immune system are relatively well studied but much less is known of their effects on the immune systems of different fish species. Here we show that CpG ODNs significantly enhance the survival of rainbow trout (Oncorhynchus mykiss) following bacterial challenge when used both as stand-alone prophylactic agents, or as adjuvants to a commercially available vaccine. They are also capable of increasing serum lysozyme activity in vivo and stimulating the production of chemoattractant factors for rainbow trout head kidney (HK) leucocytes in vitro.

Topics: Actinomycetales Infections; Adjuvants, Immunologic; Aeromonas salmonicida; Animals; Anti-Bacterial Agents; Blood Bactericidal Activity; Chemotaxis; Fish Diseases; Macrophages; Muramidase; Oligodeoxyribonucleotides; Oncorhynchus mykiss; Survival Analysis; Time Factors; Up-Regulation

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 x 10(8) cells g(-1) of feed, JB-1 led to a reduction in mortalities to 0-13% after challenge with a range of bacterial pathogens compared to 80-100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0-16% following the challenge compared to 80-100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.

Topics: Aeromonas; Animal Feed; Animals; Anti-Infective Agents; Bacillus; Bacteria; Bacterial Infections; Erythrocyte Count; Fish Diseases; Leukocyte Count; Macrophages; Muramidase; Oncorhynchus mykiss; Probiotics; Respiratory Burst; Siderophores; Survival Analysis

Responses of toll-like receptors (TLR3 and TLR5), lysozyme, and insulin-like growth factor-I (IGF-I) to experimental challenge with virulent Edwardsiella ictaluri were measured in back-cross hybrid (F1 male (blue x channel) x female channel) catfish. The resistance levels to E. ictaluri and host response mechanisms of back-cross hybrids are unknown. Fish were challenged with virulent E. ictaluri and sampled pre-challenge, 2 h and 2, 5, 8, 14, and 21 days post-challenge. Levels of mRNA expression of two toll-like receptors (TLR3 and TLR5) in liver, kidney, spleen, and stomach, plasma lysozyme activity, and circulating IGF-I levels were measured at each timepoint. Throughout challenge, TLR3 was expressed at higher levels than TLR5 in liver (P=0.0011) and kidney (P=0.0007) whereas TLR5 was more highly expressed than TLR3 in stomach (P=0.0032). TLR3 was upregulated in comparison to non-exposed controls in liver (P=0.0015) and stomach (P<0.0001) on day 14 and TLR5 was upregulated in liver (P=0.0175) on days 2 through 8. Plasma lysozyme activity peaked on day 5 (P<0.001) and IGF-I levels significantly decreased on days 2 through 14 (P<0.0001). TLR expression patterns suggest that both TLR3 and TLR5 may play a role in host response to bacterial challenge. Plasma lysozyme activity also increased and circulating IGF-I decreased in response to the presence of the pathogen.

Topics: Animals; Catfishes; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Gene Expression Profiling; Hybridization, Genetic; Insulin-Like Growth Factor I; Muramidase; RNA, Messenger; Time Factors; Toll-Like Receptors

Achyranthes aspera seed was incorporated in the diets (at 0.01%, 0.1% and 0.5%) of Labeo rohita, rohu fingerlings (3.0+/-0.4 g). After 2 weeks, the fish were immunized with heat-killed Aeromonas hydrophila, and after a further 2 weeks the rohu were experimentally infected with Aeromonas hydrophila (ATCC 49140). After 7 days blood and serum were sampled to determine superoxide anion production, bactericidal activity, lysozyme, serum protein, albumin, globulin, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP). Superoxide anion production, serum bactericidal activity, lysozyme, ALP, serum protein, albumin:globulin ratio (A/G) were enhanced in Achyranthes treated groups compared to the control group. SGOT and SGPT levels were elevated in control group, but in Achyranthes treated groups the levels were similar to the uninfected-control group. Higher cumulative mortalities were observed in the control group (77%) up to day-9 after infection. This gradually decreased with increasing dose of Achyranthes, 66% mortality in 0.01% group, 57% mortality in 0.1% group and 28% mortality in 0.5% group. These results indicate that Achyranthes aspera stimulates immunity and increases resistance to infection in L. rohita.

Topics: Achyranthes; Aeromonas hydrophila; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Blood Proteins; Cyprinidae; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Phytotherapy; Seeds; Serum Albumin; Serum Globulins; Superoxides

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Blood Chemical Analysis; Cyprinidae; Dose-Response Relationship, Drug; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Injections, Intraperitoneal; Leukocytes; Linear Models; Muramidase; Phagocytosis; Superoxides

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Blood Glucose; Blood Proteins; Carps; Complement System Proteins; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunologic Factors; Injections, Intraperitoneal; Leukocyte Count; Lymphokines; Muramidase; Phagocytosis; Serum Bactericidal Test; Superoxides; Time Factors; Tuftsin

This study investigated the effects of short and prolonged administration of a yeast beta-glucan on non-specific immune parameters, growth rate and the disease resistance of Asian catfish, Clarias batrachus. Fish fed with a basal diet (control) and test diet (basal diet supplemented with 0.1% glucan) for 1, 2 and 3 weeks were assayed for superoxide production, serum myeloperoxidase (MPO) content, natural haemagglutinin level, complement and lysozyme activities. Fish were weighed at weekly intervals and specific growth rate (SGR, % increase in body weight per day) was determined. After each week, fish were challenged with Aeromonas hydrophila to measure the level of protection. Results showed that glucan administration at 0.1% in feed, significantly (P<0.05) enhanced MPO and lysozyme levels, superoxide production, haemagglutination titre and level of protection against A. hydrophila challenge, irrespective of length of exposure. The alternative complement activity and SGR were not affected by the dietary supplementation of yeast glucan. As glucan feeding at 0.1% for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, short-term feeding might be used in farmed catfish diets to enhance disease resistance.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Catfishes; Complement System Proteins; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination; Immunity, Innate; Muramidase; Nitroblue Tetrazolium; Peroxidase; Random Allocation; Respiratory Burst; Time Factors

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Chromium; Environmental Exposure; Fish Diseases; Gram-Negative Bacterial Infections; Hemolytic Plaque Technique; Immunity, Innate; Male; Muramidase; Nitric Oxide; Tilapia; Time Factors

The innate and adaptive immune responses against Enteromyxum scophthalmi was studied in turbot (Scopthalmus maximus (L.)) experimentally exposed to the parasite by cohabitation. Haematological, histopathological, cellular and humoral factors were determined in samples taken from control (CTRL) and recipient (RCPT, naïve fish cohabited with donor infected fish) animals at 0, 20, 29, 40 and 43 days post exposure (p.e). Infection was first detected at day 20 p.e. and prevalence reached 100% at 40 days p.e, when first mortalities occurred. A significant reduction in weight and condition factor was found in RCPT, though no significant differences in haematocrit or serum protein levels were detected between CTRL and RCPT. Some immune effectors were clearly activated in RCPT: the percentage of circulating granulocytes was significantly increased, as well as the number of blood cells positive in the respiratory burst assay; leucocyte infiltration in intestine was found mainly on days 20 and 29 p.e.; total serum antiproteases and alpha-2-macroglobulin levels were higher in most of the samplings, with significant differences on the last sampling. Other effectors were clearly down regulated in RCPT: haematopoietic depletion appeared in head kidney from day 29 p.e. onwards, and the number of apoptotic cells and MMC increased in head kidney and spleen; the percentage of lymphocytes decreased progressively and significantly; a clear, but not statistically significant, drop in serum complement was registered at 40 days p.e.; also, a significant decrease occurred in serum lysozyme at 29 days p.e. No specific antibodies against the parasite were detected in any sampling.

Topics: alpha-Macroglobulins; Animals; Blood Cells; Complement Pathway, Alternative; Eukaryota; Fish Diseases; Flatfishes; Granulocytes; Immunity, Active; Immunity, Innate; Kidney; Muramidase; Protease Inhibitors; Protozoan Infections, Animal; Spleen; Time Factors

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.

Topics: Aeromonas; Animals; Bacteria; Blood Cell Count; DNA Primers; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Intestines; Muramidase; Oncorhynchus mykiss; Phagocytosis; Probiotics; Respiratory Burst; Survival Analysis; Time Factors; Trypsin; Yersinia; Yersinia Infections

In recent years, probiotics, especially lactic acid bacteria, have been used as dietary supplements to protect fish from various infections. Here, we examined the protective effects of Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Cumulative mortality was significantly lower in probiotic-supplemented fish than in control fish. In a histopathological survey, pyogranulomatous responses were observed at an earlier stage and to a greater extent in the probiotic-supplemented fish than in the control fish. Immunohistochemistry using an anti-E. tarda antibody revealed a larger number of positive signals in pyogranuloma-participating cells, indicating an enhanced phagocytic ability. Alternative complement activity was significantly higher in the probiotic groups than in the control. These results suggest that L. rhamnosus enhanced the alternative complement system of the fish, enabling phagocytic cell aggregation, increasing phagocytic activity and subsequently protecting the fish from acute septicemic death by E. tarda infection. Prevention of thymic necrosis by the probiotic supplement seems to minimize immunosuppression and to initiate an immune response against edwardsiellosis.

Topics: Animals; Complement Pathway, Alternative; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Immunohistochemistry; Lacticaseibacillus rhamnosus; Leukocytes; Muramidase; Probiotics; Random Allocation; Tilapia

Periods of stress are often associated with disease outbreaks in cultured fish, and stress is often characterized by the secretion of cortisol. Although stress and cortisol secretion are highly correlated in fish, the role of cortisol in affecting channel catfish (Ictalurus punctatus) pathogen susceptibility is unclear. The effects of short-term stress and exogenous cortisol administration on channel catfish susceptibility to Edwardsiella ictaluri, the etiologic agent of enteric septicemia of catfish (ESC), were investigated. Channel catfish were exposed to virulent E. ictaluri following a standardized 30-min low-water stress or administration of dietary cortisol (100 mg/kg feed) and compared to a pathogen-challenged control group of catfish. Pathogen susceptibility increased in stressed catfish (43.3% mortality) when compared to cortisol-fed catfish (26.7%) and controls (26.7%). A greater (P<0.05) percentage of stressed catfish (25.9%) tested positive for E. ictaluri relative to cortisol-fed catfish (13.0%) over the course of the study, however, average levels of circulating bacteria were not different (P>0.05) among the treatments. Catfish challenged by the low-water stress event had elevated (P<0.05) circulating levels of cortisol 1-day post-pathogen exposure and elevated (P<0.05) lysozyme activity 4 and 14 days post-pathogen exposure when compared to cortisol-fed and control-challenged catfish. Cortisol concentrations were not correlated (P>0.05) to either lysozyme activity or bacterial levels; however, lysozyme activity was positively correlated (P=0.0197) to blood bacterial concentrations. These results implicate other stress factors or pathways, separate from or possibly in conjunction with cortisol, in the stress-associated immunosuppression of channel catfish as it relates to ESC susceptibility.

Topics: Animals; Diet; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Hydrocortisone; Ictaluridae; Muramidase; Sepsis; Stress, Psychological

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mgkg(-1) body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant (P<0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.

Topics: Aeromonas hydrophila; Animals; Blood Glucose; Catfishes; Complement Pathway, Alternative; Cyclophosphamide; Fish Diseases; Gram-Negative Bacterial Infections; Hematocrit; Hydrocortisone; Immunity, Innate; Immunosuppression Therapy; Injections, Intraperitoneal; Muramidase; Nitroblue Tetrazolium; Peroxidase; Proteins; Superoxides; Survival Analysis

Sea bream broodstock were immunised 1 or 2 months before spawning with a novel photobacteriosis vaccine. Sixty-seven-day-old larvae (mean weight 22.3 mg) originating from immunised and non-immunised parents were experimentally infected with the Photobacterium damsela subsp. piscicida (Phdp). Larvae from immunised fish showed delayed onset and lower mortality (66.67%) compared with larvae from control fish (80%). Eighty-nine-day-old larvae (mean weight 162.2 mg) from both groups were bath vaccinated with Phdp and Escherichia coli lipopolysaccharides (LPS) and larval samples were collected for measurement of humoral parameters. Larvae vaccinated with Phdp and LPS showed significantly higher anti-protease activity, lysozyme activity and total immunoglobulin compared to the controls. One-hundred-and-twenty-day-old larvae (mean weight 297.85 mg) from both parental groups were challenged with (LD70) virulent Phdp bacterial cells. Vaccinated larvae from both groups showed significantly less mortality compared to the respective controls. The RPS values of larvae from immunised parents vaccinated with Phdp and LPS was 95.83% and 72.22%, respectively. The RPS values of larvae from non-immunised parents vaccinated with Phdp and LPS was 62.5% and 70.83%, respectively. Results are discussed with respect to the beneficial effect of broodstock immunisation prior to spawning and the immunisation of larvae on their survival against photobacteriosis.

Topics: Analysis of Variance; Animals; Antibody Formation; Aquaculture; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination Tests; Immunoglobulins; Larva; Lipopolysaccharides; Muramidase; Photobacterium; Sea Bream; Vaccination

An experiment was conducted to determine the effect of Neoparamoeba sp. infection on the innate immune responses of Atlantic salmon. Atlantic salmon were experimentally infected with Neoparamoeba sp. and serially sampled 0, 1, 4, 6, 8 and 11 days post-exposure (dpe). Histological analysis of infected fish gill arches identified the presence of characteristic amoebic gill disease lesions as early as 1 dpe with a steady increase in the number of affected gill filaments over time. Immune parameters investigated were anterior kidney phagocyte function (respiratory burst, chemotaxis and phagocytosis) and total plasma protein and lysozyme. In comparison with non-exposed control fish basal respiratory burst responses were suppressed at 8 and 11 dpe, while phorbol myristate acetate-stimulated activity was significantly suppressed at 11 dpe. Variable differences in phagocytic activity and phagocytic rate following infection were identified. There was an increase in the chemotactic response of anterior kidney macrophages isolated from exposed fish relative to control fish at 8 dpe. Total protein and lysozyme levels were not affected by Neoparamoeba sp. exposure.

Topics: Animals; Aquaculture; Blood Proteins; Chemotaxis; Fish Diseases; Gills; Histological Techniques; Immunity, Innate; Lobosea; Muramidase; Phagocytosis; Protozoan Infections; Protozoan Infections, Animal; Respiratory Burst; Salmo salar; Time Factors

The potential antiparasitic and immunomodulatory effect of three treatments against myxosporean parasites on the innate immune system of sharpsnout sea bream (Diplodus puntazzo) was investigated. Fish naturally infected with Myxobolus sp. (Bivalvulida/Platysporina), a histozoic parasite mainly affecting the renal interstitial tissue, were treated by oral administration of a combination of salinomycin with amprolium, Origanum essential oil or fumagillin in a small-scale field trial. Various leucocyte functions influenced by myxosporean infection were examined in order to determine treatment effects on leucocyte immunocompetence of treated fish. One month post treatment all drugs caused a significant decrease in prevalence and intensity of infection in comparison to untreated, infected fish. The effect was most prominent in salinomycin with amprolium treated fish, which 1-month post treatment contained either no cysts at all or a few spores free in melanomacrophage centres revealing almost total elimination of the parasite and the antiparasitic action of the treatment. There was no histopathological evidence of drug toxicity. Antiparasitic action was accompanied by a significant enhancement of phagocytic activity demonstrated by ingestion of large numbers of latex beads and the secretion of high levels of reactive nitrogen intermediates by phagocytes in vitro. Complete restoration of the diminished mitogenic responses and serum lysozyme secretion was also detected in salinomycin with amprolium-treated fish compared to untreated, infected fish. These data suggest that salilomycin with amprolium may be a promising treatment for myxosporean infections in intensively cultured warm-water fish, exhibiting action partially via the enhancement of host, innate immune functions and leading to parasite elimination.

Topics: Amprolium; Animals; Antiprotozoal Agents; Cell Proliferation; Cyclohexanes; Eukaryota; Fatty Acids, Unsaturated; Fish Diseases; Histocytochemistry; Kidney Diseases; Leukocytes; Mediterranean Sea; Muramidase; Oils, Volatile; Phagocytosis; Protozoan Infections, Animal; Pyrans; Reactive Nitrogen Species; Sea Bream; Sesquiterpenes

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

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.

Topics: Aeromonas; Animals; Bacterial Vaccines; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulins; Lipopolysaccharides; Muramidase; Phagocytosis; Poland

An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A. hydrophila infection in rainbow trout (Oncorhynchus mykiss). The protection conferred by the live attenuated vaccine against A. salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised. Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A. salmonicida (relative percent survival RPS >60%). This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout.

Topics: Aeromonas; Agglutination Tests; Analysis of Variance; Animals; Bacterial Vaccines; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Leukocytosis; Muramidase; Oncorhynchus mykiss; Protease Inhibitors; Vaccines, Attenuated

As an environmental protection point of view, the potential toxicity of chitosan on aquatic animal health, alone or associated with copper must be investigated. Fish possess defence mechanisms to counteract the impact of toxics. The non-cellular and non-specific immune defences (total immunoglobulin, ceruloplasmin, lysozyme and potential killing activity of phagocytic cells) can be modulated by the potential environmental pollutants but also by natural stimulants such as bacteria, viruses or parasites. In this study, we investigate the potential toxicity of copper (0.1 and 0.25 mg/L) or chitosan (75 and 150 mg/L) and the combination copper and chitosan (0.1 and 75 mg/L, respectively) on two groups of carp: healthy or parasitised by Ptychobothrium sp. Fish exposed to water-soluble chitosan for 96 h had significantly high levels of natural antibodies in plasma. Moreover, activities of lysozyme and ceruloplasmin were also increased in plasma after the same treatment. The exposition of fish to copper have shown apparently contradictory effects on the immune parameters measured but, significant increase of this bacteriolytic activity was observed, particularly in head kidney after 4 days of treatment of fish with copper. The two products may induce separately an acute, short and local inflammatory acute phase response by stimulating some components of the innate immune response of healthy fish. The mixture seems to reduce the impact of the each product due to the physical and chemical properties of chitosan to complex with copper. The responses of humoral immune factors of treated carp was modulated by the presence of the parasite, as shown by the high elevation of lysozyme activity observed in parasitised carps after exposition to copper and by increases in natural antibodies levels observed in parasitised carp treated with the copper-chitosan mixture. This could indicate an additive effect on the stress response mediated by parasite. It occurred a greater stress response in the parasitised group than healthy group exposed to the same treatment evoking an additive effect. So, it is important to specify the health status of organisms to understand responses of immunological markers in fish.

Topics: Animals; Antibodies; Antibody Formation; Carps; Ceruloplasmin; Cestoda; Cestode Infections; Chitin; Chitosan; Copper; Fish Diseases; Kidney; Lethal Dose 50; Liver; Muramidase

Immunisation of sea bream (Sparus aurata L.) broodstock with a novel vaccine mixture of Photobacterium damsela subsp. piscicida SK7 (Phdp) was performed during the period of egg development and the changes in specific and non-specific humoral immune parameters were measured. Total immunoglobulin level, specific antibody titre, anti-protease activity and lysozyme activity were significantly higher in immunised parents compared to the control. After spawning significantly higher anti-protease activity, lysozyme activity and total immunoglobulin level were detected in the eggs from immunised parents. Specific antibody titres against Phdp were only detected in the eggs from the immunised parents. The larvae from immunised parents also expressed significantly higher levels of specific and non-specific humoral immune parameters compared to the controls. A small amount of total immunoglobulin was detected in larvae decreasing gradually until day 8 post-hatching and then an increase was measured in larvae from immunised parents, whereas no immunoglobulin was detected at days 4, 6 and 8 in larvae from non-immunised parents. The specific antibody titre against Phdp was detected only in larvae from immunised broodstock until day 14 post-hatching. The higher humoral immune parameters in eggs and larvae from immunised parents in comparison to eggs and larvae from non-immunised parents, suggest transfer of maternal specific and non-specific immune factors.

Topics: Agglutination Tests; Analysis of Variance; Animals; Antibodies, Bacterial; Antibody Formation; Aquaculture; Blotting, Western; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Immunization; Immunoglobulins; Larva; Muramidase; Ovum; Photobacterium; Sea Bream; Time Factors; Trypsin

Barramundi, Lates calcarifer (Bloch), were immunized with an experimental Vibrio harveyi bacterin via intraperitoneal injection, immersion and anal intubation. Both specific and non-specific immune parameters were measured to compare responses to bacterin after delivery by various methods. Elevated antibody activities in sera were found in all treatment groups with barramundi injected intraperitoneally displaying significantly higher antibody activity than the other groups. In addition, there was evidence of memory induction with a heightened antibody response in the intraperitoneally injected group only. Bacteriostatic assays indicated activity against V. harveyi in the sera of all bacterin-treated groups; again this activity was significantly higher in the intraperitoneally injected groups. There was no enhancement noted in head kidney macrophage phagocytic activity or in serum lysozyme levels.

Topics: Analysis of Variance; Animals; Antibodies, Bacterial; Bacterial Vaccines; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Immersion; Immunologic Memory; Injections, Intraperitoneal; Intubation, Gastrointestinal; Muramidase; Perciformes; Phagocytosis; South Australia; Vibrio; Vibrio Infections

The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).

Topics: Animals; Enzyme Inhibitors; Fish Diseases; Flatfishes; Luminescent Measurements; Macrophages; Muramidase; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Vibrio; Vibrio Infections

There has been increasing interest in the use of probiotics in aquaculture to control fish diseases (e.g. Douillet Langdon 1994; Gildberg, Mikkelsen, Sandaker & Ringo 1997; Kennedy, Tucker, Neidic, Vermeer, Cooper, Jarrell & Sennett 1998; DeSchrijver & Ollevier 2000; Robertson, O'Dowd, Burrells, Williams & Austin 2000), and a diverse range of Gram-positive (such as Carnobacterium inhibens) (Robertson et al. 2000) and Gram-negative bacteria, including Vibrio alginolyticus (Garriques & Arevalo 1995), have been evaluated at various times. However, the possible use of probiotics to control diseases of ornamental fish has been neglected. Consequently, following previous success with live (Irianto & Austin 2002) and inactivated probiotics in salmonids (Irianto & Austin, in press), research has focused on the usefulness of inactivated bacterial preparations for the control of atypical Aeromonas salmonicida infections in goldfish, Carassius auratus (L.). Goldfish, of average 40-50 mm in length, were obtained from a commercial farm in England, and maintained in aerated re-circulating dechlorinated fresh water at 17 degrees C. Their health status was examined immediately upon arrival in the aquaria and at 1-2-week periods thereafter (after Austin & Austin 1989).

Topics: Administration, Oral; Aeromonas hydrophila; Animals; Aquaculture; Erythrocyte Count; Fish Diseases; Formaldehyde; Goldfish; Gram-Negative Bacterial Infections; Leukocyte Count; Macrophages; Muramidase; Probiotics

Topics: Aeromonas; Animal Feed; Animals; Aquaculture; Erythrocyte Count; Fish Diseases; Leukocyte Count; Muramidase; Oncorhynchus mykiss; Probiotics; Skin Diseases, Bacterial; Vibrio

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.

Topics: Alkaline Phosphatase; Animals; Copepoda; Disease Susceptibility; Ectoparasitic Infestations; Fish Diseases; Host-Parasite Interactions; Mucus; Muramidase; Oncorhynchus kisutch; Oncorhynchus mykiss; Phagocytosis; Salmo salar; Skin; Species Specificity

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.

Topics: Aeromonas; Animals; Antibodies, Bacterial; Bacterial Vaccines; Breeding; Conservation of Natural Resources; Fish Diseases; Gram-Negative Bacterial Infections; Injections, Intraperitoneal; Muramidase; Seasons; Temperature; Time Factors; Trout; Vaccination

Atlantic salmon parr were injected intraperitoneally with salmon pancreas disease virus (SPDV) grown on CHSE-214 cells. The viraemia, the histopathological changes in target organs and some immune parameters were taken at intervals up to 30 days post-infection (dpi). The earliest kind of lesion was necrosis of exocrine pancreas, appearing as soon as 2 dpi. It progressed towards complete tissue breakdown at 9 dpi before resolving gradually. Concurrent to this necrosis, a strong inflammatory response was in evidence from 9 dpi in the pancreatic area for a majority of fish. A necrosis of the myocardial cells of the ventricle occurred in infected fish mainly at 16 dpi and it faded thereafter. The monitoring of the plasma viral load showed a rapid haematogenous spreading of SPDV, peaking at 4 dpi, but also the absence of a secondary viraemia. No interferon (IFN) was detected following the infection of parr with SPDV, probably owing to an IFN activity in Atlantic salmon below the detection level of the technique. Neutralising antibodies against SPDV were in evidence from 16 dpi and they showed a time-related increasing titre and prevalence. The phagocytic activity in head-kidney leucocytes was always significantly higher in the infected fish than in the control fish, being particularly high by 9 dpi. Lysozyme and complement levels were both increased and they peaked significantly in the infected fish at 9 and 16 dpi respectively. These results demonstrated that an experimental infection of Atlantic salmon parr with SPDV provoked a stimulation of both specific and non-specific immunity with regards to the viraemia and the histopathology.

Topics: Alphavirus; Alphavirus Infections; Animals; Antibodies, Viral; Cells, Cultured; Complement Pathway, Classical; Fish Diseases; Immunohistochemistry; Injections, Intraperitoneal; Interferons; Muramidase; Necrosis; Pancreas; Pancreatic Diseases; Phagocytosis; Salmo salar; Time Factors; Viremia

The importance of genetic variation in the non-specific immune responses of Nile tilapia (Oreochromis niloticus L.) clones was investigated. Fully inbred clones (IC) of Nile tilapia, produced using gynogenesis and sex reversal, and crosses between these lines (outbred clones) were used in this study. Non-specific immune responses were compared between the ICs, including serum lysozyme activity and phagocytosis, and significant differences were observed between the different groups. Their natural resistance to Aeromonas hydrophila infection was also assessed by bacterial challenge. A positive correlation was observed between the level of infection obtained and the non-specific immune parameters measured. Cumulative mortalities of fish obtained in the study showed that when a IC susceptible to A. hydrophila was crossed with a resistant IC, the resulting progeny exhibited intermediate levels of resistance to that of their parents.

Topics: Animals; Cloning, Organism; Fish Diseases; Genetic Predisposition to Disease; Genetic Variation; Immunity, Innate; Muramidase; Tilapia

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum and Aeromonas salmonicida, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C(18) cartridge, gel filtration, and reverse-phase chromatography on a C(18) column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible Salmonella enterica serovar Typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses.

Topics: Aeromonas; Animals; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Fish Diseases; Fish Proteins; Flounder; Gram-Negative Bacterial Infections; Histones; Mucus; Muramidase; Oncorhynchus kisutch; Peptides; Proteins; Vibrio; Vibrio Infections

Two strains of freshwater-reared coho salmon Oncorhynchus kisutch were compared for differences in the activity of selected non-specific immune factors before and after lethal and non-lethal immersion challenges with the marine bacterial pathogen Vibrio anguillarum (Vang). Two disease challenge experiments were performed. The first experimental challenge resulted in no mortality; however, significant strain and challenge treatment effects were detected at Day 16 post-challenge. Strain differences in plasma lysozyme activity were found in pre-challenge samples. The second challenge experiment compared the same strains of coho salmon following immersion challenges in different doses of Vang. The fish were sampled at Days 0, 2, 7, and 18 post-challenge and mortality, plasma lysozyme, and anterior kidney phagocyte respiratory burst activity were compared. There were significant strain differences in mortality in the high dose group. The more disease-resistant strain was found to have higher levels of plasma lysozyme and anterior kidney phagocyte respiratory burst activity. These strain differences were detected at various times in the lethal (high dose) and non-lethal challenge groups. There was a clear relationship between the enhanced survival of the more disease-resistant strain and a more sustained, elevated non-specific immune response following the experimental disease challenges. The results of this study suggest that the basis for strain differences in innate disease resistance is related to the ability of the fish to respond quickly to the initial infection and to maintain the response until the infection is quelled.

Topics: Animals; Colony Count, Microbial; Dose-Response Relationship, Immunologic; Female; Fish Diseases; Immunity, Innate; Male; Muramidase; Oncorhynchus kisutch; Phagocytes; Respiratory Burst; Time Factors; Vibrio; Vibrio Infections

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

The changes in the activities of mucus hydrolytic enzymes and plasma cortisol levels were examined following infection of Atlantic salmon Salmo salar with the salmon louse Lepeophtheirus salmonis and these changes were compared with those resulting from elevated plasma cortisol. Salmon were infected at high (Trial 1; 178 +/- 67) and low (Trial 2; 20 +/- 13) numbers of lice per fish and the activities of proteases, alkaline phosphatase, esterase and lysozyme in the mucus, as well as plasma cortisol levels were determined. At both levels of infection, there were significant increases of protease activity over time (1-way K-WANOVA; Trial 1, p = 0.004; Trial 2, p < 0.001). On several sampling days, generally on later days in the infections, the mucus protease activities of infected fish were significantly higher than control fish (Student's t-tests; p < 0.05). In addition, zymography experiments demonstrated bands of proteases at 17 to 22 kDa in the mucus of infected salmon that were absent in the mucus from non-infected fish and absent in the plasma of salmon. The intensity of these protease bands increased in the mucus over the course of both infections. However, plasma cortisol levels were elevated only in the heavily infected fish from the first trial. At high infection levels (Trial 1), alkaline phosphatase activity was higher in the mucus of infected fish at all days (t-test, p < 0.05). However, at the lower infection level (Trial 2), the mucus alkaline phosphatase activity did not differ significantly between infected and non-infected fish. Esterase and lysozyme activities were very low and did not change with time nor between non-infected and infected salmon in either challenge. Mucus enzyme activities of cortisol-implanted salmon did not change over time, nor were there any differences in activities between cortisol-implanted and control salmon. The present study demonstrates biochemical changes resulting from sea lice infection of Atlantic salmon occurring at the site of host-pathogen interaction, the mucus layer. However, the origin of these enzymes, whether host or pathogen, remains to be determined.

Topics: Alkaline Phosphatase; Analysis of Variance; Animals; Crustacea; Drug Implants; Ectoparasitic Infestations; Endopeptidases; Esterases; Fish Diseases; Hydrocortisone; Mucus; Muramidase; Salmo salar; Skin

Protein-polysaccharide conjugate was prepared as a functional biopolymer using protein and polysaccharide via a Maillard-type reaction. Ovalbumin and lysozyme were conjugated with galactomannan under controlled heating and humidity conditions. The antioxidant effect of ovalbumin and the antimicrobial activity of lysozyme were enhanced by the glycosylation. The emulsifying properties of the egg protein were also significantly improved by the modification. The increase in lipid affinity due to the conjugation resulted in the enhancement of the radical scavenging ability of ovalbumin. The effectiveness of lysozyme and its glycosylated derivative in restricting the activity of a Gram-negative pathogen, Edwardsiella tarda in fish was also investigated.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; Biopolymers; Carps; Crystallization; Edwardsiella; Electrophoresis, Polyacrylamide Gel; Emulsions; Fish Diseases; Galactose; In Vitro Techniques; Lethal Dose 50; Maillard Reaction; Mannans; Muramidase; Mutagens; Ovalbumin; Rats

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Krøyer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.

Topics: Animals; Crustacea; Ectoparasitic Infestations; Female; Fish Diseases; Hematocrit; Leukocytes; Male; Muramidase; Oncorhynchus mykiss; Spleen; Stress, Physiological

Utilization of natural immunostimulants in fish culture offers a wide range of attractive methods for inducing and building protection against diseases. Lysozyme is an enzyme with bacteriolytic properties and is ubiquitous in its distribution among living organisms. This enzyme has antiviral, antibacterial and anti-inflammatory properties. In nature, lysozyme is found as a monomer. Lysozyme dimer is significantly less toxic than its monomer, and its high biological activity has been ascertained in cases of both viral and bacterial infections. In our study, we examined the influence of dimerized lysozyme (KLP-602) on the nonspecific cellular and humoral defense mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss). We have analyzed the immunomodulatory effects of KLP-602 after experimental infection by Aeromonas salmonicida. Application of dimerized lysozyme (KLP-602) by injection stimulated the cellular and humoral defense mechanisms and provided protection against furunculosis. By contrast, mortality rate was reduced to 45% (one injection) and 25% (three injections) using 10 or 100 microg/kg KLP-602. Mortality in the untreated control group was 85%.

Topics: Adjuvants, Immunologic; Aeromonas; Animals; Cytotoxicity, Immunologic; Dimerization; Fish Diseases; Furunculosis; Gram-Negative Bacterial Infections; Immunity, Cellular; Immunoglobulins; Muramidase; Oncorhynchus mykiss; Phagocytes; Respiratory Burst; Time Factors

The protective effect of lysozyme-galactomannan or lysozyme-palmitic acid conjugates orally administered to carp, Cyprinus carpio L. was investigated using a virulent strain of Gram-negative Edwardsiella tarda isolated from an infected fish. Lysozyme-galactomannan conjugate was prepared through controlled Maillard reaction. Lysozyme-palmitic acid conjugate was prepared through base-catalyzed ester exchange using N-hydroxysuccinimide ester of palmitic acid. The conjugates provided substantial protection to carp infected with a Gram-negative bacteria fish pathogen E. tarda NG 8104. Lytic activities of lysozyme conjugates with galactomannan and palmitic acid were about 80 and 71% of native lysozyme using Micrococcus lysodeikticus as a substrate. Feeding with lysozyme conjugates, for 8 days, significantly enhanced fish protection against E. tarda infection. The survival rate was 30% for lysozyme-galactomannan conjugate treated fish and 20% for lysozyme-palmitic acid conjugate treated fish after 6 days cultivation while all control fish died within 3 days. On the other hand, a recovery rate of 40% after 6 days was observed in the fish group that were fed lysozyme-palmitic acid conjugate 3 and 2 h before and after E. tarda challenge, respectively, and for 6 consecutive days. The results of this work show the possibility of utilizing lysozyme conjugates with galactomannan or palmitic acid as a therapeutic for infection in fish.

Topics: Animals; Anti-Bacterial Agents; Fish Diseases; Galactose; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Maillard Reaction; Mannans; Microbial Sensitivity Tests; Muramidase; Palmitic Acid; Time Factors; Virulence

Topics: Animals; Complement Fixation Tests; Cyprinidae; Erysipelas; Fish Diseases; Muramidase; Phagocytosis; Properdin