bassianolide and Streptococcal-Infections

Streptococcus iniae poses a serious threat to finfish aquaculture operations worldwide. Stringent regulatory standards limit the use of antibiotics to treat S. iniae infections; improved vaccination strategies are thus of great interest. We investigated the potential for efficient, non-injectable batch vaccination via the use of live attenuated vaccines. Three attenuated S. iniae strains with genetic mutations eliminating the production of virulence factors--capsular polysaccharide (delta cpsD), M-like protein (delta simA), and phosphoglucomutase (delta pgmA)--were evaluated in parallel with an adjuvanted, formalin-killed, whole-cell S. iniae bacterin. Juvenile hybrid striped bass (HSB; Morone chrysops x M. saxatilis) were vaccinated through intraperitoneal (i.p.) injection or bath immersion and held for 800 degree-days prior to challenge with a lethal dose of the virulent wild-type (WT) S. iniae parent strain. The delta cpsD, delta pgmA, and bacterin vaccines provided the highest level of vaccination safety (0% mortality), whereas the delta simA mutant, although it caused 12 to 16% vaccination-related mortality, was the only vaccine candidate to provide 100% protection in both i.p. and immersion delivery models. Our studies demonstrate the efficacy of live attenuated vaccines for prevention of S. iniae infection, and identify immersion delivery of live vaccines as an attractive option for use in commercial aquaculture settings.

Topics: Administration, Topical; Animals; Bass; Injections, Intraperitoneal; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Time Factors; Vaccines, Attenuated

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

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

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

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

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

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

Interleukins are critical cytokines that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. Here, we identified and analyzed IL-12p40 from the Chinese sea bass Lateolabrax maculatus (LmIL-12p40). The LmIL-12p40 gene is expressed as a 1386-base pair transcript that encodes a polypeptide of 321 amino acids. Transcriptional expression analysis indicated that LmIL-12p40 mRNA was ubiquitously expressed in all tested tissues and had a comparatively high expression level in immune-associated tissues (head-kidney and intestines). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments showed that, after Vibro harveyi and Streptococus agalactiae infection, LmIL-12p40 mRNA expression was significantly up-regulated in the spleen, liver and head-kidney. To further clarify the immune function of LmIL-12p40 after bacterial challenge, the recombinant LmIL-12p40 protein was acquired using a prokaryotic expression method. Furthermore, the LmIL-12p40 dimer (LmIL-12p80) could be produced via protein-protein interactions by incubating p40 monomer expressed from the pET28a vector (pET28a-LmIL-12p40) with p40 monomer expressed from the pGEX4T-1 vector (pGEX4T-1-LmIL-12p40). The antimicrobial activity of the purified LmIL-12p40 and LmIL-12p80 proteins were further studied in vitro using a bacterial growth inhibition test (for both liquid and solid cultures) and in vivo (using a bacterial growth inhibition test with the head-kidney tissues). Furthermore, BL21 (DE3) E. coli cells transformed with the recombinant pET28a-LmIL-12p40 vector were dramatically protected in response to metal toxicity and H

Topics: Animals; Bacteria; Bass; Cell Movement; Escherichia coli; Fish Diseases; Fish Proteins; HEK293 Cells; Humans; Hydrogen Peroxide; Interleukin-12 Subunit p40; Metals, Heavy; Streptococcal Infections; Stress, Physiological; Vibrio Infections

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Topics: Animals; Bass; Fish Diseases; Polymerase Chain Reaction; Streptococcal Infections; Streptococcus agalactiae

Topics: Animals; Anti-Bacterial Agents; Bass; Cichlids; Drug Resistance, Bacterial; Fish Diseases; Fisheries; Malaysia; Streptococcal Infections; Streptococcus agalactiae; Tilapia

Streptococcus iniae is a Gram-positive bacteria that causes invasive infections with severe septicemia and meningitis, producing high economic losses in marine and continental aquaculture. Head kidney leukocytes of European sea bass (Dicentrarchus labrax) were used to measure the differential innate immune response upon infection with S. iniae. The complete inhibition in the production of intracellular superoxide radicals and total peroxidase content was observed in infected cells. This study also elucidates changes in the relative expression of some immune-related genes. Interleukin 1β, tumor necrosis factor-α and interleukin-6 reached a peak of expression at 4-8 h post-infection, subsequently decreasing significantly up to 48 h post-infection. However, interleukin-10 and Mx protein increased over time, reaching the pick of expression at 48 h post-infection, whereas caspase-3 showed down regulation until 48 h post-infection. The in vivo study of immune related genes show the same kinetics of mRNAs expression as in vitro experience. The proinflammatory cytokines mRNA transcription levels peaked at an earlier time in vivo than in vitro system. Our findings indicate that there is a direct relationship between the dissemination of bacteria and the resulting infection-associated inhibition of respiratory burst, apoptosis, and the pro- and anti-inflammatory gene expression profiles.

Topics: Animals; Bass; Cytokines; Fish Diseases; Head Kidney; Immunity, Innate; Leukocytes; Peroxidases; Respiratory Burst; Streptococcal Infections; Streptococcus

Vaccination is an important strategy in the protection of aquaculture species from major diseases. However, we still do not have a good understanding of the mechanisms underlying vaccine-induced disease resistance. This is further complicated by the presence of several lymphoid organs that play different roles when mounting an immune response. In this study, we attempt to elucidate some of these mechanisms using a microarray-based approach. Asian seabass (Lates calcarifer) were vaccinated against Streptococcus iniae and the transcriptomic changes within the spleen and head kidney at one and seven days post-vaccination were profiled. We subsequently challenged the seabass at three weeks post-vaccination with live S. iniae and similarly profiled the transcriptomes of the two organs after the challenge. We found that vaccination induced an early, but transient transcriptomic change in the spleens and a delayed response in the head kidneys, which became more similar to one another compared to un-vaccinated ones. When challenged with the pathogen, the spleen, but not the head kidneys, responded transcriptomically at 25-29 hours post-challenge. A unique set of genes, in particular those involved in the activation of NF-κB signaling, was up-regulated in the vaccinated spleens upon pathogen challenge but not in the un-vaccinated spleens. A semi-quantitative PCR detection of S. iniae using metagenomic DNA extracted from the water containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. This result indicated that vaccination not only led to a successful immune defense against the infection, but also reduced the chances for horizontal transmission of the pathogen. In conclusion, we have provided a transcriptomic analysis of how the teleost spleen and head kidneys responded to vaccination and subsequent infection. The different responses from the two organs are suggestive of their unique roles in establishing a vaccine-induced disease resistance.

Topics: Animals; Bass; Fish Diseases; Kidney; Spleen; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Transcriptome; Vaccination

Vaccination is the most effective means of preventing infectious diseases; however, few vaccines are effective against Streptococcus iniae (S. iniae) in grouper. This work presents an efficacious and safe vaccine against S. iniae infections in the grouper Epinephelus coioides. The vaccine candidate was the S. iniae GSI-310 strain. The vaccination was administered by intraperitoneal injection, and consisted of formalin-inactivated antigens combined with an AS-F or ISA763A adjuvant. Peripheral blood samples were collected for RT-qPCR and phagocytosis and agglutination assays. Our results indicated that immunoglobulin M (igm) was maximally expressed in the two vaccinated groups at 3 months post-secondary vaccination (PSV). A significant upregulation of mRNA expression for interleukin-1β (il-1β) and tumor necrosis factor-α (tnf-α) was also observed in fish treated with antigens combined with ISA763A, which peaked at 3 months PSV. In fish treated with antigens combined with AS-F, il-1β and tnf-α expression peaked at 14 days post-primary vaccination (PPV). Phagocytic activity and index increased significantly in the two vaccinated groups. Furthermore, fish in the two vaccinated groups exhibited significantly elevated agglutination titers compared to fish in the control group, in which almost no agglutination reaction was detected. In the efficacy test, the vaccinated and control groupers were treated with S. iniae at 1, 3, and 6 months PSV. The relative percentage survival (RPS) values of antigens with AS-F and antigens with ISA763A were both 100% at 1 and 3 months PSV; at 6 months PSV, the RPS values for these groups were 100% and 97.7%, respectively. Furthermore, the level of protection observed in the field trial closely resembled that achieved on a laboratory scale. Therefore, the proposed vaccine mixed with AS-F or ISA763A improved immune responses and provided safe and long-lasting protection in farmed groupers.

Topics: Agglutination Tests; Animals; Antibodies, Bacterial; Bass; Fish Diseases; Immunization; Immunoglobulin M; Injections, Intraperitoneal; Interleukin-1beta; Leukocytes, Mononuclear; Phagocytosis; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Survival Analysis; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

Streptococcosis is a common cause of pathology and mortality in fishes resulting in significant economic losses for the aquaculture industry. One etiologic agent of the disease, Streptococcus parauberis, has been associated with fish mortalities in Spain and Korea. Here we report the first identification of S. parauberis in wild finfish in Chesapeake Bay, USA. Gram-positive cocci were isolated from the spleens of striped bass, Morone saxatilis, and identified via species-specific primers and 16S rRNA gene sequencing. Biochemical characterization and antibiotic susceptibility tests were used to compare local isolates to isolates infecting aquacultured fishes and dairy cattle. This is also the first report of a plasmid in S. parauberis from any host.

Topics: Animals; Animals, Wild; Bass; Cattle; Fish Diseases; North America; Spleen; Streptococcal Infections; Streptococcus

Streptococcus iniae causes invasive infections in fresh and saltwater fish and occasional zoonoses. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. A potential target for serologically cross-protective vaccines is the M-like protein SiMA, an essential virulence factor in S. iniae that is highly conserved amongst virulent strains. The present study determined how SiMA is regulated and investigated potential as a cross-protective vaccine for fish. Electrophoretic mobility shift suggested that SiMA is regulated by the multigene regulator Mgx via a binding site in the -35 region of the simA promoter. Moreover, expression of simA and mgx was highly correlated, with the highest level of simA and mgx expression during exponential growth under iron limitation (20-fold increase in relative expression compared to growth in Todd-Hewitt broth). Based on these results, a vaccination and challenge experiment was conducted in barramundi (Lates calcarifer) to determine whether SiMA is protective against S. iniae infection and cross-protective against a different capsular serotype. The challenge resulted in 60% mortality in control fish. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection, whereas bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Moreover, recombinant SiMA protein was not protective against the challenge strain in spite of eliciting specific antibody response in vaccinated fish. Specific antibody did not increase oxidative activity or phagocytosis by barramundi macrophages. Indeed incubating S. iniae with antisera significantly reduced phagocytosis. Lack of specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous vaccine suggests that other immune parameters result in protection of challenged fish.

Topics: Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Bass; Cross Protection; Fish Diseases; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Vaccination; Virulence Factors

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

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.

Topics: Animals; Bass; Cell Line; Colony Count, Microbial; Fish Diseases; Hemolysis; Injections, Intraperitoneal; Phagocytosis; Random Allocation; Species Specificity; Streptococcal Infections; Streptococcus; Virulence Factors

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Topics: Animals; Bacterial Capsules; Bass; Carbohydrates; Cell Line; Epithelial Cells; Fish Diseases; Gene Expression Regulation, Bacterial; Macrophages; Molecular Sequence Data; Phagocytosis; Streptococcal Infections; Streptococcus; Virulence

Streptococcus iniae is a leading pathogen of intensive aquaculture operations worldwide, although understanding of virulence mechanisms of this pathogen in fish is lacking. S. iniae possesses a homolog of streptolysin S (SLS), a secreted, pore-forming cytotoxin that is a proven virulence factor in the human pathogen S. pyogenes. Here we used allelic exchange mutagenesis of the structural gene for the S. iniae SLS precursor (sagA) to examine the role of SLS in S. iniae pathogenicity using in vitro and in vivo models. The isogenic Delta sagA mutant was less cytotoxic to fish blood cells and cultured epithelial cells, but comparable to wild-type (WT) S. iniae in adherence/invasion of epithelial cell monolayers and resisting phagocytic killing by fish whole blood or macrophages. In a hybrid striped bass infection model, loss of SLS production led to marked virulence attenuation, as injection of the Delta sagA mutant at 1000x the WT lethal dose (LD80) produced only 10% mortality. The neutralization of SLS could represent a novel strategy for control of S. iniae infection in aquaculture.

Topics: Animals; Bacterial Proteins; Bass; Brain; Carps; Cell Line; Colony Count, Microbial; Fish Diseases; Gene Expression Regulation, Bacterial; Genetic Engineering; Hemolysis; Kaplan-Meier Estimate; Mutation; Streptococcal Infections; Streptococcus; Streptolysins; Time Factors; Virulence; Virulence Factors

An experimental feeding trial was performed to evaluate the efficacy of florfenicol (FFC) in controlling Streptococcus iniae infection in sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis). Five dosage levels of FFC in medicated feed were administered daily: 0, 5, 10, 15, and 30 mg of active ingredient/kg of fish body weight. Treatment was started within 22-24 h postchallenge by waterborne exposure to virulent S. iniae. The FFC medication was continued for 10 consecutive days, followed by a 25-d posttreatment observation. At the conclusion of the experiment, FFC treatment significantly increased the survival of S. iniae-challenged sunshine bass from 4.2% in the nonmedicated (positive control) group to 69.2% in the 5-mg/kg dosage group, 86.7% in the 10-mg/kg group, and 94.2% in the 15- and 30-mg/kg groups. Survival was significantly higher in the 15- and 30-mg/kg treatment groups than in the 5-mg/kg treatment group; differences among the 10-mg/kg and higher dosage groups were not significant. Survival curve analysis using a log-rank test indicated no significant difference between curves for the 10- and 15-mg/kg groups but a significant difference between curves for the 5- and 10-mg/kg groups. At the end of the experiment, no carriers were detected in any challenged group receiving an FFC-medicated diet, but the bacterium was recovered from the nonmedicated challenged survivors of the infection. The results of the experiment suggest that the optimum therapeutic daily dose of FFC is between 10 and 15 mg/kg body weight for 10 d.

Topics: Animals; Anti-Bacterial Agents; Bass; Dose-Response Relationship, Drug; Fish Diseases; Kaplan-Meier Estimate; Streptococcal Infections; Streptococcus; Thiamphenicol; Time Factors; Treatment Outcome

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bass; Fish Diseases; Fish Proteins; Gene Deletion; Genes, Bacterial; Molecular Sequence Data; Mutation; Phosphoglucomutase; Streptococcal Infections; Streptococcal Vaccines; Streptococcus; Virulence; Virulence Factors

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

To determine the prevalence of Streptococcus iniae in tilapia (Oreochromis spp), hybrid striped bass (Morone chrysops X M saxatilis), and channel catfish (Ictalurus punctatus) on commercial fish farms in the United States.. 1,543 fish (970 tilapia, 415 hybrid striped bass, and 158 channel catfish).. The dry-swab technique was used for collection of specimens for streptococcal isolation. Specimens were shipped by overnight delivery and processed by use of standard bacteriologic techniques.. Streptococcus iniae was not isolated from market-size channel catfish. Prevalence in tilapia and hybrid striped bass was 37 of 970 (3.81%) and 30 of 415 (7.23%), respectively. Prevalence by farm ranged from 0.0 to 27.4% for tilapia and 0.0 to 21.6% for hybrid striped bass. In tilapia, prevalence was lowest in market-size and nursery fish (4 of 239 [1.67%] and 3 of 339 [0.88%], respectively), with an increase in prevalence for fish in the grow-out stage (30 of 337 [7.96%]). For hybrid striped bass, prevalence was lowest in nursery and market-size fish (3 of 96 [3.12%] and 1 of 47 [2.12%], respectively) and highest in fish in the grow-out stage (26 of 272 [9.56%]). Prevalence in market-size tilapia and hybrid striped bass was 5 of 286 (1.75%).. Results of this study do not support the contention that S iniae is a serious public health threat associated with commercially raised fish; rather, it represents a limited risk for older or immunocompromised people who incur puncture wounds while handling and preparing fish.

Topics: Animals; Bass; Colony Count, Microbial; Fish Diseases; Fisheries; Fishes; Ictaluridae; Prevalence; Streptococcal Infections; Streptococcus; Tilapia; United States

Streptococcus iniae was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single S. iniae clone. Wild fish are therefore potential amplifiers of pathogenic S. iniae strains.

Topics: Animals; Animals, Domestic; Animals, Wild; Bass; Fish Diseases; Perciformes; Polymerase Chain Reaction; Sensitivity and Specificity; Streptococcal Infections; Streptococcus