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 ⌬PGM mutant cells reaching sizes ϳ3-fold larger than those of the wild type (WT). The ⌬PGM 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 ⌬PGM 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 ⌬PGM mutant. In analysis of the aborted infectious process, we found that ⌬PGM 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 ⌬PGM 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 ⌬PGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.With increased development of intensive operations, disease has become a significant hurdle to the profitable culture of fish and shellfish. Streptococcal infections in fish, in particular those produced by the pathogen Streptococcus iniae, have increased markedly with intensification of aquaculture practices (37). S. iniae causes a fatal meningoencephalitis and is associated with large-scale mortality in a wide variety of marine and freshwater cultured fish species, as well as in wild species (5,39,46). More than 30 species of fish have documented susceptibility to S. iniae disease, including trout (10), yellowtail (19), tilapia (39), barramundi (6), and hybrid striped bass (HSB) (11). S. iniae is distributed globally and is estimated to cause yearly economic losses of hundreds of millions of dollars. Occasionally, S. iniae can cause serious zoonotic infections in humans who injure themselves while handling infected fish (20,42).Relatively little is known of the genetics of S. iniae or of the pathogenic mechanisms underlying its virulence. Here, we describe a severely attenuated mutant of S. iniae, identified through random transposon mutagenesis and direct screening for virulence in HSB. Analysis of the transposon insertion site revealed a disruption of an open reading frame (ORF) with similarity to bacterial phosphoglucomutase (pgm) genes. The enzyme phosphoglucomutase (PGM) interconverts glucose-6-phosphate an...

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops ؋ Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel ␤-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, ␣-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4 -5 orders of magnitude above base-line 24 -48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue.

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Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.