28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Molecular Characterization Of Virulence Factors Expressed By Marine Vibrios Ben D. Tall1, J. Arnold2, S.R. Monday1, M. Ramos-Valle1, G. Modderman1, E. Ayala-Diaz1, V. Vegas1,  A. Johnson1, A. Nyarko1, I. Agba1, S. Taherkhani1, T. DeSiano1, R. Gonzalez-Nieves1, and M.H. Kothary3 1Div. of Microbiolical Studies. and 3Virulence Assessment, CFSAN, USFDA, 5100 Paint Br. Parkway, College Park, MD 20740 and the 2Clin. Lab., National Aquarium, Baltimore, East Pratt Street, Baltimore, MD 21202 Vibrio species cause both systemic infections in seafood species and gastroenteritis, wound infections, and septicemia in humans. A collaborative study was initiated to characterize a subset of 69 Vibrio strains from the collection reported last year by Arnold and Gargan for the presence or absence of known Vibrio virulence factors by phenotypic and molecular methods such as the polymerase chain reaction (PCR). The collection included 17 Vibrio alginolyticus (Val), 7 Vibrio anguillarum (Van), 12 Photobacterium damselae (Pdam), 2 Vibrio fluvialis (Vf), 17 Vibrio logei (Vlog), 2 Vibrio ordalii (Vor), 2 Vibrio parahaemolyticus (Vpa), 4 Listonella pelagia (Lpel), 6 V. vulnificus (Vv) strains. Growth on Sheep blood agar showed that approximately 70% of the strains were hemolytic. Among the strains, there were several hemolysin phenotypes noted; which ranged from complete hemolysis to diffuse zones of clearing. A PCR assay to identify Val strains was designed using putative Val-specific primers for gyrB and toxR genes. These PCR results showed that a predicted 501 bp gyrB product and a 508 bp toxR product were amplified in 16 of 17 Val strains and in all Val strains, respectively. However, these PCR gene products were also amplified in 68% and 88% of the other Vibrio strains suggesting that these primers were not species-specific. Interestingly, this is the first report to suggest that both Lpel and Vlog possess a toxR homolog. Using primers that we developed from the published DNA sequence for the damselysin gene of Pdam and existing PCR primers published for the Pdam 16s rRNA gene, a duplex PCR assay was developed that detected both the 16s rRNA gene and the damselysin gene of Pdam. Of the 12 Pdam strains analyzed only1 was found to possess the damselysin gene. In comparison, the 16s rRNA gene target was only found in Pdam strains. These results suggested that other hemolysins besides damselysin are produced by Pdam and that the16s rRNA gene target was very specific for Pdam.  In conclusion, several hemolysin phenotypes were noted among the strains. ToxR PCR primers identified this gene in Lpel and Vlog strains, but were not specific for Vals. A PCR assay that simultaneously detected the damselysin gene and the16s rRNA gene in Pdam showed that other hemolysins besides damselysin were produced and that the 16s rRNA gene target was specific for Pdam. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page