TWENTY-FIFTH ANNUAL EASTERN FISH HEALTH WORKSHOP MARCH 10-13, 2000   An Efficacious Vaccine For Photobacterium damsela Subsp.  piscicida                                            Ronald L. Thune1,2, John P. Hawke1 and Denise H. Fernandez2   Departments of Veterinary Science1, Louisiana State University Agricultural Center and Veterinary Microbiology and Parasitology2, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803, USA     Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino acid biosynthesis, or the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases.  In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damsela subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damsela genomic library by complementation in an aroA mutant of E. coli.  The complementing plasmid was isolated and the nucleotide sequence of the P. damsela genomic insert was determined.  Subsequent analysis of the DNA sequence data demonstrated that the identified plasmid contained 3464 bp of P. damsela DNA, including the complete P. damsela aroA gene. Following deletion of a 144 base pair MscI fragment, the kanamycin resistance gene from transposon Tn903 was ligated into the MscI site.  This ?aroA::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damsela by conjugation and allelic exchange.  One selected mutant, LSU-P2, was confirmed to require supplementation with aromatic metabolites for growth in minimal media and was confirmed genotypically by PCR and DNA sequencing.  To evaluate vaccine efficacy, four replicated groups of fish were immersed for 15 minutes in 107 colony forming units (CFU)/ml of LSU-P2 and eight groups were sham-vaccinated for 15 minutes in diluent water only. All of the vaccinated fish and four groups of the sham vaccinated groups were challenged after 6 weeks by immersion in 9000 CFU/ml of the wild-type parent P. damsela 91-197.  Mean percent mortality was significantly higher (P<0.01 in ANOVA) in the sham vaccinated groups (± standard deviation) 91.65 ± 6.9, compared to 15.02 ± 11.0 in the vaccinated group.  No fish died in the sham-vaccinated, non-challenged controls.    Return to 25th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page