ATTENUATION, PERSISTENCE, AND VACCINE POTENTIAL OF AN EDWARDSIELLA-ICTALURI PURA MUTANT

In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine eff icacy were evaluated. A clone containing the purA gene was isolated fr om an E. ictaluri genomic library, sequenced, and shown to have an ove rall sequence identity of 79.3% at the nucleotide level and 85.7% at t he amino acid level with the Escherichia coli purA gene. The cloned E. ictaluri purA gene Was mutated by deleting a 598-bp segment of the ge ne and inserting the kanamycin resistance gene from Tn903 into the gap . The Delta purA::Km(r) gene was subcloned into the suicide plasmid pG P704, and the resulting plasmid was used to deliver the modified gene into a virulent strain off. ictaluri by conjugation. Homologous recomb ination replaced the chromosomal purA gene with the mutated gene to cr eate an adenine-auxotrophic strain (LSU-E2). Compared to wild-type E. ictaluri, LSU-E2 was highly attenuated by the injection, immersion, an d oral routes of exposure. Ey the injection route, LSU-E2 had a 50% le thal dose (LD50) that was greater than 5 logs(10) higher than the LD50 for wild-type E. ictaluri. In a tissue persistence study, LSU-E2 was able to invade channel catfish by the immersion route and persist in i nternal organs for at least 48 h. Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lo wer (P < 0.01) following a wild-type E. ictaluri challenge than that o f nonvaccinated fish.