Attenuation, persistence, and vaccine potential of an Edwardsiella ictaluri purA mutant

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Abstract
In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine efficacy were evaluated. A clone containing the purA gene was isolated from an E. ictaluri genomic library, sequenced, and shown to have an overall sequence identity of 79.3% at the nucleotide level and 85.7% at the 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 gene and inserting the kanamycin resistance gene from Tn903 into the gap. The delta purA::Km(r) gene was subcloned into the suicide plasmid pGP704, and the resulting plasmid was used to deliver the modified gene into a virulent strain of E. ictaluri by conjugation. Homologous recombination replaced the chromosomal purA gene with the mutated gene to create an adenine-auxotrophic strain (LSU-E2). Compared to wild-type E. ictaluri, LSU-E2 was highly attenuated by the injection, immersion, and oral routes of exposure. By the injection route, LSU-E2 had a 50% lethal dose (LD50) that was greater than 5 logs10 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 internal organs for at least 48 h. Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lower (P < 0.01) following a wild-type E. ictaluri challenge than that of nonvaccinated fish.