Development and in Vivo Evaluation of a purA Mutant Strain of the Channel Catfish Pathogen Edwardsiella Ictaluri.

Abstract

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is the most serious disease affecting the channel catfish industry. Killed vaccines have been inconsistent in providing protection against ESC, and no live attenuated vaccines have been described. The purpose of this study was to develop a nutritionally attenuated mutant strain of E. ictaluri and evaluate its virulence and vaccine efficacy. An E. ictaluri genomic library was constructed, and a clone containing the purA gene was isolated and sequenced. The E. ictaluri purA gene was found to have several features in common with the E. coli purA gene, and overall sequence identity with the E. coli purA gene was 79.3% at the nucleotide level and 85.7% at the amino acid level. The cloned E. ictaluri purA gene was mutated by deleting a 598 base pair segment of the gene and cloning a gene conferring resistance to kanamycin into this site. The $\Delta purA$::Km$\sp{\rm r}$ 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, which was designated LSU-E2. LSU-E2 was characterized phenotypically and genotypically and evaluated in channel catfish. 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 an LD$\sb{50}$ that was greater than five log$\sb{10}$ higher than the LD$\sb{50}$ 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 2 days. The gills appeared to be a more important route of entry than the intestine for E. ictaluri following immersion exposure. Finally, the effectiveness of LSU-E2 as a live attenuated vaccine was evaluated. Channel catfish that were vaccinated with a single dose of LSU-E2 by immersion had significantly lower mortalities following a wild-type E. ictaluri challenge than non-vaccinated fish.