An aroA Mutant of Edwardsiella ictaluri Is Safe and Efficacious as a Live,Attenuated Vaccine

Abstract

The aroA gene of Edwardsiella ictaluri was cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroA gene. The mutated gene was transferred into a virulent, wild-type E. ictaluri strain by conjugation and allelic exchange. Putative aroA mutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroA deletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU-E1, was passed 30 times in nonselective media with no reversion to the wild-type following screening of 1.6 × 10¹¹ colony-forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs₁₀ compared with the wild-type E. ictaluri strain, and it was avirulent by immersion and oral routes. Tissue persistence studies indicated that the mutant maintained the ability to invade following immersion exposure, but no viable cells were detected after 48–72 h. Significant levels of protection from disease were demonstrated following immersion vaccination of channel catfish Ictalurus punctatus.