Edwardsiella ictaluri type III and type VI secretion system mutant strains as candidates for live attenuated vaccines

ABSTRACT Objective Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, causes substantial economic losses to Channel Catfish Ictalurus punctatus producers in the United States. This study evaluates three E. ictaluri strains, each carrying a markerless deletion mutation in a type III or type VI secretion system gene, as candidates for a live attenuated vaccine against enteric septicemia of catfish in Channel Catfish. Replication in Channel Catfish cells, in vivo invasion and persistence, virulence, and the ability to provide single-dose protection against a wild-type E. ictaluri strain were evaluated for each mutant. Methods In this study, three isogenic mutants were constructed that introduced deletions in esrC, eseG, and evpC. EsrC is a T3SS-encoded protein that regulates expression of multiple virulence genes, including the T3SS effector EseG and the T6SS structural protein EvpC. Each mutant strain was evaluated for its ability to replicate in Channel Catfish ovary cells and head-kidney-derived macrophage cells. Channel Catfish were also challenged with the mutant strains to evaluate if mutation affected invasion, colonization, or persistence within the head kidney, attenuated mortalities in Channel Catfish, or induced protection against disease following subsequent wild-type E. ictaluri exposure. Results Each mutant maintained the ability to replicate within head-kidney-derived macrophage cells and Channel Catfish ovary cells, as well as invade and colonize the head kidney. Although able to replicate intracellularly and successfully colonize catfish tissue, all three mutants were significantly attenuated in their ability to persist in tissues and cause mortality. A single immersion in mutant strains 28 d prior to exposure to wild-type E. ictaluri resulted in significantly lower mortality than fish immersed in sterile broth, with relative percent survival ranging from 95% to 100%. Conclusions The findings demonstrate the potential for the development of live attenuated E. ictaluri vaccine strains through the targeted mutation of one or more E. ictaluri T3SS and T6SS genes.