Generation and evaluation of virulence attenuated mutants of Edwardsiella tarda as vaccine candidates to combat edwardsiellosis in flounder (Paralichthys olivaceus)

• Five double- and two triple-genes deletion mutants of Edwardsiella tarda were constructed.
• E. tarda mutants attenuated virulence by over 2–3 logs in the 50% lethal dose.
• Three E. tarda mutants provided protection in blue gourami with RPS rates of 52.2–82.6%.
• The ΔaroAΔesrB mutant provided effective protection in flounder.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. The development of a live attenuated vaccine may be an effective approach for preventing this disease in fish. In this study, we introduced deletions of esrB, esaC, evpH, rpoS, and purA into the E. tarda LSE40ΔaroA strain, thereby generating five double-gene mutants (ΔaroAΔesrB, ΔaroAΔesaC, ΔaroAΔrpoS, ΔaroAΔevpH, and ΔaroAΔpurA) and two triple-gene mutants (ΔaroAΔesrBΔevpH and ΔaroAΔesaCΔevpH). When blue gourami (Trichogaster trichopterus) was used as a fish model for the primary screening and evaluation of the vaccine candidates, all mutants were attenuated significantly by more than 2 to 3 logs in terms of the 50% lethal dose (LD50). Five double-gene mutants yielded relative percentage survival (RPS) rates of 26.1–82.6% after challenge with wild-type E. tarda. The ΔaroAΔesrB mutant that conferred the highest RPS (82.6%) in blue gourami was also evaluated in flounder (Paralichthys olivaceus). After vaccination via intramuscular (i.m.) injection or immersion, this mutant could persist in the flounder for 14–35 days and it induced higher serum antibody titers than the control fish (P < 0.01). Flounder vaccinated via i.m. injection at doses of 103–107 CFU/fish had RPS rates of 14.3–66.7% after i.m. challenge with 104 CFU/fish using wild-type E. tarda. Flounder vaccinated via immersion at a dose of 107 CFU/ml exhibited 100% RPS against immersion challenge with 107 CFU/ml using wild-type E. tarda. These results indicate that the ΔaroAΔesrB mutant could be used as an effective live vaccine to combat edwardsiellosis in flounder.