Protection of olive flounder (Paralichthys olivaceus) against Edwardsiella tarda infection by oral administration of auxotrophic mutant E. tarda (Δalr Δasd E. tarda)

A study was undertaken to evaluate the efficacy of oral vaccination of olive flounder (Paralichthys olivaceus) with two auxotrophic gene-mutated Edwardsiella tarda (Δalr Δasd E. tarda) that habors antibiotic resistant gene-free plasmids (pG02-ASD-EtPR-GFP) equipped with expression cassettes for green fluorescent protein (GFP) and aspartate semialdehyde dehydrogenase (asd) genes. In a persistence and invasiveness experiment, orally administered live Δalr Δasd E. tarda were recovered only from the intestine till 24 h post-administration, and no colonies were found from the head kidney, spleen, and liver. In challenge experiments, fish orally immunized with 109 CFU/fish of Δalr Δasd E. tarda were 100% protected against challenge through either i.p. injection or immersion, even in fish administered only once. Two doses (primary and boost) of 108 CFU of the mutated bacteria given orally led to similar protection rate to administration of 109 CFU of the bacteria. In the immunized fish, intestinal and skin mucus showed no agglutination activity and no ELISA titers, whereas serum showed agglutination activity and ELISA titers. GFP expressed in the cytoplasm of Δalr Δasd E. tarda failed to induce antibody responses. The present results suggest that oral immunization of olive flounder with the auxotrophic mutant E. tarda can induce protective immunity against virulent E. tarda challenge through injection and immersion routes. To use the present auxotrophic mutant E. tarda as an oral combined vaccine, new approaches that can strengthen antigenicity of foreign antigens should be conducted.