A multivalent killed whole-cell vaccine induces effective protection against Edwardsiella tarda and Vibrio anguillarum

Edwardsiella tarda, Vibrio anguillarum, Streptococcus iniae, and Vibrio harveyi are important aquaculture pathogens that affect a wide range of fish species. In this study, we examined in a Japanese founder (Paralichthys olivaceus) model the possibility of developing simple, killed whole-cell vaccines with protective effect against two or more of the above pathogens. For this purpose, we utilized four pathogenic fish isolates: E. tarda TX1, V. anguillarum C312, S. iniae SF1, and V. harveyi T4D. Flounder were immunized with inactivated monovalent TX1, C312, SF1, or T4D, or with different combinations of these strains in the form of (i): TX1, C312, SF1, and T4D mix (M4); (ii) TX1, C312, and SF1 mix (M3); (iii) TX1 and C312 mix (M2). Fish were challenged at two months post-vaccination with E. tarda, V. anguillarum, S. iniae, or V. harveyi and monitored for mortality. The results showed that the monovalent vaccines produced low protections, with relative percent survival (RPS) rates between 33.3% and 53.9%. Compared to monovalent vaccines, M2, M3, and M4 all induced significantly higher levels of protection against E. tarda. In addition, M2 and M4 also effected much higher (∼19%) RPS rates against V. anguillarum. Serum antibody production and bactericidal activity analysis showed a correlation between protection efficacy and antibody level. These results suggest a humoral immunity-based mechanism of protection induced by inactivated whole-cell vaccines, and that there exists a mutual and specific immunostimulatory effect between E. tarda TX1 and V. anguillarum C312, which enables the divalent M2 to induce effective protective immunity against E. tarda and V. anguillarum.