Evaluation of Protective Efficacy of Formalin-Killed and Auxotrophic-Live Vibrio anguillarum Against Homologous and Heterologous Strains in Rainbow Trout (Oncorhynchus mykiss).

Vibrio anguillarum infection poses a significant threat to freshwater and marine aquaculture. Although whole-cell killed and live attenuated vaccines are used to control vibriosis, their comparative efficacy against homologous and heterologous V. anguillarum strains remains limited. Here, we compared the protective efficacy and antibody responses of a formalin-killed V. anguillarum (FKC) and an alanine racemase gene knockout auxotrophic-live V. anguillarum (Δalr1Δalr2 V. anguillarum, LAV), both derived from the V. anguillarum JJ strain, in rainbow trout against homologous (JJ strain) and heterologous (V. anguillarum 90-11-287) strains. The LAV conferred superior protection compared to the FKC against both strains under high-dose challenge conditions. Despite this, ELISA antibody levels were comparable between vaccine groups and did not correlate with protective outcomes. No agglutination activity against the JJ strain was detected in vaccinated fish or rabbit antisera, suggesting limited assay sensitivity rather than absence of antibody binding. Analysis of the O-antigen biosynthesis-related genes revealed no genetic differences between strains; however, the JJ strain exhibited a shorter O-antigen, which may contribute to the reduced agglutination of the JJ strain. In conclusion, our results underscore the need for a comprehensive evaluation of vaccine performance, extending beyond antibody quantification, when developing effective vaccination strategies for aquaculture.