Heat-inactivated Mycobacterium marinum as a vaccine adjuvant and a trained immunity inducer in rainbow trout (Oncorhynchus mykiss).

The increasing need for effective disease prevention strategies in aquaculture highlights the importance of developing novel vaccine adjuvants and immunostimulants. This study aimed to investigate the immunomodulatory potential of heat-killed Mycobacterium marinum in rainbow trout (Oncorhynchus mykiss), with a focus on its dual role as a vaccine adjuvant and an inducer of trained immunity. To this end, we evaluated the effects of co-immunizing fish with heat-killed M. marinum and a live attenuated Vibrio anguillarum vaccine. This combination significantly enhanced protective efficacy, as demonstrated by increased survival rates, elevated serum antibody titers, and early upregulation of pro-inflammatory cytokines and immune-related genes. In addition, heat-killed M. marinum alone conferred non-specific protection against V. anguillarum infection, characterized by earlier bacterial clearance, heightened inflammatory gene expression, and epigenetic modifications, including increased H3K4me3 and H3K27ac at cytokine gene promoters. These findings support the use of heat-killed M. marinum as both an effective vaccine adjuvant and a promising inducer of trained immunity in aquaculture species.