Bacillus coagulans controls grass carp overwintering syndrome through the intestinal microbiota-metabolite-immunity network.

Abstract

Grass carp winter syndrome is a low-temperature disease that has severely impacted freshwater fish farming in China in recent years. Characterized by rapid onset, high transmissibility, and elevated mortality, it proves difficult to control with conventional treatments. Bacillus coagulans, which combines the acid-producing antibacterial properties of Lactobacillus with the heat resistance and feed pellet compatibility of Bacillus subtilis, has gained attention as an alternative to antibiotics in livestock and poultry. This study represents the first integrated multi-dimensional analysis combining gut morphology, immunological enzyme activity, gut microbiome and metabolomics. It systematically elucidates the mechanism by which feeding grass carp with a bacterial suspension of Bacillus coagulans BC66 at a concentration of 100 billion CFU/g for 20 days ameliorates wintering syndrome through regulation of the gut metabolism-immune network. The findings reveal the therapeutic efficacy of this approach for grass carp wintering syndrome. The results demonstrated that B. coagulans BC66 improved the survival rate of grass carp affected by winter syndrome and promoted the repair of intestinal tissue. Antioxidant enzyme activities, such as those of SOD, CAT, and GSH-Px, were significantly elevated, while the expression of inflammatory genes (IL-8, TNF-α, NF-κB, and IL-12) decreased and that of IL-10 increased. A significant enrichment of Firmicutes, Bacteroidota, and Actinobacteriota occurred, with a notable increase in beneficial bacterial phyla, indicating a shift towards a more balanced microbial ecosystem. Meanwhile, pathogenic genera such as Aeromonas, Escherichia-Shigella, and Vibrio were markedly reduced. Metabolite analysis revealed downregulation of SDMA, Dibutylamine, and Involucrin, suggesting a reduction in inflammatory signaling and pathological metabolites. Conversely, Bifidobacterium-associated phospholipids (PC-O-15:0), pristanic acid, britannilactone derivatives, and antioxidant phenols were upregulated, alongside activation of the ABC transporters metabolic pathway and suppression of neuroactive ligand signaling and receptor interactions. These findings indicate that B. coagulans BC66 partially restored detoxification of exogenous toxins, distribution of endogenous metabolites, pathogen resistance, and osmoregulatory functions in grass carp. These findings provide scientific evidence supporting the application of Bacillus coagulans BC66 as an effective green alternative to antibiotics for preventing and controlling winter syndrome in grass carp, offering new strategies for improving health management in freshwater aquaculture.