Dietary supplementation of Bacillus velezensis B8 enhances immune response and resistance against Aeromonas veronii in grass carp.

The heavy use of prophylactic antibiotics in aquaculture leads to elevated antibiotic residues, posing a huge hidden danger in aquaculture products and other natural aquatic environments. Therefore, this study aims to isolate probiotics that can replace antibiotics from the gut of grass carp for disease control. Bacillus velezensis B8 was isolated from the gut of grass carp and showed broad-spectrum antimicrobial activity against several fish pathogenic bacteria, including Aeromonas hydrophilis, Aeromonas veronii, Vibrio parahaemolyticus, Escherichia coli, Edwardsiella tarda and Vibrio mimicus. The safety evaluation showed that the strain B8 was non-toxic to grass carp, had no hemolytic activity, and was sensitive to most antibiotics. In vitro study indicated that strain B8 was viable at pH 2-7, had weak tolerance to 0.1% (w/v) bile salt, and could grow at 10°C-40 °C. The grass carps were fed with diets containing 0 (control), 107, and 109 cfu/g of strain B8 for 4 weeks. Various immune parameters were measured at 1, 2, 3, and 4 weeks of post-feeding. The results of non-specific immunoassay showed that diets supplemented with B8 significantly increased alkaline phosphatase (AKP) and superoxide dismutase (SOD) activity in serum samples (p < 0.05). The expression levels of immune-related genes in the kidney and spleen of grass carp were measured. Among them, the expression levels of IgM and TNF-α both in spleen and kidney were significantly increased after 3 and 4 weeks of post-feeding (p < 0.05). The expression of IgD and MHCI in kidney was significantly upregulated in high-dose groups after 2 and 3 weeks of feeding, respectively (p < 0.05). In addition, after 7 days of challenging with A. veronii, the high-dose group and low-dose group had 48% and 53% survival compared to 25% survival for the control group. These results suggest that B. velezensis B8 has the potential to be developed into a microecological preparation for the alternatives of antibiotics in aquaculture.