Identification and evaluation of potential probiotics against skin-ulceration disease in the Chinese tongue sole (Cynoglossus semilaevis)

Abstract

In this study, three highly pathogenic bacterial strains (Vibrio harveyi TB6, Vibrio alginolyticus TN1, and Vibrio parahaemolyticus TN3) were isolated from skin ulcers and intestines of diseased Chinese tongue sole (Cynoglossus semilaevis). The bacteria were investigated using hemolytic activity tests, in vitro co-culture with intestinal epithelial cells, and artificial infection of C. semilaevis. A further 126 strains were isolated from the intestines of healthy C. semilaevis. The three pathogens were used as indicator bacteria, and the antagonistic strains were identified from the 126 strains. The activities of exocrine digestive enzymes in the strains were also tested. Four strains with antibacterial and digestive enzyme activities were obtained and the best strains, Bacillus subtilis Y2 and Bacillus amyloliquefaciens Y9, were selected according to their ability to protect epithelial cells from infection. In addition, the effects of strains Y2 and Y9 at the individual level were investigated, finding that the activities of the immune-related enzymes superoxide dismutase, catalase, acid phosphatase, and peroxidase were significantly increased in the sera of the treatment group compared with the control group (p<0.05). The specific growth rate (SGR, %) was also increased, especially in the Y2 group, and was significantly higher compared with the controls (p<0.05). The result of the artificial infection test showed that the cumulative mortality within 72h in the Y2 group was the lowest (50.5%), and in the Y9 group (68.5%) it was significantly lower than that in the control group (100%) (p<0.05). Analysis of the intestinal microbial communities indicated that Y2 and Y9 could alter the composition of the intestinal flora, increasing both species richness and evenness, and inhibiting the growth of Vibrio in the intestine. These results suggested food supplemented with Y2 and Y9 could improve both immune function and disease resistance, as well as have a positive effect on the growth performance and the intestinal morphology of C. semilaevis.