Isolation and identification of pathogens of ulcer disease in Cynoglossus semilaevis

In this study, 40 bacterial strains were isolated from the intestinal tract and surface lesions of the diseased Cynoglossus semilaevis with 2216E and TCBS medium. The hemolytic activity of the strains was tested, and three strains i.e. Vibrio harveyi, V. alginolyticus and V. parahaemolyticus with the strongest hemolysis activity were screened and identified as the three potential pathogenic strains in vitro. By co-culturing the three kinds of potential pathogenic bacteria with the intestinal epithelial cells of the healthy C. semilaevis, the relative expression of the immune genes and the apoptosis rate of the intestinal epithelial cells after co-culture were detected separately to evaluate the pathogenicity of these strains at the cellular level. The results showed that the expression of interleukin-10 gene (IL-10) in intestinal epithelial cells was significantly up-regulated after being stimulated with V. harveyi, suggesting that V. harveyi elicited the most intense immune response after co-culturing, and the apoptosis rate of the intestinal epithelial cells after co-culture with V. harveyi, V. alginolyticus and V. parahaemolyticus was 48.3%, 36% and 34.5%, respectively. It is speculated that V. harveyi is a highly pathogenic strain of C. semilaevis ulcer disease. Finally, the results of the artificial infection showed that the mortality rates of V. harveyi, V. alginolyticus and V. parahaemolyticus were 100% and 95% and 75% respectively on the sixth day after artificial re-infection of C. semilaevis, which is consistent with the results of the cellular level test. The study indicated that the three Vibrio strains screened in this experiment had strong toxicity and pathogenicity, especially the pathogenicity of V. harveyi was the strongest, and it is speculated that C. semilaevis ulcer disease may be caused by co-infection of various pathogenic bacteria.