Integrated analysis of histopathology, immunoglobulin responses, and gut microbiota reveals protective effects of Ban Xia Xie Xin Tang in Streptococcus agalactiae-infected hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂)

This study evaluated the protective effects of Ban Xia Xie Xin Tang (BXT) in Streptococcus agalactiae -infected hybrid tilapia using integrated in vitro and in vivo experiments. In the in vitro experiment, BXT at 250 mg/L showed the highest activity in enhancing respiratory burst, phagocytic activity, and phagocytic index. The in vitro gene expression showed significantly upregulated immune related genes ( lgb , ontlr5s , tnfα , il1β , and tp3 ) in multiple tissue after incubation with BXT and S. agalactiae , with the strongest responses generally observed at 250 mg/L. In the in vivo experiment, hybrid tilapia was divided into three groups: negative control (NC, basal feed), positive control (PC, basal feed), and BXT250 (basal feed supplemented with 0.25 g/kg of BXT) and fed their respective diets throughout a 14-day experimental period including before and after S. agalactiae challenge. Following infection, PC fish exhibited severe hepatic vacuolation, splenic hemolysis, and gill lamellar damage, whereas BXT250 fish showed tissue morphology largely comparable to NC, with only mild lesions. The BXT250 group also displayed significantly elevated igm and igt expression in systemic and mucosal tissues, along with gut microbiota profiles and predicted metabolic pathways more similar to NC than PC. Notably, BXT250 enhanced biotin metabolism, which may improve biotin availability and support B-cell differentiation and antibody production. Survival after infection reached about 90% in BXT250 fish compared with marked mortality in PC. Overall, BXT reduced S. agalactiae -induced tissue damage, improved immunoglobulin responses, modulated gut microbiota, and increased survival, supporting its potential as a dietary immunostimulant for tilapia culture.