To evaluate the possible dietary application of live and heat-inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 AE 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 9 10 8 CFU g À1 live (T1) and heat-inactivated B. pumilus SE5 (T2). The heat-inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat-inactivated probiotic for 60 days (P < 0.05). Furthermore, the heatinactivated probiotic remarkably up-regulated expression of TLR2 and pro-inflammatory cytokines (IL-8 and IL-1b) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat-inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.

No abstract

The health benefits of probiotics are thought to occur, at least in part, through an improved intestinal microbial balance in fish, although the molecular mechanisms whereby probiotics modulate the intestinal microbiota by means of activation of mucosal immunity are rarely explored. In this study, the effects of viable and heat-inactivated probiotic Bacillus pumilus SE5 on the intestinal dominant microbial community and mucosal immune gene expression were evaluated. The fish were fed for 60 d with 3 different diets: control (without probiotic), and diets T1 and T2 supplemented with 1.0 × 10⁸ cells g⁻¹ viable and heat-inactivated B. pumilus SE5, respectively. Upregulated expression of TLR1, TLR2 and IL-8, but not MyD88 was observed in fish fed the viable probiotic, while elevated expression of TLR2, IL-8 and TGF-β1, but not MyD88 was observed in fish fed the heat-inactivated B. pumilus SE5. The induced activation of intestinal mucosal immunity, especially the enhanced expression of antibacterial epinecidin-1, was consistent with the microbial data showing that several potentially pathogenic bacterial species such as Psychroserpens burtonensis and Pantoea agglomerans were suppressed by both the viable and heat-inactivated probiotic B. pumilus SE5. These results lay the foundation for future studies on the molecular interactions between probiotics, intestinal microbiota and mucosal immunity in fish.

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat-inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10 8 CFU/g live (T1) and heatinactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat-inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed.Moreover, the heat-inactivated B. clausii DE5 significantly improved the expression of TLR5, pro-inflammatory cytokines (IL-8 and IL-1β) and TGF-β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune-related genes in head kidney and intestine. These results indicate that dietary supplementation of heat-inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides. K E Y W O R D SBacillus clausii, Epinephelus coioides, growth performance, heat-inactivated probiotic, intestinal immunity, systemic immunity