Usnic acid and tannic acid as inhibitors of coccidia and Clostridium perfringens: alleviating necrotic enteritis and improving intestinal health in broiler chickens

Necrotic enteritis (NE) in broiler chickens leads to significant economic losses in poultry production. This study examined the inhibitory effects of usnic acid and tannic acid on coccidia, sporozoite, and Clostridium perfringens and assessed their influence on growth performance and intestinal health in NE-challenged broilers through in vitro and in vivo experiments. The in vitro experiment included 5 treatment groups: the negative control (NC), 2 μmol/L diclazuril (DZ), 30 μmol/L usnic acid (UA), 90 μmol/L tannic acid (TA), and 15 μmol/L usnic acid + 45 μmol/L tannic acid (UTA) groups. The in vivo experiment involved 320 broilers divided into four groups: PC (NE-challenged), SA (500 mg/kg salinomycin premix + NE-challenged), UA (300 mg/kg usnic acid + NE-challenged), and UTA (300 mg/kg usnic acid + 500 mg/kg tannic acid + NE-challenged) groups. In the in vitro study, the UA, TA, and UTA treatments significantly increased apoptosis in coccidian oocysts and sporozoites, lowered the mitochondrial membrane potential (P < 0.05), and disrupted the oocyst structure compared with those in the NC group. UA and TA had inhibitory effects on C. perfringens, with the strongest inhibition observed in the UTA group. The in vivo results demonstrated that the SA group presented significantly improved growth performance on d 13, 21, and 28 (P < 0.05), whereas the UA and UTA groups presented improvements on d 13 and 21 (P < 0.05). The SA, UA, and UTA treatments reduced the intestinal lesion scores by d 28 and the fecal coccidian oocyst counts from d 19 to 21 (P < 0.05). Compared with the PC group, the UA and UTA groups presented lower intestinal sIgA levels and CD8+ cell percentages (P < 0.05), with a trend toward a reduced CD3+ cell percentage (P = 0.069). The SA, UA, and UTA treatments significantly reduced the serum diamine oxidase activity, crypt depth, and platelet-derived growth factor levels in the intestinal mucosa while increasing the villus height to crypt depth ratio and number of goblet cells (P < 0.05). The UTA treatment also significantly increased the acetate and butyrate concentrations in the cecum (P < 0.05). With respect to the gut microbiota, significant changes in β diversity in the ileum and cecum were observed in the SA, UA, and UTA groups, indicating that the microbial community compositions differed among the groups. Romboutsia dominated the SA group, Bacillales dominated the UA group, and Lactobacillales and Lachnospirales dominated the UTA group in the ileal microbiota. In the cecal microbiota, Lactobacillus, Butyricicoccus, and Blautia abundances were significantly elevated in the UTA group (P < 0.05). Usnic acid and tannic acid induce apoptosis in coccidia and sporozoites by lowering the mitochondrial membrane potential. Both usnic acid alone and in combination with tannic acid alleviate NE-induced adverse effects in broilers by modulating intestinal immunity, altering the microbial composition, and improving intestinal barrier function. Compared with usnic acid alone, the combination of usnic acid and tannic acid had superior effects, providing a promising basis for the development of effective feed additive combinations.