Abstract
Antibiotic growth promoters (AGPs) are increasingly subject to global regulatory restrictions and consumer pressure, driving the poultry industry toward antibiotic-free production systems. This shift has accelerated the search for effective alternatives, including innovative microbial additives, organic acids, phytogenics, and other bioactive compounds capable of supporting digestive function and enhancing immune competence in poultry. The present study reported the isolation and characterization of a novel Bacillus velezensis strain, BV-OLS1101, possessing robust probiotic attributes and a distinctive capacity to produce a serine protease subtilisin. It was hypothesised that as a direct-fed microbial (DFM) BV-OLS1101, which was isolated from the soil of poultry farm and taxonomically identified by 16 s rRNA sequencing, might improve performance and mucosal immune status in broiler chickens particularly when the birds were enterically challenged with Clostridium perfringens (CP). BV-OLS1101 exhibited high thermotolerance (89 % survival at 80 °C), strong acid and bile resistance, broad antibiotic susceptibility, and in vitro antagonism against CP. Subtilisin production was confirmed through protease activity assays, phenyl methyl sulfonyl fluoride inhibition, and SDS-PAGE analysis. A 42-day in vivo trial was conducted using 432 male Ross 308 broilers randomly assigned to six groups in a 2 × 3 factorial design: unchallenged or CP-challenged birds, each with or without BV-OLS 1101 at 250 or 500 mg/kg feed (equivalent to 6 and 12 × 108 CFU/g of feed respectively as a premix). Birds were fed an AGP-free maize - soybean meal diet. Clostridium challenge (10⁹ CFU/bird/day) was induced for 3 consecutive days starting from 18 days of age, following a coccidia priming on 14 days of age. Growth performance was assessed at weekly intervals, while intestinal lesion scores and the expression of cytokines and mucosal markers were assessed in birds at 28 days of age. In comparison to the unchallenged birds, CP challenge impaired feed conversion ratio (FCR) by 8.26 % (P = 0.005). Supplementation with BV-OLS1101 improved FCR by up to 7.9 % (P = 0.01) compared to the challenged birds. BV-OLS1101 reduced cecal and ileal lesions (P < 0.05), downregulated expression of IL-1β, IL-6, IFN-γ, and upregulated that of TGF-β, MUC2, and sIgA. Based on the present findings, subtilisin is proposed as a key immunomodulatory factor, supported by its observed expression and literature-documented bioactivity. Therefore, BV-OLS1101 may be considered a promising next-generation probiotic, which, by virtue of its heat stability and capacity to produce subtilisin, presents a novel biological strategy to reduce reliance on AGPs while sustaining gut health and improving performance in broiler chickens.