Effects of early-life Enterococcus faecium spray application and dietary Bacillus-based probiotic supplementation on gut integrity and immune responses in broilers challenged with BCO lameness.

Probiotics have gained attention as alternatives to antibiotic growth promoters in broiler production due to their ability to support gut health, immune function, and disease resistance. Bacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chickens and is commonly associated with bacterial translocation from the gastrointestinal tract. Strategies that improve intestinal integrity and host responses may help mitigate this condition. This study evaluated the efficacy of a probiotic program comprising an Enterococcus faecium-based spray (2 × 10⁹ CFU/bird at hatch) and a triple-strain Bacillus-based feed additive (B. subtilis 597, B. subtilis 600, and B. amyloliquefaciens 516 at 500 g/t feed from day 1 to 56), applied alone or in combination. A wire-flooring challenge model was used to simulate BCO transmission. A total of 1,560 Cobb 500 broilers were randomly assigned to four treatment groups: NC (negative control, 6 replicate pens), GalliPro® Hatch (E. faecium spray only, 6 replicate pens), GalliPro® Fit (Bacillus-based feed supplement only, 6 replicate pens), and Hatch/Fit (combined treatment, 6 replicate pens). Two additional wire-floor seeder pens served as the infection source for the BCO challenge. Tight junction gene expression (CLDN1, CLDN5, OCLN, ZO-1, and ZO-2) was assessed in the ileum and trachea at d 7, 28, and 56, along with serum IgM, IgA, and serotonin concentrations. Probiotic supplementation significantly influenced ileal gene expression, with significant effects of treatment, age, and treatment × age interaction for selected tight junction genes (P < 0.05). OCLN and ZO-1 were upregulated at d 7 (P< 0.001), followed by sustained increases at d 56, particularly in the Hatch/Fit group (p < 0.05). Tracheal responses were variable. Plasma IgA concentrations increased with age (P < 0.001), reflecting normal immune maturation. Notably, serum serotonin concentrations were markedly elevated in the Hatch/Fit group, indicating microbiota-driven modulation of the gut-brain axis. Bacterial isolation identified a polymicrobial profile dominated by Staphylococcus spp. Collectively, these findings demonstrate that combined probiotic supplementation modulated tight junction gene expression and serum serotonin concentrations in BCO-challenged broilers. These results support evaluation of combined probiotic strategies in commercial production systems.