Salmonella infection remains a significant concern in the poultry production industry. It causes substantial economic losses worldwide, including decreased growth rates, increased mortality, and a heightened risk of foodborne transmission to humans. The objective of this study was to isolate and characterize Bacillus species from broiler feces and to evaluate the probiotic potential of Bacillus amyloliquefaciens BS32 (BaBS32) isolate, with particular emphasis on its capacity to improve broiler health and to prevent the adverse effects of Salmonella infection. Following antimicrobial testing of 40 isolates against Staphylococcus aureus and Pseudomonas aeruginosa, the BaBS32 isolate demonstrated the largest inhibition zones, measuring 30mm and 26 mm, respectively. Morphological, biochemical, and 16S rDNA sequencing analyses revealed that BS32 shared 99% similarity with Bacillus amyloliquefaciens DSM 25840. The BaBS32 isolate exhibited several key probiotic characteristics, including robust survival at pH 2.5 (85.2%) and in 0.3% bile salt (78.6%), strong biofilm-forming ability, phosphate solubilization, and indole-3-acetic acid production. Antibiotic susceptibility testing indicated that the BaBS32 isolate is safe, exhibiting broad sensitivity and no multidrug resistance. In vitro assays demonstrated that BaBS32 possesses dose-dependent antioxidant and antibacterial activities, with DPPH radical scavenging reaching 90% at 320 mg/mL and an inhibitory zone of 8.5-34 mm against pathogenic bacteria. Dietary supplementation of broilers with BaBS32 isolate at doses of 80-320 mg/kg resulted in improved growth performance, enhanced antioxidant enzyme activities, and strengthened immunity, meanwhile, decreasing liver enzyme levels and markers of oxidative stress. Additionally, BaBS32 treatment reduced the expression of inflammatory and apoptotic genes induced by Salmonella, notably BAX, and Caspase- 3 (up to 2.6 and 1.85- fold, respectively), and upregulated genes associated with tight junction integrity and immune response. On the other hand, microbial count decreased, where E. coli and Salmonella populations by 61%, and an enrichment of beneficial lactic acid bacteria by 55%. Histopathological examination indicated that BaBS32 isolate mitigated intestinal damage caused by Salmonella, restoring villus height and improving mucosal structures. Bacillus amyloliquefaciens BS32 isolate exhibits significant probiotic potential, capable of enhancing broiler health and resistance against Salmonella infection.

To Cite This Article: Aminah Allohibi, 2025. The influence of Bacillus amyloliquefaciens BS32 isolate in enhancing growth, immune response, and regulating intestinal gene expression in Salmonella-challenged broilers. Pak Vet J, 45(4): 1638-1649. http://dx.doi.org/10.29261/pakvetj/2025.332