Prospective Application of Nanoencapsulated Bacillus amyloliquefaciens on Broiler Chickens’ Performance and Gut Health with Efficacy against Campylobacter jejuni Colonization

**Background:** The emergence of multidrug-resistant bacterial strains has necessitated alternatives to antibiotic growth promoters in poultry production. Probiotics, particularly Bacillus-based strains, have shown promise but face limitations due to harsh gastrointestinal conditions that reduce their bioavailability. Nanoencapsulation offers a potential solution to protect probiotics and enhance their efficacy. Campylobacter jejuni is a major foodborne pathogen for which poultry serve as the primary reservoir, colonizing the avian cecum at levels of 10^6–10^8 CFU/g without causing clinical illness in birds but posing significant human health risks.

**Methods:** Two hundred Ross 308 male broiler chickens were randomly divided into four groups (5 replicates of 10 birds each): a control group fed a conventional diet, and three groups fed the conventional diet supplemented with BNPs at doses of 2.5 × 10^5 (BNPs I), 5 × 10^5 (BNPs II), or 7.5 × 10^5 (BNPs III) CFU/g of feed for 35 days. BNPs were prepared by incorporating B. amyloliquefaciens CECT 5940 into chitosan (0.4% w/v) nanoparticles. Growth performance parameters were recorded throughout the study. At 30 days of age, all birds were orally challenged with 10^8 CFU/mL of a pandrug-resistant, multi-virulent field C. jejuni strain resistant to 24 antimicrobials across 10 categories. Gene expression analysis was performed using RT-qPCR for digestive enzymes (AMY2A, PNLIP, CELA1, CCK), barrier function genes (DEFB1, FABP-2, MUC-2), and proinflammatory cytokines (IL-1β, TNF-α). Intestinal microbial populations and C. jejuni colonization/shedding were quantified using qPCR. Statistical analysis employed one-way ANOVA with Tukey's test at p < 0.05.

**Key Results:** BNPs II and BNPs III groups exhibited significantly higher body weight gain (2499 and 2569 g/bird, respectively) compared to control (2297 g/bird) and BNPs I (2430 g/bird) (p < 0.001). Feed conversion ratio was significantly improved in BNPs III (1.48) and BNPs II (1.50) compared to control (1.82) (p < 0.001). Mortality was significantly reduced in BNPs II and BNPs III groups (1% each) versus control (3%) (p = 0.025). The BNPs III group showed the highest mRNA expression of digestive enzyme genes: AMY2A (1.69-fold), PNLIP (1.49-fold), CELA1 (1.33-fold), and CCK (1.29-fold) relative to control. Beneficial microbiota (Lactobacillus and Bifidobacterium) were significantly increased in BNPs-fed groups, with BNPs III showing the highest counts, while Clostridium and Enterobacteriaceae were reduced. Barrier function genes (MUC-2, FABP-2, DEFB1) were significantly upregulated in a dose-dependent manner, and proinflammatory cytokines (IL-1β, TNF-α) were significantly downregulated in BNPs-fed groups. At 7 days post-infection, C. jejuni counts in BNPs III group were significantly reduced to 2.93 CFU/g (fecal) and 4.22 CFU/g (cecal) compared to control (6.79 and 8.16 CFU/g, respectively).

**Clinical Implications:** This study demonstrates that chitosan nanoencapsulation of B. amyloliquefaciens enhances probiotic bioavailability and efficacy in broiler chickens, improving growth performance and gut health while significantly reducing C. jejuni colonization and shedding. The findings support BNPs as a viable antibiotic alternative for poultry production, with potential to interrupt the transmission cycle of campylobacteriosis through the food chain. The dose-dependent effects suggest that the highest tested dose (7.5 × 10^5 CFU/g feed) provides optimal benefits. However, the study was limited to a single probiotic strain and nanoparticle formulation, and the mechanisms underlying probiotic-mediated regulation of digestive enzyme secretion require further elucidation. Long-term safety studies and commercial-scale trials would be needed before practical application.