Prophylactic Administration of Engineered Bacillus subtilis Expressing Mucosal Repair Factors Alleviates Pullorum Disease in Chicks.

Salmonella pullorum (S. pullorum) remains a major enteric pathogen in young chicks, causing high mortality and severe economic losses in poultry production. This study evaluated a prophylactic strategy using engineered Bacillus subtilis (B. subtilis) expressing gallus trefoil factor 2 (gTFF2) and epidermal growth factor (gEGF) to protect chicks against S. pullorum infection. In vitro, gEGF significantly promoted UMNSAH/DF-1 (DF-1) cell proliferation, whereas both gTFF2 and gEGF enhanced epithelial cell migration. In vivo, oral administration of recombinant B. subtilis significantly reduced mortality from 75.0% and 66.7% in the PBS and pHT43 groups to 50%, 33.3%, and 25.0% in the gTFF2, gEGF, and gTFF2+gEGF groups, respectively. Cecal colonization of S. pullorum was reduced by 2-4 log10 CFU/g, accompanied by improved average daily gain and immune organ indices. Treatment also significantly decreased serum IL-6 levels and increased TGF-β levels (p < 0.05), indicating attenuation of the inflammatory response. Histological analysis showed significantly increased villus height (0.94-1.58-fold) and villus height-to-crypt depth ratio (1.47-3.25-fold) compared with the infected controls, together with markedly alleviated hepatic and intestinal lesions. The combined administration of gTFF2- and gEGF-expressing strains consistently exhibited the greatest protective efficacy. Collectively, these findings demonstrate that engineered probiotic-mediated delivery of mucosal repair factors represents a promising antibiotic-alternative strategy for preventing pullorum disease and improving intestinal health in poultry production.