Phage therapy: A novel strategy to combat drug-resistant Salmonella Pullorum infection in chickens

Salmonella Pullorum, a significant pathogen causing high mortality in chicks and economic losses in poultry, faces increasing antibiotic resistance challenges. This study isolated and characterized four novel virulent bacteriophages from chicken feces and farm effluent, using Salmonella Pullorum CVCC 528 as the host. Transmission electron microscopy (TEM) revealed that all phages possessed contractile tails, a morphology historically associated with the family Myoviridae. Host range analysis revealed lytic activity against Salmonella Pullorum and Salmonella Typhimurium strains, but no activity against non-target bacteria. The phages exhibited robust environmental stability, maintaining high activity between -20°C and 37°C and across pH 6-10. They also tolerated osmotic stress but were sensitive to ethanol, acetone, SDS, and benzalkonium bromide. Phage vB_SpuM_18 demonstrated superior replication kinetics with a notably short latent period and high titer. All phages significantly inhibited and eradicated mature biofilms formed by strong biofilm-producer Salmonella Enteritidis 50335 within 16 h. In vivo efficacy was evaluated in a chick infection model. Treatment with vB_SpuM_18, vB_SpuM_23, or a phage cocktail (each at 1 ×10¹⁰ PFU per chick per dose, administered orally twice daily) significantly improved outcomes compared to PBS controls. vB_SpuM_18 and the cocktail achieved 100% survival with no clinical signs, while vB_SpuM_23 showed 83.3% survival. Phage therapy drastically reduced Salmonella load in anal swabs and cecal contents, and significantly lowered serum inflammatory cytokines. Gross and histopathological examination confirmed marked mitigation of infection-induced hepatic swelling, hemorrhage, necrosis, and intestinal damage in phage-treated groups. These findings demonstrate that the isolated Salmonella Pullorum phages, particularly vB_SpuM_18, possess excellent biological stability, potent biofilm disruption capabilities, and significant therapeutic efficacy against avian Pullorum disease.