Bacteriophage vB_SalS_KY05 controls Salmonella in poultry without disrupting cecal microbiota composition

Salmonellosis is a major food safety challenge in poultry production, requiring effective antibiotic alternatives. We isolated a polyvalent Tequintavirus, vB_SalS_KY05, from a Taiwanese poultry farm and evaluated its biological properties and in vivo efficacy against Salmonella enterica serovars Typhimurium and Enteritidis, and Escherichia coli. The objective of this study was to comprehensively characterize vB_SalS_KY05 and assess its potential as a biocontrol agent for poultry farming. Genome analysis confirmed a lytic lifestyle with no detectable virulence or antimicrobial resistance genes. The phage remained stable at pH 4–10, at 41 °C, and in water for 7 days. For improved biosafety, large-scale amplification was achieved by switching the propagation host to non-pathogenic E. coli K12. In vivo, SPF chickens challenged with S. Typhimurium received low-dose (105 PFU/mL) or high-dose (108 PFU/mL) phage via drinking water. Low-dose treatment reduced splenic Salmonella, improved albumin-to-globulin ratios, and enriched beneficial taxa like Lactobacillus crispatus and Blautia coccoides. In contrast, high-dose treatment resulted in phage–bacteria coexistence and increased potentially harmful taxa including Erysipelatoclostridium. Overall, cecal microbiota composition remained largely unchanged. These findings indicate vB_SalS_KY05 is a promising biocontrol candidate, highlighting the importance of dose optimization to enhance pathogen control while preserving gut microbiota stability in poultry production.