Bacteriophage vB_SalS_KY05 controls <i>Salmonella</i> 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 <i>Tequintavirus</i>, vB_SalS_KY05, from a Taiwanese poultry farm and evaluated its biological properties and <i>in vivo</i> efficacy against <i>Salmonella enterica</i> serovars Typhimurium and Enteritidis, and <i>Escherichia coli</i>. 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 <i>E. coli</i> K12. <i>In vivo</i>, SPF chickens challenged with <i>S.</i> Typhimurium received low-dose (10⁵ PFU/mL) or high-dose (10⁸ PFU/mL) phage <i>via</i> drinking water. Low-dose treatment reduced splenic <i>Salmonella</i>, improved albumin-to-globulin ratios, and enriched beneficial taxa like <i>Lactobacillus crispatus</i> and <i>Blautia coccoides</i>. In contrast, high-dose treatment resulted in phage-bacteria coexistence and increased potentially harmful taxa including <i>Erysipelatoclostridium</i>. 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.