Isolation and identification of a chicken-derived Salmonella phage VB-SalS-XND03, Its biological characteristics and application effect against Salmonella infection in chicks.

INTRODUCTION: Salmonellosis, a zoonotic disease caused by Salmonella spp., poses a major threat to poultry production and public health. The increasing prevalence of antimicrobial resistance has reduced the effectiveness of conventional antibiotics, highlighting the need for alternative strategies such as phage therapy. METHODS: A lytic phage, VB-SalS-XND03, was isolated using multidrug-resistant S. Enteritidis J3 as the host strain. Its morphology, host range (43 strains), biological characteristics, and whole-genome sequence were characterized. To mimic the natural fecal-oral transmission route, prophylactic and therapeutic efficacy was evaluated in a healthy chick oral gavage model (n = 8 per group for survival analysis; n = 20 per group for mechanistic studies) using survival analysis, quantitative polymerase chain reaction (qPCR) for inflammatory cytokines and tight junction proteins, histopathology, 16S rRNA sequencing (n = 4/group), transcriptomics (n = 3/group), and metabolomics (n = 3/group). An intraperitoneal challenge model was also included as supplementary validation. RESULTS: Transmission electron microscopy showed that VB-SalS-XND03 possesses an icosahedral morphology and belongs to the class Caudoviricetes, genus Tequintavirus. Host range analysis demonstrated serotype-preferential lytic activity, lysing 10 of 43 Salmonella strains (23%), predominantly Salmonella Enteritidis. The phage exhibited robust environmental stability (pH 4-12, temperature 4-60 °C) and a rapid lytic cycle with a latent period of approximately 40 min and a burst period of approximately 60 min. Whole-genome sequencing confirmed the absence of virulence or antibiotic resistance genes. In vivo, both prophylactic and therapeutic oral administration significantly improved survival rates (100% and 90%, respectively) compared with the challenge group (30%) and reduced intestinal expression of inflammatory cytokines (IL-1, IL-6, TNF-α), restored the expression of ZO-1 and Claudin, whereas Occludin expression remained unchanged, and attenuated histopathological damage in the intestine and heart in the oral gavage model, whereas hepatic pathology remained unaffected in the oral model; however, hepatoprotective effects were observed in the supplementary intraperitoneal challenge model. 16S rRNA sequencing, transcriptomics, and metabolomics revealed that phage treatment restored gut microbiota balance (enrichment of Firmicutes, reduction of Proteobacteria/Enterobacteriaceae), modulated host energy metabolism and protein synthesis pathways, and mitigated infection-induced metabolic disturbances. DISCUSSION: VB-SalS-XND03 exhibits potent lytic activity, environmental stability, and a favorable safety profile. Its efficacy in a physiologically relevant oral infection model, supported by integrated multi-omics analyses, highlights its potential as a promising biocontrol candidate for Salmonella infections in poultry.