Bacteriophage cocktail in drinking water suppresses systemic avian pathogenic Escherichia coli infection and pathology in laying hens.

Avian colibacillosis, caused by avian pathogenic Escherichia coli (APEC), results in high morbidity and mortality and reduces egg production in domestic poultry. Increasing antimicrobial resistance (AMR) and restriction on antibiotic use necessitate alternative disease control strategies. We evaluated the efficacy of a phage cocktail (Tequatrovirus, Sashavirus, and an unclassified Caudoviricetes) at a multiplicity of infection > 100, administered for 4 days in drinking water (DW) or as a single intramuscular (IM) treatment, in preventing experimental APEC infection (108 CFU given intravenously) in specific-pathogen-free laying hens (25-27 weeks old, n=10 per experimental group). Phage treatment reduced mortality from 20% to 0 and morbidity by 30%. Birds receiving phages in DW showed a marginal trend toward reduced weight loss (p = 0.063) than those in IM or APEC-only groups at 4 days post-infection. Macroscopic lesions were present in 6.7-11.7% of organs in APEC-only and IM groups but were absent in DW-treated hens. In the DW group, median histopathological scores were lower, with the most notable reduction (p = 0.006) in the spleen. Across organs, concentrations of recovered phages were similar between DW and IM groups (p = 0.227); however, in the DW group, 1.3-2.1 log10 PFU/g of phages were detected in all tissues except blood. Based on 16S rRNA gene sequencing, neither IM nor DW phage treatments altered the overall general bacterial structure or population in the cecum. We conclude that this phage cocktail, administered via DW, lysed APEC without disrupting gut microbiota, improved overall bird health and welfare, and protected internal organs from APEC invasion.