Background: Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78 are important serogroups for chicken health responsible for colibacillosis. With sub-therapeutic use of antibiotics being phased out in egg farms and emergence of multidrug resistant (MDR) APEC, novel approach is required to disease management.

Hypothesis: Bacteriophages, virus infecting bacteria, can be used as an alternative approach for the treatment of APEC infections in laying hens due to their efficient lytic ability against specific hosts.

Objectives: In this study, we isolated and characterized phages from hen feces and human sewage in Alberta and evaluated their potential for controlling colibacillosis in laying hens.

Methods and Results: The 7 novel APEC-infecting phages preferentially lysed APEC strains in this study and ECL21443 (O2) was most susceptible to phages (n = 5) tested. ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Comparative genomics analysis placed 6 of the 7 phages in the genus Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was unassigned a genus in the family Siphoviridae. For the bird trial, O78 targeting phage cocktail (ASO78A+ AVIO78A+ ASO78B) at multiplicity of infection (MOI >100) was introduced through oral (drinking water-DW) and intramuscular route (IM) in preventing APEC infection in experimentally challenged laying hens. Laying hens (n=35, 25 weeks) were divided in four groups 1. Medium control group (bacterial broth + phage buffer 2. DW group (phage in drinking water (4 days prior to inoculation + APEC), IM group (Phage +APEC) and APEC positive control (APEC + buffer). The mortality rate was decreased from 20% to 0% in DW and IM phage group when compared with APEC only group. Regarding body weight (BW), except for the medium control group, birds from all other experimental groups experienced weight loss over time. The group that received drinking water tended to exhibit lesser weight loss compared to the IM and APEC-only groups. The clinical score, bacterial recovery, and macroscopic lesion were greater (P < 0.05) in APEC followed by IM and DW group. Surprisingly, there were comparable (P > 0.05) moderate to severe microscopic organ lesions between the APEC only and IM groups. In contrast, all organ lesions at microscopic level were markedly attenuated from DW group. For APEC enumeration, only few APEC were recoverable from spleen of DW group, whereas greater number of bacteria were found from different organs of APEC only and IM groups. When it came to phage recovery, regardless of the phage delivery route, the spleen consistently exhibited the highest presence of phages. Furthermore, in the DW group, phages were recoverable from the liver, lungs, and heart; However, in the IM group, the phage titer decreased significantly by 7 log₁₀ PFU/mL in blood samples collected 24 h after inoculation and phages were not detectable from other organs at the end of experiment. This decrease in phage effectiveness in the IM group could possibly be attributed to the rapid clearance of phages from the bloodstream and the recovery of phage mutants that displayed slightly reduced sensitivity to the phage cocktail compared to the wild type APEC.

Conclusions: Various type of endogenous phages were capable of lysing MDR APEC. Phage given in water may be valid approach for protecting laying hens form APEC infection.