Development of a Bacteriophage-Based Feed Additive to Prevent Bovine Liver Abscesses

Abstract In the US, beef cattle feedlots commonly use antibiotic feed additives, such as tylosin and virginiamycin, to prevent liver abscesses, which can cause significant economic losses and animal welfare concerns. However, growing public and regulatory pressure to curb the proliferation of antibiotic resistant bacteria has prompted a need to reduce or eliminate their use. As an alternative, our group is working on a bacteriophage-based feed additive that targets Fusobacterium necrophorum, the primary cause of bovine liver abscesses, with the aim of reducing or eliminating the dependence on antibiotic feed additives. Using a combination of phage isolation techniques, we identified and characterized fifteen new F. necrophorum phages belonging to six genetically distinct groups. These phages exhibited a broad intraspecific host range and could infect various contemporary F. necrophorum strains isolated from different regions of the United States. All the phages were temperate, with dsDNA genomes ranging from around 36 kbp to 114 kbp. Almost all contemporary F. necrophorum isolates were found to have one or more active prophages upon bacterial genome sequencing and prophage induction. In vitro evolution resulted in the recovery of a potential virulent mutant, φBB37, which inhibited the growth of the high leukotoxin-producing F. necrophorum 8L1 strain for over 40 hours in vitro. Phage cocktails proved effective for up to 60 hours. After minimal optimization, most phages demonstrated high productivity (> 109 PFU/mL) and little yield variation during scale-up, highlighting their commercial potential. Unexpectedly, we discovered a previously unrecognized Fusobacterium species, Fusobacterium varium, is widely prevalent and frequently the most abundant member of the genus in cattle rumen. Previous studies on liver abscess control in cattle have almost exclusively focused on F. necrophorum, which may explain the lack of effectiveness observed. To elucidate the ecological role and potential pathogenicity of ruminal F. varium, we conducted genome sequencing and found these bovine strains to have multiple virulence factors and high similarity to human clinical isolates. However, phages with the ability to infect F. varium were plentiful in rumen fluid, with several demonstrating purely lytic activity. To assess the safety and in vivo dynamics of F. necrophorum and F. varium phage cocktails, a small-scale animal trial was recently conducted with ten cannulated cattle.