The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity

Background Despite widespread use of antimicrobials and vaccines, the incidence of infectious bovine keratoconjunctivitis (IBK), or pinkeye, continues to increase in North American beef cow-calf operations. Recent research suggests that there is potential for the commensal ocular microbiome to help mitigate IBK. Therefore, this study characterized the ocular microbiome of cattle with and without IBK using culturing and shotgun metagenomic sequencing and assessed the ability of commensal bacteria to inhibit Moraxella spp. in vitro . Ocular swabs (n = 143) were collected from IBK-affected (n = 102) and healthy cattle (n = 41) before antimicrobial treatment from North Dakota herds. Bacteria were cultured aerobically and anaerobically on five different media and the isolates identified. A subset of swabs (37 IBK; 12 healthy) underwent shotgun metagenomic sequencing. The genomes of 31 isolates, including Moraxella bovoculi , Moraxella bovis , and commensal bacteria, were also sequenced. Fifty-two commensal isolates were screened for inhibition of Moraxella spp. using an agar slab method, with five isolates further tested for inhibition in the presence of culturable ocular microbiome using qPCR. Results The 351 bacterial isolates taxonomically identified represented 61 genera from three phyla. The majority of isolates belonged to Bacillus (25.9%), Streptococcus (11.1%), Staphylococcus (10.1%), and Moraxella (9.4%) genera. Shotgun metagenomic analysis revealed significant differences in the ocular microbiome composition between IBK-affected and healthy cattle (R² = 0.042; P  = 0.034). Dominant bacterial species included Cutibacterium acnes , Mannheimia pernigra , Mesomycoplasma bovoculi , Moraxella bovis , and Moraxella bovoculi . Eight bacterial species, including Bifidobacterium globosum and Bacillus licheniformis , were more abundant in healthy cattle, while Arthrobacter luteus was enriched in IBK cases. Thirty-seven high-quality metagenome-assembled genomes were also recovered, with 27% classified as Mesomycoplasma bovoculi . Moraxella spp. genomes exhibited strain-specific antimicrobial resistance and virulence gene diversity. Seventeen commensal isolates inhibited Moraxella , with Weizmannia coagulans , Lentilactobacillus buchneri , and Paenibacillus polymyxa showing strong activity. Selected isolates maintained inhibitory effects in co-culture with the ocular microbiome. Conclusion The ocular surface of beef cattle is inhabited by a diverse microbiome that includes several bacterial strains that have the potential to be used as therapeutics to inhibit IBK pathogens.