168 Characterization of the Culturable Fraction of Bovine Ocular Microbiota and Its in Vitro Antimicrobial Activities Against Pinkeye Pathogens
Authors: Luecke Sarah M , Stokka Gerald , Schmidt Kaycie N , Havelka Garrett , Amat Samat 
Published: November 2023

Abstract
Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is considered one of the most important ocular diseases in cattle, causing an estimated $226 million in annual losses to U.S. cow-calf producers. While Moraxella bovis is considered the principal IBK pathogen, Moraxella bovoculi has also been associated with the pathogenesis of IBK. Thus, these Moraxella species are often the target for antibiotic and/or vaccine-based treatment and prevention strategies. Despite extensive use of antibiotics, IBK infections are challenging to treat, highlighting the increased need for an antibiotic-alternative approach to control IBK. Recent evidence suggests that the microbial community associated with the bovine oculus is important for eye health, and that the ocular microbiota can be a target for enhancing disease resistance against IBK in cattle. The objectives of this study were to characterize the ocular microbiota of healthy and IBK infected cattle using sequencing and culturing techniques, and to isolate and screen commensal ocular isolates for their antimicrobial activity against M. bovis and M. bovoculi. Ocular swabs (n = 64) were collected using eSwabs from cattle diagnosed with IBK or without IBK (healthy control) before antibiotic treatment from beef herds across North Dakota. Swabs were plated onto four different agar plates (blood, Columbia blood, MRS and multi-sliced agar plates), and cultured aerobically and anaerobically. A total of 658 bacterial isolates were obtained via culturing and cryopreserved. Taxonomic identification was performed on a subset of banked isolates (n = 277) by near full-length sequencing of the 16S rRNA gene. These isolates taxonomically belong to 6 different bacterial phyla: Bacillus (42%), Firmicutes (27%), Actinomycetota (13%), Pseudomonadota (11%), Actinobacteria (5%), and Proteobacteria (2%). A total of 55 different genera were identified with Bacillus (25%), Streptococcus (13%), Staphylococcus (10%), Moraxella (6%), Macrococcus (5%), Paenibacillus (3%), Caldibacillus (3%), and Rummeliibacillus (3%) being the most dominant genera. Next, a subset of these commensal isolates (n 53) were screened for their growth inhibition against M. bovis and M. bovoculi using an agar slab method. Among the screened isolates, 6 isolates showed inhibition against M. bovis and M. bovoculi with zones of inhibition ranging from 12 mm to 28 mm. Ocular isolates identified as Weizmannia coagulans (43Y-MRSC), Lentillactobacillus buchneri (23D-MRSA) and Paenibacillus polymyxa (785-WCF) exhibited relatively strong inhibition against both Moraxella isolates. Scanning electron microscopy revealed noticeable structural and morphological damages in M. bovis and M. bovoculi cells due to Weizmannia coagulans culture-supernatant treatment. Overall, our results indicate that ocular mucosa of beef cattle is inhabited by relatively rich and diverse culturable bacterial communities, and that some ocular commensals can inhibit IBK pathogens. This suggests the potential for the development of bacterial therapeutics based on commensal ocular bacteria to mitigate IBK in cattle.