315. Development of Ocular Probiotics to Mitigate Pinkeye in Cattle: An in Vivo Pilot Study.

Abstract 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, including our own, suggests that the ocular surface of beef cattle harbors a diverse microbiome including commensal bacterial strains with the potential to be used as therapeutics to inhibit IBK pathogens. In this study, we evaluated safety and efficacy of the five ocular probiotic strains isolated from ocular microbiota of beef cattle, previously characterized in vitro as candidates for mitigating IBK-associated Moraxella pathogens. 20 Holstein bull calves (16-23 days old) received either 1 mL of an ocular probiotic cocktail containing those strains (Bacillus pumilus, Bacillus velezensis, Lactiplantibacillus pentosus, Lentilactobacillus buchneri and Weizmannia coagulans; approximately 10⁹ CFU per strain per mL) or 1 mL saline (Control). Ocular, nasal, oral and periocular skin swabs, ruminal fluid and fecal samples from both probiotic and control calves were collected on day -1 (baseline) and days 1, 2, 7, 14, 21, and 28 after probiotic inoculation. Genomic DNA extracted from these samples was subjected to 16S rRNA gene (V3-V4) amplicon sequencing for bacterial microbiota characterization. Body temperatures and daily gain of the calves were also monitored over these 28 days. PERMANOVA revealed small but statistically significant differences in ocular (R² = 0.0207, P = 0.0017) and nasal microbiota (R² = 0.0212, P = 0.0323) between probiotic and control calves. However, oral, ruminal, fecal and periocular skin microbiotas showed no treatment-associated differences (P > 0.05). The overall ocular microbiota structure was distinctive (R² = 0.451; P < 0.0001) from that of periocular skin microbiota. Ocular alpha-diversity and richness remained stable throughout the study period. A modest difference in richness was detected at d21 based on the Chao1 index, while microbial richness (Observed ASVs) and diversity metrics (Shannon and inverse Simpson) did not differ between the two group calves. Similarly, microbial richness and diversity of microbial communities associated with oral, ruminal, fecal, and skin sites remained unchanged (P > 0.05) in response to ocular probiotic inoculum. A small number of bacterial genera were differentially abundant in the ocular microbiota of probiotic and control calves. Ocular probiotics did not influence the overall calf weight gain (control: 24.4 vs. probiotics: 23.4 kg; P = 0.488) and body temperature (P = 0.443). Ocular probiotic inoculation at the tested dose had minimal impact on microbial composition across ocular, oral, nasal, and gut sites, with microbiome variation primarily driven by age-related maturation. Probiotic administration did not affect body weight or body temperature, indicating safety and no adverse effects on calf health. However, the limited microbiome response suggests that the inoculation method and/or dosing strategy should be optimized to enhance efficacy.