The culturable ocular bacterial microbiota of beef cattle and its commensal members that can inhibit pinkeye-associated pathogens

In this study, we cultured a wide range of bacterial species using both aerobic and anaerobic culture conditions and five different growth mediums from ocular swabs obtained from beef cattle with (n = 35) and without (n = 29; healthy control) pinkeye infections. We taxonomically identified a subset of these bacterial isolates using near-full length 16s rRNA gene sequencing and tested a subset of these isolates for their ability to directly inhibit the growth of Moraxella bovis and Moraxella bovoculi, the primary pinkeye pathogens. We identified 6 bacterial isolates that can inhibit the growth of M. bovis and M. bovoculi in vitro. Using scanning electron microscopy (SEM), we further investigated the morphological and structural damage that occurred to M. bovis and M. bovoculi cells after incubation with culture supernatants of selected isolates that demonstrated growth inhibition in vitro. The SEM imaging provided clear indication of damage to Moraxella cell structure. Together, these commensal ocular bacteria that displayed antimicrobial activity against Moraxella in vitro are viable candidates for the development of bacterial therapeutics to treat and prevent pinkeye in cattle.

Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is a highly contagious disease of the eye and is one of the most significant health challenges impacting producers in the Midwest (Martin et al., 2019) with an estimated $150 million in annual losses to US beef producers (Bartenslager et al., 2021). The development of pinkeye in cattle is multifactorial, and it is commonly believed that irritation to the eye from face flies, tall grass, and ultraviolet (UV) light predisposes animals to pinkeye development. However, the primary ecological agents that are known to contribute to pinkeye infection are the bacterial pathogens Moraxella bovis and Moraxella bovoculi. Currently, pinkeye vaccinations against these pathogens are limited in their efficacy, and as such, producers are left with very little protection from outbreaks. Challenges in preventing and treating pinkeye infections may be a result of the knowledge gap surrounding the ocular microbiome (Bartenslager et al., 2021). To date, there are very few studies that have investigated the bacterial community of the bovine eye using culture independent metagenomic sequencing-based techniques (Cullen et al., 2017; Bartenslager et al., 2021). Research suggests that the bovine eye does harbor bacterial communities and that they may be important in ocular health. A relatively rich and site-specific bacterial community has recently been reported in the eyes of healthy newborn calves (Luecke et al., 2023). The genera Moraxella was well represented in those samples, and it is currently unclear if early colonization of the eye acts to prime the newborn’s immune system against pathogens or if it predisposes them to infection. Given this information, we conducted the present study to 1) characterize the ocular microbiota of healthy and pinkeye-infected beef cattle using sequencing and culturing techniques, and 2) isolate and screen commensal ocular bacterial isolates for their antimicrobial activity against M. bovis and M. bovoculi.

Ocular swabs from the cornea and conjunctiva of cattle exhibiting IBK symptoms (n = 35) as well as control swabs from healthy animals (n = 29) were collected from multiple herds across North Dakota as well as from the NDSU Beef Cattle Research and Teaching Center and from the NDSU Veterinary Diagnostic Laboratory (Table 1). Ocular swabs were collected using Puritan Opti-Swabs with the Liquid Amies Collection and Transport System (Puritan, Guilford, ME) and were stored on ice for transport to the lab. Once in the lab, samples were aliquoted and spread on up to five types of agar plates (Blood, Columbia Blood, De Man, Rogosa and Sharpe (MRS), Wilkins-Chalgren, and Multi-slice agar), all with various growth mediums to support the growth of a wide range of microorganisms. Plated samples were incubated both aerobically and anaerobically for 24 – 48 h. Bacterial colony growth was quantified, and unique colonies were sub-streaked onto fresh media and incubated for 24 h. Isolated bacteria were then cryopreserved (n = 658). Genomic DNA was extracted from a subset of preserved bacterial isolates (n = 351) and used for taxonomic identification by the near-full length 16S rRNA gene sequencing. Of the 351 identified isolates, 53 were tested for growth inhibitory effects against M. bovis and M. bovoculi using the agar slab method as described previously (Amat et al., 2019). Following the agar slab experiments, a selection of candidate bacteria that exhibited relatively strong inhibition of Moraxella growth were used to evaluate changes to cell morphology of Moraxella by inoculating Moraxella into the cell free supernatant of the candidate bacteria, incubating for 14 h, and observing changes using scanning electron microscopy (SEM) as described previously (Amat et al., 2019).

Table 1: Number of swabs collected and total number of isolated bacteria that were cryopreserved.

The 351 bacterial isolates identified using near-full length 16s rRNA gene sequencing were represented by 6 different bacterial phyla and 61 different bacterial genera. Bacterial phyla included Bacillota (44%), Firmicutes (23%), Actinomycetota (13%), Pseudomonadota (13%), Actinobacteria (5%), and Proteobacteria (2% Figure 1A). Of the 61 bacterial genera, Bacillus (26%), Streptococcus (11%), Staphylococcus (11%), Moraxella (9%), and Macrococcus (4%) were the most prevalent (Figure 1B). A total of 33 Moraxella isolates were identified, and they consisted of M. bovis and M. bovoculi. Of the 53 isolates tested for inhibition against Moraxella using the agar slab method (Figure 2), 6 isolates showed zones of inhibition ranging from an average of 13 mm to 25.7 mm (Table 2). Weizmannia coagulans (43Y MRS-C), Lactobacillus fermentum (ATTC 9338), and Paenibacillus polymyxa (42G WC-F) showed relatively strong inhibition against Moraxella, while Alkalihalobacillus rhizosphaerae (25F CB-B) and Lentilactobacillus buchneri (23D MRS-A) showed medium growth inhibition and Weissella paramesenteroides (23D MRS-F) displayed weak growth inhibition. Weizmannia coagulans (43Y MRS-C), Lactobacillus fermentum (ATTC 9338), and Lentilactobacillus buchneri (23D MRS-A) cell-free culture supernatants were incubated with M. bovis and M. bovoculi for 14 h and prepared for SEM imaging. The W. coagulans and L. fermentum isolates exhibited the greatest amount of cell damage to M. bovoculi. Complete cell lysis was observed, indicating that W. coagulans and L. fermentum effectively inhibit the growth of M. bovoculi. Lentilactobacillus buchneri exhibited only minor cell damage to M. bovoculi, with few structural damages to the M. bovoculi cells (Figure 3). When the cell-free culture supernatant of W. coagulans and L. fermentum was incubated with M. bovis, noticeable cell damage occurred, but it was not to the extent of the damage that occurred to M. bovoculi (Data not shown). Irregular cell shape and damages to the cell wall of M. bovis were observed, which indicates that W. coagulans and L. fermentum may be viable candidates for the development of bacterial therapeutics against M. bovis.

Table 2: Six bacterial isolates that exhibited antimicrobial activity against M. bovis and M. bovoculi when tested using the agar slab method.

These results indicate that the bovine oculus harbors relatively diverse culturable bacteria. In addition, some of the commensal bacterial isolates can inhibit the growth of M. bovis and M. bovoculi, potentially through the production of antimicrobial agents that can damage the cell structure and cell morphology of the pathogens. This information adds to the current understanding of the bovine ocular microbiota and indicates that commensal bacterial species within the ocular microbiota may be able to be harnessed to combat against pinkeye pathogens and modulate ocular microbiome-mediated eye health in cattle.

This study was supported by the North Dakota State Board of Agricultural Research and Education (award number 22-14-0231). A special thank you to the veterinarians and producers who assisted in sample collection. The authors express gratitude to Kelli Maddock at the NDSU Veterinary Diagnostic Laboratory for assistance with sample collection as well as Jayma Moore at the NDSU Electron Microscopy Core for SEM imaging.

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