The economic stakes are enormous. Infertility in dairy cattle is estimated to cost the global industry roughly 12.6 billion US dollars every year, with conditions such as repeat breeding, postpartum anestrus, and cervicitis prolonging calving intervals and eroding lifetime productivity. In Nepal specifically, repeat breeding prevalence has been reported between 0.5 and 31 percent, while anestrus affects around 24 percent of animals. Because bacterial infections of the reproductive tract are considered a major underlying factor, and because antimicrobial resistance increasingly complicates treatment, researchers from Agriculture and Forestry University and the National Veterinary Medicine Center set out to establish the first baseline data of their kind in the country.

Between July 2024 and April 2025, the team sampled cattle in Sainamaina Municipality in Rupandehi District and Bharatpur Metropolitan City in Chitwan, including the university livestock farm and the National Cattle Research Program. Using a sterile cytobrush guided by a vaginoscope, they collected cervical and vaginal mucus from 35 animals for culture and antimicrobial testing, and from 18 animals for sequencing. The groups were carefully defined: healthy heifers under two years of age, cows with an enlarged and congested cervix consistent with cervicitis, repeat breeders that failed to conceive after three or more inseminations despite normal estrous cycles, cows within 35 days of calving, and postpartum cows that had not shown estrus after 60 days, subdivided by ultrasound into silent heat and true anestrus.

Culture results showed that bacterial growth was detectable in 30 of the 35 samples, an isolation rate of 85.7 percent, with every sampled true anestrous cow, postpartum cow, and cervicitis case yielding bacteria. Among the 54 isolates, Gram-negative organisms slightly outnumbered Gram-positive ones, and Enterobacter spp. together with Micrococcus spp. emerged as the two most frequently recovered genera. Acinetobacter, Salmonella, Bacillus, and Staphylococcus species rounded out the dominant isolates, while single isolates of E. coli, Pseudomonas, Shigella, Citrobacter, and Corynebacterium completed the picture. Many of these organisms are not primary pathogens, but the authors note that under dysbiotic conditions, when microbial balance and host defenses are disrupted, even opportunistic contaminants can become clinically relevant.

The antimicrobial susceptibility results were the most alarming component of the study. Using the Kirby-Bauer disk diffusion method against eight antibiotics spanning tetracycline, ampicillin, sulfamethoxazole, ciprofloxacin, chloramphenicol, erythromycin, ceftriaxone, and gentamicin, the researchers found that 71.7 percent of isolates qualified as multidrug-resistant, meaning resistance to at least one agent in three or more antibiotic classes. Not a single isolate was sensitive to all tested drugs. Salmonella spp. showed complete resistance to ciprofloxacin, Micrococcus spp. were fully resistant to ceftriaxone and ampicillin, and Staphylococcus spp. were uniformly resistant to ampicillin. Moreover, 91.3 percent of isolates carried a multiple antibiotic resistance index above 0.2, a threshold interpreted as signaling origin from environments with frequent antibiotic use or misuse.

Particularly concerning is the presence of ESKAPE-associated genera among the isolates. Enterobacter, Staphylococcus, and Acinetobacter belong to a group of nosocomial pathogens notorious for multidrug resistance in human medicine, and the World Health Organization classifies several of them as critical or high priority. The proportion of multidrug-resistant ESKAPE-related isolates in this cattle study actually exceeded rates reported in clinical human isolates from Nepalese hospitals, suggesting that resistant bacteria circulate widely in livestock environments and may pose zoonotic and occupational risks to farmers, veterinarians, and food handlers.

On the sequencing side, the team generated more than one million high-quality reads, yielding 2,366 operational taxonomic units at a 97 percent similarity threshold. All four sequenced groups shared 434 OTUs, pointing to a conserved core vaginal microbiota. Across all samples, Bacillota was the most abundant phylum at 37.3 percent, followed by Pseudomonadota at 27.8 percent, Fusobacteriota at 14.6 percent, Actinomycetota, Bacteroidota, and Mycoplasmatota, together accounting for 98.3 percent of total sequences. Alpha diversity did not differ significantly among groups, but beta diversity analysis revealed significant differences in community composition, with healthy heifers and cervicitis cows forming clearly distinct clusters.

Group-specific signatures emerged from linear discriminant analysis effect size screening. Cows with cervicitis were enriched for Chryseobacterium and members of Weeksellaceae, alongside Fusobacterium and Porphyromonas, both genera long associated with uterine disease. Repeat-breeding cows showed enrichment of Filifactor and the phylum Synergistota, hinting at a dysbiotic state without strong pathogen dominance. Healthy heifers, by contrast, carried the richest array of taxa, including Histophilus, Campylobacteria, Leptotrichia, and the probiotic-potential species Bifidobacterium pseudolongum, and they harbored the highest number of unique OTUs at 414. Notably, Fusobacteriota were entirely absent from healthy heifers but present in all other groups, suggesting a possible association with altered reproductive status.

Correlation analysis added another layer of ecological insight. Fusobacterium necrophorum and Porphyromonas levii showed a strong positive correlation, consistent with proposed synergism in maintaining an anaerobic niche within the inflamed reproductive tract, while both species correlated negatively with Trueperella pyogenes. A positive relationship between T. pyogenes and Proteus mirabilis echoed earlier reports of their co-occurrence in uterine infections. Intriguingly, Priestia megaterium, a bacterium with known probiotic and antimicrobial properties, correlated negatively with Helcococcus ovis, an emerging reproductive pathogen linked to metritis and abortion, raising the possibility of natural inhibition that could be explored therapeutically.

The authors are careful to frame these findings as exploratory. The cross-sectional design, modest and uneven sample sizes, convenience sampling, and the lack of full matching for age, parity, lactation status, and farm management all limit causal inference, and correlation analyses cannot establish direct ecological interactions. Nevertheless, the study delivers the first combined culture and sequencing portrait of the bovine cervico-vaginal microbiota in Nepal and lays the groundwork for defining a core vaginal microbiome tied to fertility. If larger longitudinal studies confirm these microbial signatures, vaginal community profiling could eventually become a diagnostic tool for predicting reproductive failure, while the documented resistance patterns underscore an urgent need to monitor antimicrobial use in dairy systems where the same drugs safeguard both animal and human health.

Subject of Research: Cervico-vaginal microbiota and antimicrobial resistance in cattle with different reproductive statuses in Nepal

Article Title: Cervico-vaginal microbiota and antimicrobial resistance patterns in relation to different reproductive statuses in cattle: a field study from Nepal

Article References: Bhattachan, B., Gautam, G., Raut, R., Basnet, H. B., Bhattachan, B., Khatiwada, S., & Bhattarai, R. K. (2026). Cervico-vaginal microbiota and antimicrobial resistance patterns in relation to different reproductive statuses in cattle: a field study from Nepal. Frontiers in Microbiomes, 5, Article 1900716. https://doi.org/10.3389/frmbi.2026.1900716

Image Credits: AI Generated

DOI: 10.3389/frmbi.2026.1900716

Keywords: bovine vaginal microbiota, 16S rRNA sequencing, antimicrobial resistance, multidrug-resistant bacteria, cattle fertility, cervicitis, repeat breeding, postpartum anestrus, ESKAPE pathogens, dairy cattle, Nepal, microbiome