Digital droplet PCR-based detection and quantification of ovine papillomavirus DNA from the vaginal virobiota of healthy mares

There are four genotypes of ovine papillomaviruses (OaPVs): OaPV1, OaPV2, and OaPV4, which are ovine delta papillomaviruses responsible for epithelial and mesenchymal cell infections, and OaPV3, an epitheliotropic Dyokappapapillomavirus associated with cutaneous tumors in sheep, including squamous cell carcinoma. Vaginal swabs of healthy mares were evaluated for the presence of PVs to investigate whether the vaginal virobiota of asymptomatic mares harbored OaPVs. High-performance digital droplet polymerase chain reaction (ddPCR) was used to quantitatively detect OaPV types 1-4 DNA in 94 vaginal swabs collected at the National Reference Center for Veterinary and Comparative Oncology (CEROVEC), Genoa, Italy. All samples were comparatively evaluated for OaPV DNA loading using real-time quantitative PCR. ddPCR detected OaPV DNA in 25 vaginal swab samples (26.6%), whereas qPCR revealed 13 vaginal swabs (11.7%). Differences between the two molecular protocols were determined to be statistically significant using McNemar's test (p < 0.0005). The detected OaPV types were OaPV1 and OaPV3. Both methods failed to detect OaPV2 or OaPV4 DNA, which could be attributed to the limited number of samples examined. OaPV1 is the most prevalent OaPV in equine vaginal virobiota . This study is the first to provide evidence of the presence of OaPV DNA in vaginal swabs of healthy mares. This comparative detection approach underscores the superior sensitivity of ddPCR over qPCR.