To our knowledge, this study is the first to use the sensitive ddPCR assay, a biotechnological refinement of conventional PCR, to detect and quantify highly pathogenic bovine δPVs in clinically healthy sheep by liquid biopsy. Our previous studies have shown that ddPCR has both high specificity and sensitivity for the detection and quantification of BPV DNA in healthy and diseased cattle  as well as in healthy goats . The current study provides further evidence that ddPCR is a very useful approach to detect and quantify BPV in the blood of healthy sheep and allows us to gain diagnostic and epidemiological insights into BPV presence in ovine species as data on the prevalence and types of BPVs in sheep are not currently available.
The liquid biopsy approach to the detection of circulating BPV DNA has garnered growing interest in PV studies . Indeed, PV detection in the blood can be used as diagnostic, prognostic, and epidemiological markers .
Our study showed that BPV-2 is the most prevalent BPV genotype in healthy sheep, similar to other ruminants, such as cattle and goats. The highest number of copies of this genotype was found in Sar from sheep flocks in which cross-infections by BPVs have been previously reported , which suggests that copy numbers may correlate with the risk of cutaneous and mucosal lesions that progress to cancer. Furthermore, this study reports the first detection of BPV-14 in sheep. This genotype, chronologically the last BPV type identified in cattle, has never been described in the ovine species. Furthermore, our results demonstrated a statistically significant prevalence of BPV-14 in Apu compared to Sar, Cal, Bas, and Cam, which clearly showed that BPV genotype prevalence has a territorial divergence in these regions.
We compared the sensitivity of ddPCR with that of RT-qPCR in evaluating the same liquid biopsy, demonstrating that ddPCR has superior sensitivity compared to RT-qPCR. Therefore, our results suggest that ddPCR is by far the most sensitive and accurate assay for BPV detection. It is worth noting that it has been shown that ddPCR outperforms RT-qPCR in terms of the sensitivity, specificity, and reproducibility of oncogenic human papillomavirus detection and quantification [22-24].
BPV genotype detection in the blood of healthy sheep suggests that the bloodstream can be the primary site of BPV infection. As bovine δPVs are known to infect ovine species and result in anatomoclinical diseases, it is conceivable that these viruses may spread through the blood, which could be responsible for secondary tissue localization and infection. However, as in humans , further epidemiological studies are required to enhance the understanding of BPV transmission via the bloodstream.
Here, we detected a higher percentage of BPVs in sheep that were in close contact with cattle herds, about which numerous case reports of BPV infection have been described. Indeed, the sheep flocks in our study shared bracken fern-infested lands with pasture-residing cattle for grazing. The evidence from epidemiological studies of cattle is sufficiently strong to suggest that in the presence of BPV infection, the toxic components of bracken ferns such as ptaquiloside (PT), a water-soluble norsesquiterpenoid glycoside, are ecological co-factors in the development of severe diseases due to BPVs, including chronic enzootic hematuria (CEH), a clinical syndrome caused by bladder tumors . Thus, it is conceivable that PT may also be a co-factor of diseases in sheep. Indeed, PT has recently been detected in biological matrices from healthy sheep . PT is known to hamper the immune system and may play an important role in cross-species transmission and infection of bovine δPVs. It is worth noting that outbreaks of CEH have also been reported in sheep [27, 28]. Furthermore, bovine δPV infection resulting in clinical disease are known to occur in sheep [8-10]. Therefore, the detection of bovine δPV DNA in the blood of sheep means that sheep can be infected by these PVs, which may make δPVs an additional, potential cause of ovine disease. Furthermore, our results suggest that clinically healthy sheep may represent a reservoir for bovine δPVs. Thus, it is conceivable that sheep may play a role in intra- and interspecies bovine δPV transmission and infection. In this context, very precise quantitation of very low viral copy numbers can provide more precise monitoring of latent BPV DNA reservoirs.
Finally, it is well known that PV distribution varies considerably by geography . Therefore, ddPCR may be an essential tool for improving diagnostic procedures thus allowing the identification of the genotypic distribution of BPV and a better understanding of the possible, geographical divergence of BPV prevalence in different areas. The ddPCR assay appears to possess high sensitivity and accuracy, which is valuable for addressing the molecular burden of BPV infections and useful for defining an accurate ecological epidemiology. This baseline information improves our knowledge about the molecular mechanisms of the disease and provides insights into necessary measures for reducing the risk of BPV infection and/or co-infection.
In conclusion, ddPCR is presently being used to detect very low nucleic acid concentrations and, therefore, appears to be of interest in the diagnosis of infectious diseases, including viral diseases . DdPCR has proven to be a valuable new technology and with additional improvements in prospect it is likely to become an indispensable tool in diagnostic, prognostic and epidemiological virus research . Therefore, the ddPCR method may provide a new and promising tool for evaluating the BPV viral load in clinical samples. Future PV research warrants the use of this molecular approach to assess PV type-specific pathogenetic pathways of disease, including carcinogenicity. Indeed, available evidence from BPV distribution lends strong support to the notion that the risk of an animal developing a BPV-associated disease varies substantially according to the specific BPV type with which the animal is infected. Finally, the ddPCR approach may provide a better understanding of the complex interactions between multiple BPV types during coinfections, as the possible interference resulting from multiple PV genotypes in coinfection cases remains an open question .