4. Discussion
There are no survey data on the burden of OaPVs carried by sheep. To the best of our knowledge, this study is the first systematic research on the molecular epidemiology of OaPV infection among sheep using real-time qPCR and ddPCR as diagnostic procedures. DdPCR revealed the nucleic acid of ovine Delta -PV (OaPV1, OaPV2, OaPV4), and Dyokappa- PV (OaPV3) in a very high percentage as it was able to detect OaPV DNA in 124 out of 126 positive blood samples (~ 98.4%). Our findings showed that ddPCR, which has not yet been utilized for studying OaPV epidemiology, is an advanced technology that can accurately diagnose OaPV infection with high specificity and sensitivity thus representing a promising new tool for the accurate detection and quantification of the OaPV load. qPCR failed to detect OaPV DNA in a large number of samples which, in contrast, harbored OaPV DNA, as detected via ddPCR, thus suggesting that DNA levels may be too low and traditional methods such as real-time qPCR may be faulty to detect them via liquid biopsy. Therefore, this study demonstrated that ddPCR outperforms qRT-PCR in terms of sensitivity and specificity for OaPV detection.
We showed that OaPV4 and OaPV3 are the most prevalent OaPVs in sheep flocks in southern Italy, respectively. It is worth noting that OaPV3 and OaPV4 have been identified in tumors of sheep from the Mediterranean area only (Alberti et al., 2010; Tore et al., 2017), whereas OaPV1 and OaPV2 have not previously been reported in Italy. OaPV3 and OaPV4 achieved an overall higher viral load than OaPV1 and OaPV2. DdPCR assay showed a very high sensitivity as the LoD showed values < 1 copies/μL, which are believed to be a robust marker of the high sensitivity of the ddPCR protocol for research on virus, including PVs (Lillsunde Larsson and Helenius, 2017; Tastanova et al., 2021). Furthermore, our study showed that diagnostic testing plays a critical role in addressing OaPV epidemiology and confirmed that qPCR is extremely inaccurate for detecting pathogens at very low concentrations, as previously suggested (Li et al., 2018). A higher percentage of OaPV-positive samples detected by ddPCR showed that this assay offers the potential to perform precise low-level quantification otherwise undetectable thus allowing us to assess the epidemiology profile of OaPVs and gather insights into their territorial prevalence. In this context, our study confirmed that ddPCR can be used for low-abundance nucleic acid detection and is very useful in diagnosing infectious diseases, including viral infections in comparative medicine (Li et al., 2018). In addition, ddPCR is very accurate and sensitive diagnostic assay for the detection and quantification of human papillomavirus DNA (Biron et al., 2016; Carow et al., 2017; Cheung et al., 2019) and BPV DNA (Cutarelli et al., 2021; De Falco et al., 2021; Roperto et al., 2021).
DdPCR testing is pivotal for accurate viral load measurements, OaPV epidemiological interpretations, and the health management of sheep flocks. Quantification of viral load may be very useful both as a diagnostic procedure and as a prognostic biomarker. Although the correlation between viral load and PV infection remains to be elucidated (van der Weele et al., 2016), it is believed that PV viral load is an important determinant of viral persistence (Kaliff et al. 2021). Furthermore, ddPCR significantly reduced the false negative rates of OaPV detection, which may be responsible for virus spread. It could be of epidemiological importance to know whether sheep harboring OaPVs, particularly those belonging to the Delta -PV genus, can represent a potential reservoir for intra- and inter-species transmission similar to cattle for bovine Delta -PV. Preliminary results of an ongoing study on the detection and quantification of OaPV DNA in the blood of cattle and goats appear to corroborate our assumption (Roperto, personal observations). In addition, OaPV2 DNA sequences have recently been found in a gingival sarcoid-like mass of a pig; therefore cross-species transmission of OaPVs may be possible (Munday et al., 2020). Both bovine and ovine Delta -PVs are characterized by overlapping biological properties, including cell tropism and pathogenicity (de Villiers et al., 2004; Tore et al., 2017).
As OaPVs have been detected in healthy sheep, it is conceivable that blood represents an important primary route of infection; therefore, OaPVs can disseminate to any organs via the bloodstream. Epidemiological data on the territorial genotype prevalence of OaPVs are of interest as PV diseases appear to be associated with specific genotypes both in humans (Del Rio-Ospina et al., 2015) and farm animals (Roperto et al., 2013; Roperto et al., 2016; Sykora et al., 2017).
Finally, the high prevalence of OaPVs may represent an important, yet unknown threat to ovine industries. The improvement of virus detection in livestock remains a priority in clinical practice. This study showed that accurate diagnostic methods play a crucial role in OaPV control strategies. Therefore, ddPCR may represent the diagnostic molecular tool of choice, which may ultimately provide useful insights into molecular epidemiology and field surveillance, known to be key components of the control program of any infectious disease, including viral diseases. Further studies to better understand the risks posed by the infectivity of OaPVs and manage the potential clinical impact of PV-related diseases in sheep flocks are warranted.