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.