1. Introduction
Papillomaviruses (PVs) comprise a diverse group of epitheliotropic,
double-stranded DNA viruses that infect humans and animals in a
species-specific manner (IARC, 2007). PVs have co-evolved with their
respective hosts, resulting in minimal cross-transfer between species
(Doorbar et al., 2016). Viruses such as PVs that slowly evolve with
their hosts typically cause latent infection (Antonsson and McMillan,
2006). However, persistent infection by PVs causes a variety of severe
diseases, including cutaneous and mucosal cellular proliferation (IARC,
2007).
Bovine papillomaviruses (BPVs) comprise 29 genotypes
(http://pave.niaid.nih.gov/;
Yamashita-Kawanishi et al., 2020).
Four highly pathogenic BPVs (BPV-1, -2, -13, and -14) belong to theDelta genus (δPVs). They are associated with both cutaneous and
mucosal benign and malignant tumors. Indeed, BPV-2 and BPV-13 are the
most notable infectious agents commonly responsible for bladder tumors
in some breeds of pasture-residing cattle that graze on lands rich in
bracken fern (Pteridium spp.) (Campo et al., 1992; Borzacchiello et al.,
2003).
δPVs are the only BPVs known to infect mesenchymal tissues and to show
cross-species transmission and infection (IARC, 2007). δPVs have been
detected in cutaneous wart lesions from ovines (Mazzucchelli-de-Souza et
al., 2018; Savini et al., 2020). Vertical transmission of δPVs in sheep,
resulting in oral fibropapillomatosis and epidermal hyperplasia of the
lip in newborn lambs, has also been documented (Roperto et al., 2018a).
Furthermore, δPV DNA has been detected by polymerase chain reaction
(PCR) in the peripheral blood of healthy sheep (Roperto et al., 2018b).
Although there are very limited numbers of reports describing BPV
quantification data, PV studies have traditionally used real-time
quantitative PCR (RT-qPCR) to measure the virus reservoir represented by
PV DNA in both cutaneous and bladder neoplastic samples (Yuan et al.,
2007; Pangty et al., 2010; Pathania et al., 2012; Cota et al., 2015).
Most recently, digital PCR is gaining popularity as a novel approach to
nucleic acid quantification as it allows for absolute target
quantification. Indeed, digital droplet PCR (ddPCR) is a robust PCR
technique that enables precise and accurate absolute quantification of
target molecules by diluting and partitioning the samples into numerous
compartments (Kanagal-Shamanna, 2016).
Quantification of PVs by digital PCR is proving to be a valuable
improvement over qPCR, as it has been shown to have a higher robustness
to mismatches between the primer-probe set and PV genotypes. Due to
pathogens that cause latent infection, BPV concentrations in the blood
are sometimes too low to be determined by traditional methods. In cattle
and goats, ddPCR has been found to outperform RT-qPCR in terms of the
sensitivity, specificity, and reproducibility of BPV detection, all of
which play a central role in diagnostic and epidemiological procedures
to identify the geolocalization of BPVs (De Falco et al., 2020;
Cutarelli et al., submitted).
The present study aimed to detect and quantify BPV DNA in the peripheral
blood of sheep using ddPCR and to show the potential advantages of this
molecular technology in the diagnosis and epidemiology of infectious
diseases, including viral diseases.