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.