1. Introduction
The bovine papillomaviruses (BPVs) comprise 29 genotypes within five genera: Deltapapillomavirus (δPV; BPV-1, -2, -13, and -14), Xipapillomavirus (χPV; BPV-3, -4, -6, -9, -10, -11, -12, -15, -17, -20, -23, -24, -26, -28, and 29), Epsilonpapillomavirus(εPV; BPV-5, -8, and -25), Dyokappapapillomavirus (DyoκPV; BPV-16, -18, -22), Dyoxipapillomavirus (DyoχPV; BPV-7). In addition, BPV-19, -21, and -27 remain to be classified. (http://pave.niaid.nih.gov/; Yamashita-Kawanishi et al., 2020a; 2020b).
Bovine δPVs recognize Bos taurus as their classical host. However, they are also the only papillomaviruses capable of natural cross-species transmission and infection (IARC, 2007). Indeed, beyond cattle, bovine δPVs have been extensively investigated in horses (Epperson and Castleman, 2017; Savini et al., 2019), sheep (Mazzucchelli-de-Souza et al., 2018; Roperto et al., 2018; Savini et al., 2020) and buffaloes (Somvanshi, 2011; Roperto et al., 2013). They have even been studied in wild ruminants (Savini et al., 2016).
Quantitative real time polymerase chain reaction (qPCR) is considered the assay with the highest sensitivity and specificity for detecting papillomavirus DNA (Biron et al., 2016). As such, it has long been the method of choice for estimating viral load. However, to quantify real time qPCR, standard curves are necessary, and the efficiency of the method may vary between runs and reactions (Clementi and Bagnarelli, 2015).
Droplet digital PCR (ddPCR), first described by Sykes et al. (1992), is an improved method of conventional PCR that can clonally amplify and directly quantify DNA or RNA (Li et al., 2018). The technique involves diluting and partitioning the sample in many reaction chambers or droplets. Because ddPCR can perform absolute quantitation without a well-calibrated standard or highly efficient amplification, it is useful for determining of pathogen loads being faster, more precise, and reproducible (Kuypers and Jerome, 2017). Indeed, ddPCR is currently the most accurate and sensitive method for quantifying nucleic acids of interest particularly in cases of low pathogen loads (Isaac et al., 2017). Because it is more sensitive than classical approaches, including real time qPCR, it can detect small amounts of DNA that indicate residual disease and may otherwise be undetectable. For this reason, ddPCR has been used to quantify many viruses being especially useful for precise quantification of low viral loads (Kuypers and Jerome, 2017). In particular, precise quantitation of very low viral copy numbers may permit more reliable monitoring of latent papillomavirus DNA reservoirs, allowing early diagnosis and more timely treatment of viral infections (Li et al., 2018). Furthermore, ddPCR yields significantly fewer inaccurate false negatives and positives than real time qPCR in samples with low viral load (Liu et al., 2020).
The aim of the present study was to investigate, not previously reported, viral load of bovine δPVs in blood from clinically normal goats, as well as to compare ddPCR with real time qPCR to evaluate the diagnostic value of the ddPCR assay for the detection and quantification of BPVs by liquid biopsy.