Abstract – Highly pathogenic bovine Delta papillomaviruses (δPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected bovine δPVs in 68 blood samples (66%). Bovine papillomavirus (BPV) infection by a single genotype was revealed in 59% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 41% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection. Therefore, ddPCR displayed diagnostic and epidemiological value resulting in the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.

In the present study, the highly pathogenic bovine Deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real time PCR (qPCR). Overall, ddPCR detected BPV E5 DNA in ~61.3% of the blood samples examined, while real time qPCR revealed the virus in ~10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR revealed a single BPV genotype in ~59.2% and multiple genotypes in ~40.8% of goats harboring BPV DNA, while real time qPCR detected single genotypes in ~17% and multiple genotypes in ~1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes, and six (~14%) four genotypes. In contrast, real time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that the ddPCR technique has much higher sensitivity and specificity in the detection of these viruses, and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.