Francesca De Falco

and 5 more

Ovine papillomaviruses (OaPVs) were detected and quantified, for the first time, using droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (qPCR) via liquid biopsy of 165 clinically healthy sheep. OaPV DNA was detected in 126 blood samples (~76.4%). DdPCR detected OaPV DNA in 124 samples; in only two additional samples positive for real-time qPCR, ddPCR failed to detect the presence of any OaPVs. In 70 of the positive samples (~55.6%), a single OaPV infection was observed, 12 of which were caused by OaPV1 (~17.1%) and 14 by OaPV2 (20%). OaPV3 was responsible for 19 single infections (~27.1%), and OaPV4 for 25 single infections (~35.7%). Multiple OaPV coinfections were observed in 56 (~44.4%) positive samples. OaPV coinfections caused by two genotypes were observed in 31 positive samples (~55.4%), with dual OaPV3/OaPV4 infection being the most prevalent as seen in 11 blood samples. In addition, five OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, four OaPV1/OaPV3, and three OaPV2/OaPV4 dual coinfections were also detected. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~42.8%) and only one (~1.8%) of coinfected blood samples, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent, as observed in 12 (50%) blood samples harboring triple OaPV infections. This study showed that ddPCR is the most sensitive and accurate assay for OaPV detection and quantification thus outperforming real-time qPCR in terms of sensitivity and specificity. Therefore, ddPCR may represent the molecular diagnostic tool of choice, ultimately providing useful insights into OaPV molecular epidemiology and field surveillance.

Francesca De Falco

and 5 more

Ovine papillomavirus (OaPV) comprises four genotypes; OaPV1, OaPV2, and OaPV4 are fibropapillomaviruses within the genus Delta-papillomavirus ( Delta-PV ), whereas OaPV3 is an epitheliotropic virus that belongs to the genus Dyokappa-papillomavirus ( Dyokappa-PV ). To date, all of them have been known to infect sheep only. OaPV1, OaPV2, and OaPV4 have been associated with ovine cutaneous and mucosal fibropapillomas, while OaPV3 is a key factor in the squamous cell carcinoma (SCC) pathway of the sheep skin. Peripheral blood mononuclear cell (PBMC) samples obtained from 128 cattle at public slaughterhouses were investigated using droplet digital polymerase chain reaction (ddPCR). ddPCR is a new-generation PCR technique that enables accurate and absolute quantification of target molecules with high sensitivity and specificity. All OaPVs were detected by identification and quantification of nucleic acids using specific fluorescent probes. Of 128 PBMC samples, 100 (~78%) showed OaPV infections. Further, 42, 35, and 23 PBMC samples showed single, double, and triple OaPV infections, respectively. OaPV1 was responsible for 22 single infections, OaPV2 caused 16 single infections, and OaPV3 and OaPV4 caused two single infections each. OaPV1 and OaPV2 were the most frequent ovine viruses in dual and triple infections. In many PBMC samples, both ovine Delta-PV and Dyokappa-PV were found to be transcriptionally active, as shown by the detection and quantification of E5 oncogene transcripts for OaPV1, L1 transcripts for OaPV2, E6 and E7 transcripts for OaPV3, and E6 for OaPV4. OaPVs were found in the blood samples from cattle that shared grasslands rich in bracken ferns known to contain immunosuppressant substances. Furthermore, OaPVs were also found in cattle from intensive livestock farming without any contact with sheep. Because OaPV DNA was detected in both grass hay and corn silage, it is conceivable that these feed may be the viral sources.