Molecular Detection and Quantification of Ovine Papillomavirus DNA in Equine Sarcoid

Abstract

Equine sarcoids are caused by infection with bovine papillomavirus (BPV) types 1, 2, and possibly 13. However, a number of sarcoids lack BPV DNA, and new potential etiological agents for sarcoid diseases need to be considered. High-performance digital droplet polymerase chain reaction (ddPCR) was used for the quantitative detection of ovine papillomavirus (OaPV) types 1-4 DNA from 63 sarcoid DNA samples collected in Austria. All samples were comparatively evaluated for OaPV DNA loads by qPCR. Conventional PCR and amplicon sequencing were used to validate the data. Of the 63 sarcoid DNA isolates, ddPCR was able to detect 22 samples harboring OaPV DNA (34.92%), whereas only five of the OaPV-positive samples were revealed by qPCR (22.72%). The differences in detection by ddPCR and qPCR were statistically significant (p < 0.05). The detected OaPV types were OaPV1, 3, and 4. Both methods failed to detect OaPV2 DNA, which could be due to the limited number of examined samples. Importantly, ddPCR detected multiple types of OaPV DNA in seven cases, whereas the qPCR failed to detect multiple infections. This study is the first to provide evidence of the presence of OaPV types 1, 3, and 4 DNA in a subset of equine sarcoids. The comparative detection approach underscores the superior sensitivity of ddPCR compared to that of qPCR.

Figure 1

OaPV, ovine papillomavirus; ddPCR, digital droplet polymerase chain reaction. Detection rates of OaPV single infections using ddPCR. McNemar's test indicated a statistically significant OaPV3 percentage (p ≤ 0.05).

Figure 2

OaPV, ovine papillomavirus; qPCR and ddPCR. (a) qPCR curves (left) and the relative rain plots of the ddPCR (right) for OaPV1, (b) OaPV3, and (c) OaPV4 genotypes. Positive plots are represented in blue, whereas negative droplets are in gray. For each OaPV, two positive samples from equine sarcoid, a positive control, and a negative control are also shown.

Figure 3

Detection of OaPV3. (a) virtual gel of OaPV3 obtained by the QIAxcel system showing positive samples from the equine sarcoid (Lines 1 and 2), the artificial positive (Line 3), and negative (no template) (Line 4) controls. (b) visualization of OaPV3 electropherograms after the electrophoretic run. (c) 100% identity between the sequences of the OaPV3 Seq amplicons, and the sequences reported in GenBank (accession number NC_038516.1).