Ultrasensitive detection and quantification of bovine Deltapapillomavirus in the semen of healthy horses

Abstract

BPV1, BPV2, BPV13, and BPV14 are all genotypes of bovine delta papillomaviruses (δPV), of which the first three cause infections in horses and are associated with equine sarcoids. However, BPV14 infection has never been reported in equine species. In this study, we examined 58 fresh and thawed commercial semen samples from healthy stallions. In 34 (58.6%), bovine δPV DNA was detected and quantified using droplet digital polymerase chain reaction (ddPCR). Real time quantitative PCR (qPCR) was able to identify bovine δPV DNA in 5 samples (8.6%). Of the BPV-infected semen samples, 15 were positive for BPV2 (~ 44.1%) on ddPCR and 4 (~ 11.7%) on qPCR; 12 (~ 35.3%) for BPV14 on ddPCR and 1 (~ 3%) by qPCR; 4 (~ 11.7%) for BPV1 on ddPCR, whereas qPCR failed to reveal this infection; 3 (~ 8.8%) for BPV13 on ddPCR; and BPV13 infection was not detected by qPCR. Our study showed for the first time that BPV14 is an additional infectious agent potentially responsible for infection in horses, as its transcripts were detected and quantified in some semen samples. Large-scale BPV14 screening is necessary to provide substantial data on the molecular epidemiology for a better understanding of the geographical divergence of BPV14 prevalence in different areas and how widespread BPV14 is among equids.

Keywords: Bovine papillomavirus; Commercial stallion semen; Droplet digital PCR (ddPCR); Healthy stallions; Real time quantitative PCR (qPCR); Semen.

Fig. 1

Percentages of single BPV genotype DNA detected by ddPCR. qPCR identified the DNA of two BPV genotypes, but did not reveal either BPV1 or BPV13 DNA. UL = undetectable levels.

Fig. 2

qPCR curves (A) and the relative rain plots of the ddPCR (B) for the four BPVs. QuantaSoft screenshots show the ddPCR results. Positive plots are represented in blue, whereas negative droplets are in grey. BPV1–D04(1): negative samples; H05(2): positive samples; and C05(3) is the positive control. BPV2–G04(1): negative samples; H04(2): positive samples; and A05(3) is the positive control. BPV13–A05(1): negative samples; A08(2): positive samples; and C09(3) is the positive control. BPV14–AO5(1): negative sample; B05(2): positive sample; and F02(3) is the positive control.