Digital droplet PCR for the detection and quantification of circulating bovine Deltapapillomavirus

Abstract

In this study, the digital droplet polymerase chain reaction (ddPCR) was used to quantify circulating bovine papillomavirus (BPV; genus: Deltapapillomavirus) DNA levels in blood samples from 25 clinically normal cows and 15 cows with chronic enzootic haematuria due to papillomavirus-associated bladder tumours. ddPCR detected BPV DNA in 95% of all the samples (i.e. in 24 of the clinically normal cows and 14 of the diseased animals), whereas quantitative real-time PCR (qPCR) detected it in only 57.5% of the same blood samples, with percentage differences between ddPCR and qPCR being statistically significant (p-value ≤ .05), according to chi-squared test. Furthermore, ddPCR detected BPV infections by a single genotype and by multiple genotypes in 37% and 63% of the cows, whereas qPCR detected these in 16% and 16%. Of the two assays, ddPCR was the more sensitive and accurate clinical diagnostic tool, allowing the detection of otherwise undetectable BPV genotypes, and consequently, a higher number of BPV co-infections. qPCR failed to detect many BPV co-infections by multiple genotypes. Therefore, ddPCR may be an essential tool for improving diagnostic procedures, allowing the identification of the genotypic distribution of BPV and a better understanding about the territorial divergence, if any, of the BPV prevalence in different areas. No significant differences in the blood viral load estimations were observed between the two animal groups, suggesting that the bloodstream could be a site of primary infection. Finally, as BPV DNA was detected in cows affected by non-invasive urothelial tumours, including papilloma and papillary urothelial neoplasms of low malignant potential, the circulating BPVs appeared to be independent of the status of urothelial neoplasms. Therefore, unlike in humans, circulating BPVs cannot be an actual prognostic marker of urothelial tumours in cows.

Keywords: blood; bovine papillomavirus; chronic enzootic haematuria; droplet digital polymerase chain reaction; quantitative real-time polymerase chain reaction.