4. Discussion
To our knowledge, this study was the first to detect and quantify highly pathogenic bovine δPVs from clinically normal goats using the sensitive ddPCR approach. We previously showed that this approach has the highest sensitivity and specificity for detecting and quantifying bovine δPVs in cattle (De Falco et al., 2020).
Overall, ddPCR detected BPV E5 DNA in a greater percentage of the blood samples than real time qPCR, and ddPCR quantified the number of E5 DNA copies per microliter of blood for all four bovine δPV genotypes. In particular, BPV genotype detection was significantly more common in goats That lived in close contact with cattle suffering from anatomoclinical δPV-related pathology than in those from closed pens that had no contact with cattle. This suggests that cattle are the major source of bovine δPVs in goats. Furthermore, the goats harboring a higher prevalence of δPVs in the blood lived with cattle on lands contaminated by bracken fern (Pteridium aquilinum ), so they may have ingested this plant. The major toxic substance of bracken fern is ptaquiloside, which impairs the immune system and has recently been detected in healthy goats (Virgilio et al., 2015). Therefore, bracken fern may also facilitate cross-species transmission of δPVs from cattle to goats.
We compared diagnostic sensitivity between ddPCR and real time qPCR by using them to evaluate the same liquid biopsy. The results showed that ddPCR had superior sensitivity to real time qPCR, which is believed to be the gold standard for measuring papillomavirus DNA (Isaac et al., 2017). All differences in the detection of circulating BPV E5 DNA were significant, confirming that ddPCR is a more reliable approach. Therefore, ddPCR should confer markedly improved diagnosis of BPV infection over both PCR and real time qPCR. The present study clearly demonstrated that ddPCR outperforms real time qPCR in terms of the sensitivity, specificity, and reproducibility of BPV detection, similar to recent studies that focused on oncogenic human papillomavirus (HPV) detection (Biron et al., 2016; Carow et al., 2017). Therefore, as for human samples (Cheung et al., 2019), ddPCR could be a diagnostic procedure capable of detecting otherwise undetectable BPVs.
Our study raises important questions that warrant further investigation. Outbreaks of cutaneous and bladder diseases associated with bovine δPVs occur in small ruminants such as sheep (Mazzucchelli-de-Souza et al., 2018; Roperto et al., 2018; Savini et al., 2020). No clinical manifestations of bovine δPV infections have hitherto occurred in goats. Therefore, it is unlikely that bovine δPVs can cause infections in goats that result in clinical symptoms. However, the present study suggested that goats play a major role in the epidemiology of the virus, as they may serve as an environmental reservoir. Indeed, bovine δPVs were found in goats living in close proximity with cattle more than in goats that had no contact with cattle.
It may be that ddPCR is approximately 500 times more sensitive than real time qPCR to detect low level analyte (Suo et al., 2020). As such, ddPCR can detect clinically relevant changes in viral load and could provide valuable insight to inform the development prophylactic measures and/or improve treatment outcomes. Very precise quantitation of very low viral copy numbers allows latent BPV DNA reservoirs to be monitored. Therefore, ddPCR will allow researchers to better understand BPV epidemiology. To develop appropriate prophylactic and/or therapeutic tool, as well as to determine the interspecies transmission potential and evolution of BPVs, the BPV prevalence, distribution, and clinical consequences must be identified in different animal species (Dogan et al., 2018). We believe that this type of study is remarkable for bovine δPVs, which are the only BPVs responsible for cross-species transfection and infection (IARC, 2007).
In conclusion, the ddPCR technique allows low-abundance nucleic acid detection and is useful in the diagnosis of infectious diseases. The method has excellent precision and well defined accuracy in the quantitation of ddPCR making it useful for detecting low pathogen loads. The sensitivity and specificity of ddPCR are superior to those of real time qPCR in the clinical diagnosis of infectious diseases, including viral, bacterial diseases and parasitic infections. As such, ddPCR may be a better choice for clinical and epidemiological applications in the future. DdPCR is a valuable and reliable new technology with additional improvements in prospect, it is likely to become a useful tool in future BPV research. Therefore, ddPCR could be used to improve diagnostic procedures, accurately identify the genotypic distribution of BPVs, and allow researchers to better understand the territorial divergence of BPV prevalence. Such insights will improve our understanding of the molecular and ecological epidemiology of infectious diseases, including viral ones.