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.