3. Results
In summary, OaPV DNA was found in 126 of the165 blood samples examined (~76.4%) from healthy sheep using both ddPCR and real-time qPCR protocols; 39 sheep did not harbor any OaPV DNA. DdPCR detected OaPV DNA in 124 positive blood samples (~98.4.%) whereas real-time qPCR revealed OaPV DNA in 48 positive liquid biopsies (~38.1%) (Figure 1), 46 of which were shown to harbor OaPV DNA using two methods. Differences between the two molecular protocols in detecting OaPV DNA were statistically significant, as the McNemar’s test showed a p-value <0.05. Figure 2 shows the cycle threshold (Ct) for the qPCR results for both positive and negative samples. Data from qPCR were compared to those obtained via ddPCR performed on the same samples that correlated Ct and copy number obtained using qPCR and ddPCR, respectively (Supplemental Table S1).
Single OaPV infection was observed in 70 positive samples (~ 55.6%) whereas multiple OaPV coinfections were observed in 56 positive samples (~ 44.4%). DdPCR detected single infections in 51 samples; 18 single infections were detected by both ddPCR and qPCR. In only one case, qPCR detected DNA of an OaPV genotype, causing a single infection that ddPCR did not detect (Figure 3). Overall, OaPV1 DNA was detected in 12 out of 70 single infections (~17.1%) and OaPV2 DNA in 14 (20%). OaPV3 and OaPV4 were responsible for 19 (~27.1%) and 25 (~35.7%) single infections, respectively (Figure 4). Differences in the sensitivity of OaPV DNA detection were statistically significant as the Cochran-Armitage Test showed a p-value < 0.05. Both methods detected a greater number of positive samples to OaPV3 and OaPV4 than positive samples to OaPV1 and OaPV2.
OaPV coinfections caused by the two genotypes were observed in 31 positive samples harboring multiple OaPV DNA (~ 55.4%). DdPCR detected 30 double infections, with OaPV3/OaPV4 genotype combination being the most prevalent infection, as observed in 11 blood samples. In addition, five coinfections composed of OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, three OaPV1/OaPV3, and three OaPV2/OaPV4 were also detected. qPCR detected only four dual coinfections. Three of them were shown to have triple infections by ddPCR. In only one case, qPCR revealed a double infection in which ddPCR failed to detect it. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~ 42.8%) and only one (~ 1.8%) of 56 multiple infections, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent ones being seen in 12 (50%) blood samples harboring triple OaPV infections. Neither triple nor quadruple infection was observed by real-time qPCR. Table 2 summarizes the coinfection results.
The overall quantification results showed that viral copy number/μL ranged from 0.22 to 207 for OaPV1, 0.17 to 2.85 for OaPV2, 0.18 to 4.98 for OaPV3, and 0.28 to 12.72 for OaPV4. In samples positive for both assays, the copy number of ddPCR was correlated with the Ct of real-time qPCR because the higher the copy number, the lower was the Ct of qPCR. The detailed results are summarized in Supplemental Table S1.