3. Results
Overall, ddPCR detected BPV DNA in ~61.3% (103/168) of the blood samples examined, while real time qPCR revealed the virus in ~10.7% (18/168) of the same samples (p ≤ .001). Moreover, ddPCR showed BPV DNA in ~78.8% (93/118) of blood samples from goats that were in close contact with cattle and in 20% (10/50) of blood samples from goats living in closed pens without any contact with cattle (p ≤ .001), suggesting that animal husbandry practices contribute to virus spread and, very likely, to cross-species transmission of BPVs. In addition, ddPCR revealed a single BPV genotype in ~59.2% (61/103) and multiple genotypes in ~40.8% (42/103) of the goats harboring BPV DNA, while real time qPCR detected single genotypes in ~17% (17/103) and multiple genotypes in ~1% (1/103). Furthermore, real time qPCR failed to detect BPV DNA in samples tested negative by ddPCR. BPV2 and BPV13 were the most frequent genotypes detected in single infections, while only occasionally were BPV1 and BPV14 found alone. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes, and six (~14%) four genotypes. In contrast, real time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three and four genotypes. Co-infections by BPV2 and BPV13 were the most frequent infections. Table 1 summarizes these results, while Table 2 shows the BPV genotype frequencies. Furthermore, the ddPCR assay was also carried out for quantification of BPV, with the results showing remarkable distinctions between positive FAM (blue), and VIC (gree) and negative (grey) droplets (Figure 1-4). There were also differences in the fluorescence amplitude range of the background (negative) droplets among the BPV samples, that is 3,000-9,000 for BPV-1, 4,000-16,000 for BPV-2, 4,000-16,000 for BPV-13 and 2,000-6,000 for BPV-14. Table 3 reports the numbers of BPV DNA copies per microliter of blood samples from 168 clinically healthy goats.