Results
OaPV DNA was detected in 100 of 128 PBMC samples (~78%) examined through ddPCR. Detection and quantification of OaPVs were performed by ddPCR. In particular, OaPV1 DNA quantification ranged from 0.22 to 34.4 copy number/μL; OaPV2 DNA from 0.23 to 14.92 copy number/μL; OaPV3 DNA showed a range from 0.25 to 6.22 copies per μL, and finally OaPV4 DNA from 0.25 to 12.03 copies per μL. These detailed results are listed in Figure 1 SuppInfo.
Among 100 OaPV positive samples, single infections were detected in 42 (42%) using ddPCR, whereas only 18 (18%) were detected using qPCR. Differences between the two molecular methods in detecting OaPV DNA were statistically significant, as shown by McNemar’s test (p <0.05).
OaPV1 and OaPV2 infections were the most representative being found in 22 (52.4%) and 16 (38%) single infections by ddPCR. On the other hand, qPCR revealed OaPV1 and OaPV2 infections in 9 (50%) and 8 (44.4%) samples, respectively. Hence, OaPV1 and OaPV2 infections were the most prevalent single infections as per results obtained from both the methods. OaPV3 and OaPV4 DNAs were found in two single infections (4.8%) by ddPCR, while qPCR revealed only a single OaPV3 infection (5.6%). No OaPV4 infection was detected by qPCR. Figure 1 summarizes the results obtained by ddPCR.
Differences in OaPV DNA genotype detection were statistically significant, as shown by the Cochran-Armitage test (p <0.05).
Table 2 summarizes the results of the coinfection studies. Coinfections were observed in 58 out of 100 (58%) positive samples by ddPCR. In particular, 35 (35%) were dual infections, and 23 (23%) were triple infections. Dual coinfections by OaPV1/2 were the most frequent being observed in 23/35 (~66%); coinfections by OaPV1/3, OaPV2/3, OaPV2/4, and OaPV3/4 were more rarely detected. Coinfections with OaPV1/2/3 were the most frequent triple infections, as observed in 18/23 samples (~78%). OaPV1/2/4 infections have also been reported. qPCR failed to detect most of the multiple coinfections and identified only seven dual infections. Furthermore, qPCR revealed only one genotype in many dual and triple coinfections confirmed by ddPCR. Triple coinfections were not observed by qPCR. However, ddPCR revealed that OaPV1 was the most prevalent genotype in multiple coinfections being detected in 52 of them. OaPV2, OaPV3, and OaPV4 were detected in 51, 29, and 7 samples, respectively. In the same multiple coinfections, qPCR revealed presence of the OaPV1, OaPV2, OaPV3, and OaPV4 genotypes in 13, 7, 2, and 1 samples, respectively.
PCR analysis, using DNA isolated from PBMC samples, detected amplicons using primers specific to all OaPV genotypes. Sequencing revealed the presence of DNA fragments with 100% identity with OaPV1 E5, OaPV2 L1, OaPV3 E7, and OaPV4 E6 DNAs reported in GenBank (accession number: U83594.1., U83595.1., NC_038516.1, and KX954121.1, respectively) (Figure 2).
One-step reverse transcription (RT)-ddPCR was performed on 34 randomized positive samples. We detected and quantified the transcripts of OaPV1 E5 and OaPV2 L1 as well as transcripts of OaPV3 E6, E7, and OaPV4 E6, which showed that all OaPV genotypes can be transcriptionally active in healthy cattle. The Bio-Rad system quantified mRNA in copies per μL. Samples were considered positive if they had at least three or more positive droplets at the same amplitude as the positive control (Figure 3). Details of this investigation are reported in Figure 2 SuppInfo.
Furthermore, we performed RT-PCR analysis of RNA from the PBMC samples. We detected amplicons, the sequencing of which showed 100% identity with OaPV1 E5, OaPV2 L1, OaPV3 E6 and E7, and OaPV E6 mRNAs reported in GenBank, thus validating the one step RT-ddPCR results (Figure 4).
OaPV coinfections were most prevalent in cattle that shared grasslands with sheep. In cattle from intensive dairy farms without any apparent contact with sheep, double coinfections with OaPV1 and OaPV2 were also observed (detected in 11 out of 30 examined PBMC samples). OaPV1 E5 and OaPV2 L1 DNAs were found in feed composed of grass hay and corn silage, which are known to be fed. ddPCR detected and quantified high copy numbers of OaPV1 DNA as it was found in grass hay samples (from 4.2 to 7.7 copies per µL) as well as in feed composed of maize silage (from 3.43 to 5.7/µL copy number). OaPV2 DNA was also detected in grass hay (up to 14.4 copies per µL) and corn silage samples (up to 10.9 copies per µL) (Figure 3A SuppInfo). PCR analysis performed on all these matrix samples revealed amplicons, and the sequencing of obtained DNA fragments showed 100% identity with OaPV1, and OaPV2 DNA deposited in GenBank (Figure 3B SuppInfo).