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).