2. Materials and Methods
2.1 Ethics statement
Blood samples were collected from animals in public slaughterhouses during the mandatory ante-mortem clinical examination. All procedures performed in this study followed common good clinical practices and received institutional approval from the Ethical Animal Care and Use Committee of the University of Naples Federico II (PG/2017/ 0099607). All farmers were previously informed and in agreement with the purpose and methods used.
2.2 Liquid biopsy samples and DNA extraction
Blood samples from 103 apparently healthy 1- to 3-year-old sheep were collected from the jugular vein in vacutainers containing ethylenediaminetetraacetic acid (EDTA). A total of 40 samples were obtained from sheep living in Sardinia (Sar) (20) and Campania (Cam) (20), 48 samples from Calabria (Cal) (24) and Basilicata (Bas) (24), and 15 samples from Apulia (Apu). All sheep, excluding those from Cam, were from flocks that lived and shared the bracken fern-infested lands that they grazed on with pasture-residing cattle. Sheep from Cam were from flocks living in closed pens without any contact with other animals. Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Wilmington, DE, USA), according to the manufacturer’s instructions.
2.3. RT-qPCR
RT-qPCR was performed in a final volume of 20 μL containing 10 μL of TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 900 nM of each of the forward and reverse primers (Bio-Rad Laboratories, Hercules, CA, USA), 250 nM of the probe (Bio-Rad Laboratories), and 100 ng of the DNA sample. The primers and probes for the detection of four BPV genotypes (BPV-1, -2, -13, and -14) were used as reported elsewhere (De Falco et al., 2020). The reaction was performed on the CFX96 Real-Time System of the C1000 TouchTM Thermal Cycler (Bio-Rad Laboratories). The thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 58 °C for 60 s (acquiring FAM and VIC dyes). Each sample was analyzed in duplicate, and negative controls were included in all runs. Data acquisition and data analyses were performed using CFX MaestroTM (Bio-Rad Laboratories) software. The same samples used as positive controls for ddPCR were also tested using RT-qPCR.
2.4. DdPCR
For ddPCR, Bio-Rad’s QX100 ddPCR System was used according to the manufacturer’s instructions. The reaction was performed in a final volume of 20 μL containing 10 μL of ddPCR Supermix for Probes (no dUTP 2×; Bio-Rad), 0.9 μM primer, and 0.25 μM probe together with 5 μL sample DNA (100 ng). A black hole quencher was used in combination with FAM and VIC fluorescent dye reporters (Bio-Rad Laboratories). The reaction mixture was placed into the sample well of a DG8 cartridge (Bio-Rad Laboratories). A volume of 70 μL of droplet generation oil was loaded into the oil well, and droplets were formed in the droplet generator (Bio-Rad Laboratories). After processing, the droplets were transferred to a 96-well PCR plate (Eppendorf, Hamburg, Germany). PCR amplification was carried out on a T100 Thermal Cycler (Bio-Rad Laboratories) with the following thermal profile: hold at 95 °C for 10 min, 40 cycles of 94 °C for 30 s and 58 °C for 1 min, 1 cycle at 98 °C for 10 min, and ending at 4 °C. After amplification, the plate was loaded onto a droplet reader (Bio-Rad Laboratories) and the droplets from each well of the plate were read automatically. QuantaSoft software was used to count the PCR-positive and PCR-negative droplets to provide absolute quantification of the target DNA. Therefore, the ddPCR results could be directly converted into copies/µL in the initial samples simply by multiplying them by the total volume of the reaction mixture (20 µL) and then dividing that number by the volume of DNA sample added to the reaction mixture (5 µL) at the beginning of the assay. Samples with very few positive droplets were re-analyzed to ensure that these low copy number samples were not due to cross-contamination.
2.5 Statistical analysis
Differences in the proportions of detected cases were tested using the chi-square test by Campbell and Richardson (Richardson, 2011). Furthermore, regarding the significance relative to the number of copies of BPV DNA detected in sheep in the different regions, the t-test was used after adjusting for the Bonferroni multiple comparison correction of means. P-values ≤ .05 were considered to be statistically significant. All analyses were performed using R statistical software (The R Foundation, Vienna, Austria).
3. Results
Overall, our results showed that BPV DNA was found in 68 out of 103 blood samples (66%) from healthy sheep using ddPCR. The same liquid biopsies were also investigated using RT-qPCR, which revealed BPV DNA in approximately 9% of blood samples (Fig 1). In 42 of the positive samples (61.8%), a single BPV infection was observed (Fig. 2), 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%), and BPV-1 for two single infections (4.7%) (Fig. 3). Multiple BPV infections were seen in 26 (38.2%) positive samples. BPV coinfections caused by two genotypes were seen in 22 positive cases (84.6%), with dual BPV-2/BPV-13 infection being the most prevalent. BPV coinfections by triple and quadruple genotypes were detected in 11.5% (3/26) and 3.8% (1/26) of blood samples, respectively (Fig. 4). In sheep flocks that lived and shared lands with cattle, BPV DNA was detected in approximately 53% of blood samples collected in Apu (8/15), 75% of samples acquired in both Bas and Cal (18/24), and 100% of blood samples harvested from Sar (20/20). In sheep flocks from Cam that lived in isolated and closed pens without any contact with cattle, BPV DNA was detected in 20% of blood samples examined (4/20). The percentage differences in BPV infections in all sheep flocks with cattle contact were statistically significant compared to the percentage observed in sheep flocks without any contact with cattle, as the Campbell-Ricardson’s chi-square test resulted in a p-value < 0.05. Furthermore, in all geographical areas except for Apu, BPV-2 was the most prevalent genotype. BPV-13 and BPV-14, as well as BPV-1 were also observed. Furthermore, Apu BPV-14 showed very high numbers of copies/µL (mean value of 895.2); it was the most prevalent BPV genotype at a detection level of 40% in the examined samples (6/15) and a statistically significant Campbell-Ricardson’s chi-square test p-value < .05.
The overall quantification results showed that viral copy numbers/µL ranged from 76 to 568 for BPV-1 (mean value = 183.6 ), 65 to 3768 for BPV-2 (mean value = 397.1), 66 to 2112 for BPV-13 (246.5), and  76 to 1768 for BPV-14 (mean value = 447.5). When sheep flocks from Sar alone were considered, BPV-2 showed the highest copy numbers/µL with a mean of 1162. Using t-tests, the differences between the copy numbers of BPV-2 in Sar compared to the other means found in Cal, Cam, Bas, and Apu were statistically significant with p-values < .05. Indeed, after adjusting for the Bonferroni multiple comparison correction, their p-values were .003 (Sar-Cal), .04 (Sar-Cam), .002 (Sar-Bas), and .01 (Sar-Apu), respectively. Table 1 summarizes the quantitative data using QuantaSoft software, demonstrating the numbers of copies/µL in blood samples from sheep flocks located in the five regions in South Italy.