Cy415

BLV-infected cattle with high proviral load (HPL) in blood were selected for this study. It was reported that BLV-infected cattle with HPL in blood were considered as cattle at high risk to be BLV spreaders and might be one of the factors of disease progression [20 (link)]. BLV proviral load was measured by using 100 ng of WBC DNA by real-time PCR (qRT-PCR). The amplification was carried out in a reaction mixture containing 12.5 µL of 2× CycleavePCR Reaction Mix (CY510, Takara Bio), 5 µL of probe/primer/positive control for BLV (CY415, Takara Bio), 5 µL of a template DNA sample, and PCR grade water to increase the volume up to 25 µL. For the proviral quantification, BLV tax gene was used as a control from the kit (CY415, Takara Bio) and BLV proviral DNA was measured by a Thermal Cycler Dice Real Time System III (TP970, Takara Bio) according to the manufacturer’s instructions. After the measurement, BLV proviral copies of > 5000/100 ng of WBC DNA was considered HPL in BLV-infected cattle (Table 1). Hematology test, detection of serum antibodies against BLV, detection of BLV provirus, and measurement of BLV proviral load were conducted by the Gifu Central Livestock Hygiene Service Center (Gifu, Japan).