Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Anti-BLV antibodies in whey correlate with bovine leukemia virus disease progression and BoLA-DRB3 polymorphism.

Introduction: Bovine leukemia virus (BLV) belongs to the family Retroviridae and is a causative agent for enzootic bovine leucosis, the most common neoplastic disease affecting cattle worldwide. BLV proviral load (PVL) is associated with disease progression and transmission risk but requires blood collection and quantitative PCR testing. Anti-BLV antibodies in whey have been used as a diagnostic tool for BLV infection; however, quantitative utilization has not been fully investigated. Furthermore, bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and PVL, but its effect on anti-BLV antibody levels in whey from BLV infected dams is unknown. Therefore, we aimed to investigate whether it is possible to correctly predict PVL in the blood and milk based on the amount of anti-BLV antibodies in milk, and whether the BoLA-DRB3 alleles associate with the amount of anti-BLV antibodies in milk. Methods: We examined whey from 442 dams from 11 different dairy farms located in 6 prefectures in Japan, including susceptible dams carrying at least one BoLA-DRB3* 012:01 or * 015:01 allele related with high PVL, resistant dams carrying at least one BoLA-DRB3 * 002:01, * 009:02, or * 014:01:01 allele related with low PVL, and neutral dams carrying other alleles. Results: First, our results provided compelling evidence that anti-BLV antibody levels in whey were positively correlated with the anti-BLV antibody levels in serum and with BLV PVL in blood and milk, indicating the possibility of estimating BLV PVL in blood and milk by measuring anti-BLV antibody levels in whey. Thus, our results showed that antibody titers in milk might be effective for estimating BLV transmission risk and disease progression in the field. Second, we demonstrated that anti-BLV antibody levels in whey from BLV resistant dams were significantly lower than those from susceptible and neutral dams. Discussion: This is the first report suggesting that the BoLA-DRB3 polymorphism affects anti-BLV antibody levels in whey from BLV-infected dams. Taken together, our results suggested that anti-BLV antibody levels in whey, measured by enzyme-linked immunosorbent assay, may be a useful marker to diagnose the risk of BLV infection and estimate PVL in blood and milk.