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.

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.

The proviral load (PVL) of the bovine leukemia virus (BLV) is a useful index for estimating disease progression and transmission risk. Real-time quantitative PCR techniques are widely used for PVL quantification. We previously developed a dual-target detection method, the “Liquid Dual-CoCoMo assay”, that uses the coordination of common motif (CoCoMo) degenerate primers. This method can detect two genes simultaneously using a FAM-labeled minor groove binder (MGB) probe for the BLV long terminal repeat (LTR) region and a VIC-labeled MGB probe for the BoLA-DRA gene. In this study, we evaluated the diagnostic and analytical performance of the Dual-CoCoMo assay targeting the LTR region by comparing its performance against the commercially available Takara multiplex assay targeting the pol region. The diagnostic sensitivity and specificity of the Liquid Dual-CoCoMo assay based on the diagnostic results of the ELISA or original Single-CoCoMo qPCR were higher than those of the Takara multiplex assay. Furthermore, using a BLV molecular clone, the analytical sensitivity of our assay was higher than that of the Takara multiplex assay. Our results provide the first evidence that the diagnostic and analytical performances of the Liquid Dual-CoCoMo assay are better than those of commercially available multiplex assays that target the pol region.

Bovine leukemia virus (BLV), the most prevalent neoplastic disease of cattle worldwide, is the causative agent of enzootic bovine leukosis. Polymorphisms in the bovine leukocyte antigen (BoLA)-DRB3 gene can influence host immune responses to pathogens, including BLV. However, the associations between specific BoLA-DRB3 alleles, BLV proviral load (PVL), a useful index for estimating disease progression and transmission risk, and BLV infection in Chinese cattle remain unknown. In this study, we identified 28 previously reported alleles in 289 Holstein cattle from Shandong Province, China, using polymerase chain reaction sequence-based typing. We further investigated whether BoLA-DRB3 polymorphisms influenced infection status and identified BoLA-DRB3*011:01 as an allele associated with susceptibility to BLV infection. An association analysis of allele frequencies between cattle with high and low PVL demonstrated that BoLA-DRB3*014:01:01 was significantly associated with low PVL. Farms with a higher frequency of cattle carrying BoLA-DRB3*014:01:01 had lower mean PVL values than farms with a lower frequency, indicating that resistant alleles are linked to low PVL levels. To our knowledge, this is the first study to demonstrate that BoLA-DRB3 polymorphisms associate with differential susceptibility to BLV infection and PVL in Holstein cattle in China. These findings may contribute to BLV control and eradication efforts through genetic selection.

Bovine leukemia virus (BLV) is widespread globally and causes economic losses in the cattle industry. BoLA-DRB3 is a polymorphic gene associated with the BLV proviral load (PVL), which correlates with disease progression and transmission risk. However, the distribution of BoLA-DRB3 alleles in semen and their potential impact on the PVL of progeny remain unclear. Here, we investigated whether BLV susceptibility linked to BoLA-DRB3 alleles in semen is inherited by progeny. We analyzed 178 commercial semen samples from Japanese Black sires and identified 20 BoLA-DRB3 alleles and 70 genotypes. The susceptible allele DRB3*016:01 was the most frequent (26.4%), whereas resistant alleles DRB3*011:01 (5.3%) and DRB3*009:02 (0.6%) were rare. Subsequently, we collected blood samples from 200 progeny produced by artificial insemination using 36 of the 178 semen samples. Progeny derived from semen carrying at least one susceptible allele and no resistant alleles had significantly higher PVL in the blood than those derived from semen containing at least one resistant allele. These findings demonstrate that BLV susceptibility is inherited via BoLA-DRB3 alleles in semen and highlight the potential of BoLA-DRB3 alleles as valuable markers in breeding strategies aimed at mitigating BLV infection and transmission.

Bovine leukemia virus (BLV), an oncogenic retrovirus of the genus Deltaretrovirus, causes enzootic bovine leukosis (EBL), the most prevalent neoplastic disease in cattle and a major source of economic loss. While BLV prevalence has been studied in commercial breeds, data on native Latin American cattle remain limited. This study assessed BLV infection and proviral load in 244 animals from six native breeds: Argentine Creole (CrAr), Patagonian Argentine Creole (CrArPat), Pampa Chaqueño Creole (CrPaCh), Bolivian Creole from Cochabamba (CrCoch), Saavedreño Creole (CrSaa), and Siboney (Sib), sampled across Argentina, Bolivia, Paraguay, and Cuba. BLV-CoCoMo-qPCR-2 assay detected BLV provirus in 76 animals (31.1%), with a mean load of 9923 copies per 10⁵ cells (range: 1–79,740). Infection rates varied significantly by breed (9.8% in CrAr to 83.8% in CrPaCh) and country (15.6% in Argentina to 83.8% in Paraguay) (p = 9.999 × 10⁻⁵). Among positives, 57.9% exhibited low proviral load (≤1000 copies), and 13.2% showed moderate levels (1001–9999), suggesting potential resistance to EBL progression. This is the first comprehensive report of BLV proviral load in Creole cattle across Latin America, offering novel epidemiological insights and highlighting the importance of native breeds in BLV surveillance.