Circulating Cell-Free DNA of Bovine Leukemia Virus: A Promising Biomarker for Enzootic Bovine Leukosis

Bovine leukemia virus (BLV) causes Enzootic bovine leukosis (EBL) in approximately 1%-5% of infected cattle after a long latent period. Few biomarkers effectively distinguish non-EBL from EBL cattle. Given the rapid turnover of tumor cells, we hypothesized that cell-free DNA (cfDNA) in plasma could serve as a more effective biomarker for EBL diagnosis. We measured the proviral load (PVL) in whole blood and plasma by quantitative PCR targeting LTR and pol. Consistent with previous reports, PVL levels in whole blood in EBL cattle were generally higher than those in non-EBL with some overlap between these two groups. In contrast, PVL in plasma clearly distinguished non-EBL from EBL ones. The receiver operating characteristic analysis showed plasma PVL perfectly discriminated EBL from non-EBL (100% sensitivity and specificity), while whole-blood PVL achieved 70% sensitivity and 30% specificity. Additionally, length of PCR products played a role in PVL detection sensitivity in plasma. We compared the complete BLV sequence between genomic DNA from lymphoma tissue and cfDNA in plasma and found that the predominant BLV sequences were highly similar between them. By assessing the major tumor clone burden based on unique integration sites, we found that BLV cfDNA derived more from tumor clones in the tissues than from peripheral blood mononuclear cells (PBMCs). These data support the idea that BLV in cfDNA primarily originates from tumor cells in EBL cattle. These findings demonstrated that cfDNA could be a better indicator for EBL diagnosis, improving early detection and more timely intervention to reduce the economic loss in the meat and dairy industry.