BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status

Abstract

BackgroundBovine leukemia virus (BLV) is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests.ResultsBLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lew et al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA)-DRB3 genotypes.ConclusionsOur results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is useful tool for evaluating the progression of BLV-induced disease. BLV-CoCoMo-qPCR allows us to monitor the spread of BLV infection in different viewpoint compared with classical serotest.

Detection and molecular characterization of bovine leukemia virus in Philippine cattle

“…Second, the present results clearly indicate that the sensitivity of BLV-CoCoMo-qPCR-2 was higher than that of nested PCR because we found a number of cattle on Leyte, Iloilo and Luzon Islands that were BLVpositive as determined by the BLV-CoCoMo-qPCR-2 but were negative for BLV provirus as determined by nested PCR. This result showed the same tendency as discussed in our previous publications [7,12], showing that our original BLV-CoCoMo-qPCR is highly specific and sensitive and able to detect BLV in samples that are negative by the nested PCR assay. In addition, our previous paper [7] demonstrated that the positive rate for the nested PCR in cattle correlated with the proviral load determined by the original BLV-CoCoMo-qPCR as follows: 1) positive rates for the nested PCR ranged from 62.9 % to 98.5 % among animals with proviral copy numbers ranging from 10 0 to 10 4 copies per 10 5 cells, and 2) the positive rate for nested PCR was 100 % in cattle with high proviral loads ([10 4 copies per 10 5 cells).…”

“…This result showed the same tendency as discussed in our previous publications [7,12], showing that our original BLV-CoCoMo-qPCR is highly specific and sensitive and able to detect BLV in samples that are negative by the nested PCR assay. In addition, our previous paper [7] demonstrated that the positive rate for the nested PCR in cattle correlated with the proviral load determined by the original BLV-CoCoMo-qPCR as follows: 1) positive rates for the nested PCR ranged from 62.9 % to 98.5 % among animals with proviral copy numbers ranging from 10 0 to 10 4 copies per 10 5 cells, and 2) the positive rate for nested PCR was 100 % in cattle with high proviral loads ([10 4 copies per 10 5 cells). Therefore, cattle that were BLVpositive as determined by the BLV-CoCoMo-qPCR-2 but were negative for BLV provirus as determined by nested PCR may have a very low copy number of BLV, which cannot be detected by nested PCR.…”

“…The above findings suggest that the BLV provirus remains integrated in cellular genomes [6,7], even in the absence of detectable BLV antibodies. After infection, BLV expression in cattle is blocked at the transcriptional level during the latent period [8][9][10].…”

“…Detection and genetic variation analysis of BLV isolates are important for diagnostic purposes and vaccine development. Therefore, we recently developed a new quantitative real-time PCR method using coordination of common motifs (CoCoMo) primers to measure the proviral load of both known and novel BLV variants in BLV-infected animals [7,12,13]. The assay was highly effective in detecting BLV in cattle from a number of international locations.…”

BLV-CoCoMo-qPCR-2: improvements to the BLV-CoCoMo-qPCR assay for bovine leukemia virus by reducing primer degeneracy and constructing an optimal standard curve

“…BLV-infected cows produce less milk, with industry losses in the US dairy industry estimated at $525 million per year [14]. BLV infection in cattle is usually identified using serological methods such as enzyme-linked immunosorbent assay (ELISA), Passive hemagglutination test (PHA), or agar-gel immunodiffusion tests (AGID) [7]. However, these tests cannot allow judgments as to the degree of BLV-induced disease progression [7].…”