A field evaluation of the polymerase chain reaction procedure for the detection of bovine leukaemia virus proviral DNA in cattle

Abstract

A polymerase chain reaction (PCR) procedure that detects proviral bovine leukaemia virus (BLV) in peripheral blood mononuclear cell DNA was evaluated. Blood samples from all animals (164) in a commercial dairy herd with a 30% prevalence of BLV infection, and from 194 animals from BLV free herds were tested. The absence of any positive PCR results in animals from BLV free herds confirmed the specificity of the assay. Initial testing of the infected herd using a single amplification PCR (SA-PCR), detected BLV infection in 62 of 72 adult animals that were seropositive by the agar gel immunodiffusion (AGID) test and in one persistently seronegative cow. Infection in this cow was confirmed by sheep bioassay. Subsequent testing of the SA-PCR negative, seropositive animals using a double amplification PCR (DA-PCR) detected proviral BLV in eight of nine animals that were available for retesting. The PCR assay was also able to distinguish BLV infected calves from uninfected calves that were serologically positive because of the presence of colostral antibody. Lymphocytes from all seropositive animals were cultured for determination of BLV antigen expression. Cultures from 37 of 62 SA-PCR positive animals produced detectable quantities of viral antigens. However, antigen expression was not detected in cultures from seropositive animals that were negative in the SA-PCR. In addition, in experimental transmission tests, inoculation of more than 106 lymphocytes from these cows was required for sheep to become seropositive to BLV. These results suggest that the failure of the PCR assays to detect some seropositive animals was due to a low proportion of lymphocytes being infected with BLV in these animals. The DA-PCR detected BLV infection with a sensitivity comparable to that of the AGID test and the sheep bioassay. PCR assays may be an alternative to the sheep bioassay as an adjunct to serological testing for use in situations where it is essential to detect all infected cattle. However, the stringent precautions found to be essential to prevent false positive results due to contamination of samples with PCR product are likely to preclude the routine use of PCR for diagnosis of BLV infection.