A FIELD-EVALUATION OF THE POLYMERASE CHAIN-REACTION PROCEDURE FOR THEDETECTION OF BOVINE LEUKEMIA-VIRUS PROVIRAL DNA IN CATTLE

A polymerase chain reaction (PCR) procedure that detects proviral bovi ne 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 i n 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 s eropositive by the agar gel immunodiffusion (ACID) test and in one per sistently seronegative cow. Infection in this cow was confirmed by she ep bioassay. Subsequent testing of the SA-PCR negative, seropositive a nimals using a double amplification PCR (DA-PCR) detected proviral BLV in eight of nine animals that were available for retesting. The PCR a ssay was also able to distinguish BLV infected calves from uninfected calves that were serologically positive because of the presence of col ostral antibody. Lymphocytes from all seropositive animals were cultur ed for determination of BLV antigen expression. Cultures from 37 of 62 SA-PCR positive animals produced detectable quantities of viral antig ens. However, antigen expression was not detected in cultures from ser opositive animals that were negative in the SA-PCR. In addition, in ex perimental transmission tests, inoculation of more than 10(6) lymphocy tes from these cows was required for sheep to become seropositive to B LV. 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 infe ction with a sensitivity comparable to that of the ACID test and the s heep 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 preca utions found to be essential to prevent false positive results due to contamination of samples with PCR product are likely to preclude the r outine use of PCR for diagnosis of BLV infection.