Development of a PCR to diagnose BLV genome in frozen semen samples.

The sanitary and economic impact of BLV infection is associated with the interference in the international movement of cattle and their germ plasm. Although experimental data support the improbability that semen from BLV-positive bulls could infect recipient cows, restriction for commercialization of semen from infected animals is still present. The objective of this work was to standardize a PCR assay to diagnose the presence of BLV genome in frozen semen samples. The developed methodology involves the amplification of an internal fragment of gag gene. The limit of detection of this technique was six viral particles, using gag-PCR followed by hybridization analysis. Frozen semen samples from seropositive bulls were analyzed. It was possible to detect proviral DNA in 9 out of 173 samples. Additionally, a biological test in susceptible sheep was performed in order to evaluate the transmission of BLV genome by semen from seropositive animals. This data strongly suggest that semen from seropositive bulls that resulted negative by PCR can be used for artificial insemination (AI), accompanied by proper collection protocols. The development of this PCR assay constitutes a valuable diagnostic tool to determine the BLV-free status of frozen semen samples used for AI.