Nested Polymerase Chain Reaction Validated for Sensitive Detection of Bovine Leukemia Virus in Blood Samples From Brazilian Cattle Herds: Comparison With Conventional Elisa and Agar Gel Immunodiffusion Methods

Abstract

A sensitive and specific nested polymerase chain reaction (nested PCR) for detection of bovine leukemia virus (BLV) in blood samples was evaluated as a reliable assay for accurate diagnosis of naturally infected animals under field conditions. The standardized nested PCR relies on the identification of BLV DNA fragments of 340 or 444 bp produced by applying 2 different pairs of primers sets to the proviral env gene. This protocol was validated by testing 40 bovine blood samples from a BLV-positive dairy herd, and by comparing results with serological diagnosis provided by agar-gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). The nested PCR protocol was able to discriminate infected animals from that ones serodiagnosed as negative. This method also showed applicability for other body fluids or tissue samples. Investigation of 40 blood samples from a dairy herd showed that 37 animals were positive in ELISA, 36 in nested PCR, and only 25 in AGID. The nested PCR has demonstrated a good agreement in the results with ELISA up to 95%. These results demonstrated that nested PCR could be indicated for an early, sensitive and direct detection of BLV-infection in naturally infected cattle. Compared to the serological tests, this method proved to be the most suitable for identification of BLV-infected cattle with low, transient or without BLV-antibody titers. Moreover, this assay can be used to confirm or exclude BLV-infection in asymptomatic cattle or with doubtful serological results. This nested PCR is a valuable assay for prompt epidemiological investigations of BLV as well as for surveillance of animal health for bovine leukosis.