A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

Abstract

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID 50. BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID 50 per 50 μl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.