A multiplex PCR for viruses associated with exanthematic and vesicular disease in cattle

Exanthematic and papulo-vesicular lesions in the udder and teats of milking cows are fairly common in some Brazilian dairies, especially those with poor sanitary conditions and hand milking. The orthopoxvirus Vaccinia virus (VACV) and the parapoxviruses Pseudocowpox virus (PCPV) and Bovine popular stomatitis virus (BPSV) have been frequently associated with such conditions. Elsewhere, Bovine herpesvirus 2 (BoHV-2) has also been associated with similar clinical signs. Thus, we herein describe a conventional multiplex PCR designed to detect the genome of these viruses in clinical samples while differentiating among them by amplicon size. For this, primer sets targeting the orthopoxvirus vascular growth factor (amplicon size 292bp), PCPV (374bp) and BSPV (607bp) B2L genes, and the BoHV-2 DNA polymerase gene (138bp) were selected. The chosen primers anneal within the same temperature range and do not interfere with each other during the PCR amplification. PCR conditions were initially standardized for each agent in individual PCR reactions firstly using the target virus as positive control followed by using a mixture of all four virues. Lastly, a multiplex PCR containing the four sets of primers was set up to amplify all four targeted viruses in one reaction. The multiplex PCR was able to detect DNA extracted from cell culture supernatants containing 20 TCID₅₀ of BoHV-2 and 50 TCID₅₀ of VACV. Further, the test could detect the viral genomes in 1:10, 1:50 and 1:1000 dilutions of total DNA extracted from clinical specimens (e.g. scabs, crusts) of natural cases (PCPV, VACV and BPSV) and 1:10 dilutions of DNA extracted from scabs collected from BoHV-2 experimentally infected cattle. A possible amplification of other orthopoxviruses, predicted by in silico analysis, was considered to not represent an important pitfall since these are exotic in Brazil, very rare, or viruses not associated with cattle. For definitive agent identification amplicon sequencing needs to be conducted. Thus, this multiplex PCR seems suitable for initial detection and identification of the agents involved in exanthematic and vesicular disease, providing a sensitive and specific diagnosis for such conditions in dairy cows.