Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program

A bovine viral diarrhoea/mucosal disease (BVD/MD) control and eradication program was introduced in Lower Austria in 1996, according to the Swedish model. An important risk factor for BVD transmission under local conditions is communal grazing where susceptible pregnant cattle from several herds may be mixed with unrecognised persistently infected (PI) animals. A reliable system for identification of PI animals is therefore essential for BVD eradication and steps were taken to improve a commercially available antigen-capture ELISA (Ag-ELISA) by modifying the method for leukocyte preparation and adjusting the negative cut-off value. A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) employing panpestivirus 324/326 primers targeting the 5'-untranslated region of the virus genome was also simplified and used on pooled blood samples to facilitate larger sample throughputs. RT-PCR positive pools were analysed individually to identify infected animals. Seven hundred eighty-six samples were tested by Ag-ELISA according to the instruction manual and 5324 samples with the modified method. All 6110 samples were retested by RT-PCR. The percentage of RT-PCR positive results with doubtful and negative Ag-ELISA samples significantly diminished using the modified method (from 4.71 to 0.82%). Selected BVD viruses were genetically typed by PCR product sequencing; special attention being paid to RT-PCR amplicons from samples which were negative or doubtful by ELISA. However, no correlation was found between the phylogenetic grouping of the viruses and the Ag-ELISA results.