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

A bovine viral diarrhoea/mucosal disease (BVD/MD) control and eradication p rogram was introduced in Lower Austria in 1996, according to the Swedish mo del. An important risk factor for BVD transmission under local conditions i s 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 erad ication and steps were taken to improve a commercially available antigen-ca pture ELISA (Ag-ELISA) by modifying the method for leukocyte preparation an d 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 simplifi ed and used on pooled blood samples to facilitate larger sample throughputs . RT-PCR positive pools were analysed individually to identify infected ani mals. 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 geneti cally typed by PCR product sequencing; special attention being paid to RT-P CR 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. (C) 2001 Elsevier Science B.V. All rights reserv ed.