Genetic diversity of bovine viral diarrhoea viruses (BVDV) in Denmark during a 10-year eradication period.

A 243 base-pair fragment of the 5'- untranslated region (5'-UTR) of bovine viral diarrhoea virus (BVDV) was RT-PCR amplified from tissue samples (after one passage) or from plasma collected from Danish cattle in 1962 (1), 1993 (7), or in 2002-03 (28) when BVD was almost extinct as a result of a 6-year eradication programme. The PCR products were sequenced and phylogenetically analysed. All 36 samples were BVDV species 1 (BVDV-1), 29 sequences belonged to the BVDV 1d subtype, 6 to the BVDV 1b subtype, and one sequence to the BVDV 1e subtype. While all samples from 1993 and 1962 were of 1d subtype, the samples collected in 2002-2003 belonged to 1d (22 samples), 1b (5 samples) and 1e (1 sample) subtypes. In five herds, materials from two animals were obtained for PCR analysis. In four of five herds the sequences of the two viruses were identical, but in one herd the obtained sequences belonged to two different subtypes. Routine analysis detected 11 PI calves older than 2 months of age. For early detection of infected calves it is recommended that antigen ELISA be replaced by PCR detection. Here we present the first sequence analysis of Danish BVDV strains.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at -5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%-67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

A retrospective evaluation of a Bovine Herpesvirus-1 (BHV-1) antibody ELISA on bulk-tank milk samples for classification of the BHV-1 status of Danish dairy herds.

Bulk-tank milk samples analysed in a Bovine Herpesvirus-1 (BHV-1) blocking ELISA are still in use in the Danish BHV-1 programme as a tool to classify dairy herds as BHV-1 infected or BHV-1 free herds. In this retrospective study, we used data from the Danish BHV-1 eradication campaign to evaluate performance characteristics of the BHV-1 blocking ELISA in 1039 BHV-1-seropositive and 502 repeatedly BHV-1-negative dairy herds using the results of blood testing of the individual animals as the true infection status. At a cut-off value of 30% blocking reaction, the herd-level relative sensitivity and relative specificity were 82 and 100%, respectively. The herd-level relative sensitivity depended on the within-herd prevalence of seropositive cows and the cut-off value in the assay, but not on the time interval (up to 90 days) between the collection of the bulk-tank milk sample and the individual serum samples. The BHV-1 blocking ELISA on bulk-tank milk could detect seropositive herds (few), with prevalence proportions as low as one seropositive cow out of 70 cows.

Reintroduction of bovine herpes virus type 1 into Danish cattle herds during the period 1991-1995: a review of the investigations in the infected herds.

In Denmark a programme for the systematic eradication of bovine herpes virus 1 (BHV-1) was completed during the years 1984 to 1991, but outbreaks due to new introductions of BHV-1 were seen. Between January 1991 and May 1994, 22 herds became infected with BHV-1, all located closely to the German border. In 1995, 61 herds were detected BHV-1 antibody positive, but they were situated in many different parts of Denmark. In order to find the source of infection owners of infected herds were interviewed, and restriction fragment pattern analysis (RFP-analysis) was performed on virus isolates from the herds with clinical outbreaks. Isolates from clinical outbreaks up to 1995 were identified as a Cooper-like strain, while 2 of those in 1995 had characteristics of a "new" strain, which had never before been identified in Denmark or elsewhere in Europe. In the described situation different transmission routes for virus seemed possible. One being a sporadic introduction of virus due to accidental contact with infected cattle near the German border or maybe due to an airborne transmission of virus over longer distance. The other, presumably a result of import of an infected animal despite the national regulations. The latter, due to an extensive trade pattern, resulted in the introduction of infected cattle into 51 BHV-1 seronegative cattle herds.

A contribution to the systematization of bovine herpesvirus 1 based on genomic mapping by restriction fragment pattern analysis.

Fourteen isolates of bovine herpesvirus 1 (BHV-1) found representative of more than 100 isolates studied, were compared by restriction fragment pattern analyses and molecularly characterized. A number of evolutionary links between the variants originally associated with infectious bovine rhinotracheitis and the variants originally associated with infectious pustular vulvovaginitis were identified. These findings, as well as the lack of any correlation between genome type and clinical manifestation, confirm that there is no phylogenetic basis for a distinction between groups of strains associated with genital and respiratory disease. Two attenuated vaccine strains can be identified as deviating from field isolates.

Mycotic and algal bovine mastitis in Denmark.

A one-year examination of mammary secretions (n = 2,896) from Danish cattle with clinical or subclinical mastitis revealed 45 strains of fungi and algae. The strains originated from 44 mammary secretions of 42 cows in 40 herds. The following species of fungi were identified: Candida catenulata (n = 2), Candida kefyr (n = 6), Candida krusei (n = 17), Candida rugosa (n = 6), Candida tropicalis (n = 3), Candida valida (n = 1), Geotrichum capitatum (n = 5). The algal species Prototheca zopfii was demonstrated in five samples.