Sample processing and serological analysis:
Serum was obtained from blood after allowing clotting at room temperature for 2-5 hours, then centrifuged for 10 minutes at 1700x g to separate the serum from the cellular fractions. Serum samples were aliquoted and frozen at -20ºC until subsequent analyses.
The IFAT was carried out according to the protocol in Viro Vet Diagnostik laboratory (Giessen, Germany). Noninfected Madine Darby canine kidney (MDCK) as well as MDCK cells persistently infected with BoDV H1766 (MDCK/-BoDV) were cultured for 3 to 4 days with medium produced in-house [4,5 g/l Dulbecco’s Modified Eagle Medium (DMEM), 200μM L-Alanine, 225μM LAspartate, 933μM Glycine, 510 μM L-Glutamate, 217 μM, L-Proline, 184 μM, Hypoxanthine, 0,1 mg/l Biotine, 44mM NaHCO3] supplemented with 10% fetal bovine serum (FBS). Cells were trypsinized, suspended in the medium, diluted to a final amount of 10^6 cells / ml and spotted onto 10-well glass slides and cultured overnight. The slides were washed with PBS, fixed with anhydrous acetone at -20°C for 2 - 4 hours and dried. MDCK as well as MDCK/BoDV slides were incubated with horse sera diluted 1:10, 1:40, 1:160, 1:640 and 1:2560 in a humidity chamber at 37°C for 35 minutes. After three washes with PBS, Fluorescein isothiocyanate conjugated goat anti horse IgG (Dianova, Germany) was added to each well and the slide was incubated in a humidity chamber at 37°C for 35 minutes. The slides were washed with PBS and Aqua dd, mounted with PBS-glycerol under cover slips and examined for characteristic nuclear fluorescent pattern with a fluorescence microscope. The cut-off titre for a positive sample was 1:160, when the sample yielded no fluorescent pattern in the non-infected MDCK cell wells (Herzog, 1980). Titres of 1:10 to 1:40 are often considered questionable; however, they were regarded as negative for this study in order to easy the analysis of results.