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.