Serological analysis
All the serum samples were tested in
duplicate with PPD-B-ELISA as previously described (de Val Perez et al.,
2011; Garcia-Bocanegra et al., 2012; Perez de Val et al., 2017). The
96-well plate was coated with 100 μL bovine PPD (PPD-B, Harbin
Pharmaceutical Group Bio-vaccine Co. Ltd., China) which was prediluted
with carbonate/bicarbonate buffer to a final concentration of 2 μg/mL
and incubated overnight at 4°C. After 45-min blockade at 37°C with PBS
containing 0.05% Tween 20 (PBST) and 0.5% casein, serum samples
(diluted at 1/200 in PBST containing 1% casein) were added into 96-well
plate in duplicate and incubated for 1 h at 37°C. Then, 96-well plate
was added with protein A and protein
G conjugated peroxidase (Sigma, America) and incubated for 1 h at 37°C.
The protein A and protein G conjugated peroxidases were prediluted in
PBST containing 1% casein to final concentrations of 50 ng/mL and 100
ng/mL, respectively. The plate was washed six times with PBST after each
step during this procedure. Afterward, the plate was added with 100 μL
TMB substrate (Beyotime, China) and incubated for 20 min in the dark,
and then the reaction was stopped with 100 μL 1 N HCl. After incubation,
the Optical Density at 450 nm (OD450) was calculated as
the sample OD450 value minus the background
OD450 value (nonspecific absorbance in wells with no
antigen added). A sample was defined
as positive when the average OD450 value was higher than
the cut-off value (calculated as the mean of the background
OD450 plus 3 folds of standard deviation). All the
positive sera and 20 randomly selected negative sera were further tested
with PPD-A-ELISA which was the same as PPD-B-ELISA but coated with avian
PPD (PPD-A, Harbin Pharmaceutical Group Bio-vaccine Co. Ltd., China).