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).