Then, diluted serum samples were added to each well and the plates were incubated for 2 h at 37◦C. Chicken antibod-ies were detected with an AP conjugated rabbit anti-chicken IgY(Sigma). The plates were washed four times and the reactionwas developed with a substrate solution consisting of 1 mg/mL p-nitrophenylphosphate (Sigma) in diethanolamine buffer, pH 9.8.Absorbance was measured at 405 nm in an ELISA reader (Tekan,Salzburg, Austria).