Splenic lymphocytes collected form the 8 vaccinated chickens were plated at 10 6 well -1 in 24-well plates with RPMI 1640 medium, before stimulation for 48 h using 100 mg mL -1 of the nine peptides generated from NP and the unrelated peptide N 71-78 . Cells were then collected and centrifuged at 1000 rpm for 5 min. The cell culture supernatants were then analysed to determine the chicken IFN-c using chicken IFN-c CytoSets TM (Invitrogen), according to the manufacturer's instructions [35] . The extinctions were measured at 450 nm with a micro ELISA reader (Bio-Rad). For each peptide, two copies of splenic lymphocytes from one chicken were treated and analysed simultaneously.