Tracheal and cloacal swabs were taken from each bird at days 2, 4, 6, and 8 after challenge and they were placed in a 3.0 mL UVT tube (Becton, Dickinson, NJ). Blood samples from each bird were collected before each booster as well as two weeks after the second booster prior to challenge. Each blood sample was collected in a separating blood tube and serum was separated by placing the tubes at 37 • C for 1 h then at room temperature overnight followed by a 5 to 10 min centrifugation at 1000 rpm at 4 • C. Then, the collected serum samples were stored at −20 • C until analysis.