Animal studies were conducted at the Central Veterinary Institute (CVI), Lelystad, under BSL3 conditions and after approval by the Animal Ethic Committee. In the first chicken experiment, 6 weeks old SPF white Leghorn chickens (Lohmann, Cuxhaven, Germany) were used (10 chickens per group). One group of animals was immunized twice (on day 0 and 21) by intramuscular (i.m.) injection (0.5 ml) of 10 mg For the next experiment, 70 one-day-old layer hens (white Leghorn) were purchased from a local breeder. The chickens had been vaccinated against Newcastle disease virus and infectious bronchitis virus at the age of one day according to the farm's routine and were raised in CVI's animal facility. At the age of 6 weeks, the birds were transported to the BSL-3 facility and allocated to 7 experimental groups of 10 birds each. The animals of six groups were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection of 10, 2 or 0.4 mg sH5 3 antigen adjuvanted with Stimune. When immunized twice, the same doses were given on day 0 and 21. As a challenge control, one group was mock-vaccinated twice (on day 0 and 21) with PBS in Stimune. Four weeks after vaccination (day 49), blood samples were taken and the chickens were challenged as above and observed daily for clinical signs during 14 days. Trachea and cloaca swabs were taken from each chicken on day 2, 4 and 7 p.i.