After all challenges, the swabs from all the birds were tested individually. to 30% in turkey poults (6). It is now known that the European isolates of APV belong to subgroups A, B, and D and that the US isolates belong to subgroup C (7C9). Since killed vaccines against APV were found not to be effective (10), the main focus in the US has been on the development of live, attenuated vaccines. Our laboratory has reported on the development of live, attenuated APV vaccines with 2 different approaches: serial propagation of APV in cell culture (11) and cold adaptation of APV (12). Experimentally, the cold-adapted strain protected 2-wk-old turkeys against virulent APV challenge Cefamandole nafate (12). In this paper we report on the duration of immunity afforded by a single dose of coldadapted APV vaccine. The APV strain APV/MN/turkey/1-a/97 (also called APV/MN-1a) was isolated from a respiratory disease outbreak in Minnesota (1) and used to develop the cold-adapted strain, as described earlier (12). Briefly, APV/MN-1a was blind-passaged in chicken embryo fibroblasts (7 passages) and Vero cells (34 passages). The virus, designated P41 (13), was developed as a cold-adapted strain Cefamandole nafate through growth at successively lower temperatures (35C, 33C, and 31C) for 8 passages each, until it was finally able to grow Cefamandole nafate at 31C (12). The cold-adapted virus was plaque-purified (to a titre of 106.3 plaque-forming units [pfu]/mL) before use in this study. A virulent strain of APV (APV/Mn-2a) at 13th cell culture passage in Vero cells (titre 104.3 tissue culture infective dose [TCID]50/mL) was used as the challenge virus. We obtained 140 tom turkeys from a commercial APV-free hatchery. The birds were reared in animal housing facilities at the College of Veterinary Medicine, University of Minnesota, St. Paul, throughout the study. At 1 wk of age, 120 birds were randomly divided into 2 groups of 60 birds each (groups 1 and 2). To determine APV serostatus before the experiment, we took blood from the remaining 20 birds, which were then killed. The birds in group 1 were inoculated with 50 L of vaccine in each eye and each nostril (200 L/bird); thus, each bird received 3.9 105 pfu. The birds in group 2 were inoculated with mock-infected Vero cell culture fluid. At 3, 7, 10, and 14 wk after inoculation, 15 birds from each group were transferred to another 2 rooms and challenged by inoculation with the virulent virus (50 L in each eye and nostril). Ten days after each challenge, blood was drawn and the birds were then killed. The 3 main criteria for protective efficacy of the vaccine after challenge were absence of clinical signs in the Cefamandole nafate vaccinated birds, lack of demonstrable APV-RNA in the vaccinated birds 5 d after challenge, and development of an anamnestic antibody response in the vaccinated birds 10 d after challenge. After vaccination and after each challenge, the birds were monitored for the appearance of clinical signs. Clinical scores were assigned as described previously (11). At 5 d after vaccination, choanal swabs from all 120 birds in groups 1 and 2 were collected in Eagles minimum essential medium containing 1% fetal bovine serum and antibiotics (150 IU/mL of penicillin, 150 g/mL of streptomycin, 50 g/mL TNFSF10 of neomycin, and 1 g/mL of amphotericin B). The samples were pooled (15 samples in each pool) and examined by reverse transcription polymerase chain reaction (RT-PCR) for APV-RNA (14). In addition, 5 d after each challenge, choanal swabs were collected from the vaccinated and challenged birds (= 15 at each time) and from the nonvaccinated and challenged birds (= 15 at each time).