Detection of infectious bronchitis virus strains similar to Japan in Taiwan

TSAI, Cheng-Ta; TSAI, Hsin-Fu; WANG, Ching-Ho

Eight tracheal samples positive for the IBV gene were selected for virus isolation, including S14, S41, S78, TC3, TC4, TC5, TC17 and TC38, using specific-pathogen-free (SPF) chicken embryos (Animal Health Research Institute, Chiding, Taiwan). Tracheal samples were frozen at −20°C. After thawing, the organs were homogenized in sterile TPB in a 1:10 ratio. The homogenate was then centrifuged at 1,500 rpm for 5 min. The supernatant was treated with a 1% mixture of ampicillin, streptomycin and amphotericin (Biological Industries, Kibbutz Beit-Haemek, Israel) for 1 hr at room temperature and then inoculated into the allantoic cavity of five 9-to-11-day-old SPF embryonated eggs. The eggs were incubated for 5 days and candled each day. After 5 days of incubation, the eggs were chilled at 4°C, and the allantoic fluid was collected. Each sample was given two blind passages before being considered negative. The presence of IBV in the allantoic fluid was detected using RT-PCR with NP1/NP2 primers [8] . Only two IBVs, S78 and TC3, were isolated. The IBVs were confirmed as IBV using sequencing. The IBV isolates were then used for one-direction neutralization tests with reference anti-IBV antisera.