Isolation And Preliminary Identification Of Turkey Coronaviruses

An acute, highly infectious disease was encountered in turkey poults at 15-28 days of age in a turkey farm in 2003. Clinical signs included water droppings and 10% ~ 20% mortality mostly. The lesions are limited to the hyperaemia and hemorrhage of duodenum and rectum, tumescence of cecum and yellow contents filled in intestines. It was suspected as Bluecomb disease. So in this study the viruses were isolated and identified in following methods.1. Livers, spleens and intestines obtained from the infected turkey poults were homogenized with 5 volumes of 0.01 M PBS containing 20 000 U/ml penicillin and20 000mg/ml streptomycin. Then the homogenate were incubated at 4℃ for 2 hours. After centrifuged at 5000×g for 20 minutes, the supernatant were obtained and inoculated into 15-day-old SPF chicken embryos via the allantoic cavity. The intestines and yolks were harvested separately from the second passages chicken embryos, and tested by HA with rabbit erythrocytes (titer was 2~6).2. After five successive passages, the yolk of chicken embryos were harvested 72 hours postinoculation, degreased by 2% polyethylene glycol (MW 6000), and concentrated with 10% polyethylene glycol. The intestines of chicken embryos were harvested and homogenized with 5 volumes of 0.01 M PBS containing 4 000 U/ml penicillin and 4000mg/ml streptomycin. The intestines homogenate were ultracentrifuged at 9000×g for 20 mins to remove the tissue debris. The viruses were concentrated with 10% polyethylene glycol in the same methods as above. Then the concentrations of the virus of yolk and intestine were separately added the top of a step gradient consisting 35%~55% of sucrose solutions, and ultracentrifugated at 91000×g for 2 hours. The opalescent bands with density of 1.156 to 1.260 g/cm~3 were collected and ultracentrifugated at 96 000×g for 2 hours to remove the sucrose solutions. The pellets containing viruses were obtained and stained with hosphotungstic acid, and examined by election microscopy. Several spherical or oval particles surrounded regularly by spaced petal-or pear-shaped projections were observed. The particle diameter is about 120 nm.3. The total RNA of infected chicken embryos was extracted, Turkey Coronaviruses S2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with designed primer. The products were identical with expectation.4. 1-day-old Bluecomb-susceptible turkey poults were inoculated via orally with 1ml of the intestines homogenate of the infected chicken embryos. 3 days postinoculation, the inoculated turkeys showed weight lost, watery droppings and with 20% mortality, which were the same clinical symptoms as Bluecomb disease. The control group turkeys had no any signs.These results indicated that the isolated virus was turkey coronavirus.