Identification And Pathologenicity Of Avian Infectious Bronchitis Coronaviruses Isolated From 1995 To 2004 In China

Twenty-one IBV isolates were isolated from the layer and broiler flocks suspected IB from 1995 to 2004 in China, and investigations of pathologenicity on thOese isolates were conducted in the present study.Samples collected from kidneys, proventriculuses, or oviducts with lesions were inoculated into 9-to 11-day-old specified-pathogen-free (SPF) embryonated chicken eggs after treatment. Allantoic fluids were harvested 72h post-inoculation, experiencing 2 ~7 blinded passages. The allantoic fluids from each passage were negatively stained with 2% sodium phosphotungstate and observed by electron microscopy, and haemagglutination activity of viruses in the allantoic fluids were examined by haemagglutination assay. The virus titers were tested using 9-to 11-day-old SPF embryonated chicken eggs, pathologenicity of individual isolate on ten 15-day-old SPF chichens were investigated by intranasally challenging according to its titer. Clinical signs were observed each day. Necropsies were performed on all the dead chickens and gross lesions were examined. Laryngopharyngeal swabs and blood samples were collected every 4 days lasting 28 days. Laryngopharyngeal swabs were used to inoculate embryonated chicken eggs and isolate viruses, viruses in the allantoic fluids were detected by RT-PCR. And IBV-specific antibodies were tested by indirected ELISA.Characteristic chicken embryo changes such as stunting, dwarfing or curling of embryos were observed between 2 and 7 days post-inoculation, different IBV isolates resulted in these changes between 2 and 7 passages, and typical round coronavirus morphology with sparse spikes was observed by electron microscopy. The allantoic fluids were free of other agents. Haemagglutination assays were performed on all the allantoic fluids without trypsinase treatment, all the results were negative. 30% chickens challenged with CK/CH/LHN/00 I showed mild clinical signs, overt diseases including respiratory distress, shuffledness and depression were caused by other isolates. Death occurred at 3 days after challenge with duration from 2 to 9 days in different isolates. With the exception of CK/CH/LHN/00 I and CK/CH/LSD/03I, the remaining 19 isolates resulted in death at various degree, 3 isolates caused less than 20% mortality;6 isolates caused 20%~30% mortality;10 isolates caused more than 30% mortality, among them CK/CH/LTJ/95 I and CK/CH/LJL/04 I killed all the chickens challenged by them. Tracheal hemorrhage and oedema of caecal tonsils were observed almost in all dead chickens. The kidneys from the dead chickens showed the predominant gross lesions which were pale, mottled, and swollen. The renal tubules and ureters were distended with excess urates. With the disease development, the kidneys became further swollen. Five IBV strains were isolated from the swollen proventriculuses, lesions of diseased chickens in field were not replicated, and however obvious renalIBV strain isolated from atrophic oviduct also caused renal lesions. The results indicated that the IBV strains isolated in the last 10 years in China were mainly nephropathologenic IBV (NIBV). The virulence of isolates on SPF chickens was different, but most of the isolates showed high virulence.Laryngopharyngeal swabs were inoculated into chicken embryos and allantic fluids were harvested 48h post-inoculation for RT-PCR.The results demonstrated that 9 isolates could be examined at both 4 days and 8 days post-challenge;6 isolates could be detected only at 4 days post-challenge;2 isolates could be detected only at 8 days post-challenge;one isolates could be detected at both 4 days and 12 days post-challenge;2 isolates could be detected at 4, 8, and 12 days;one isolates wasn't detected anytime post-challenge. These results showed that most chickens could excrete viruses during 8 days post-challenge, and the number of chickens which could excrete reduced dramastically at 12 days post-challenge. No virus was excreted at 16 days post-challenge.The IBV specific-antibodies in serum samples taken every 4 days post-challenge were detected with indirect ELISA. The serum samples taken from three chicken groups at 4 days post-challenge had 3(3/7), 3(3/10) and 2(2/10) samples seroconversion, respectively, but other serum samples taken at 4 days post-challenge were all negative;the serum samples taken from four chicken groups at 8 days post-challenge had 1(1/5), 5(5/9), 5(5/9) and 4(4/8) serum samples, respectively, with no IBV-specific-antibody and other serum samples taken at 8 days post-challenge were all positive;among the serum samples taken between 12 days and 28 days post-challenge, only the serum samples from the chickens challenged with CK/CH/LHLJ/04XI at 12 days and 16 days post-inoculation had 1(5/6) sample without IBV-specific antibody, other samples were all positive.