【作者】 杨杰华；

【导师】 刁有祥；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 鸡传染性支气管炎(Infectious Bronchitis, IB)是由冠状病毒科(Coronavirdae)冠状病毒属(Coronavirus)的传染性支气管炎病毒(Infectious bronchitis virus, IBV)引起的一种急性高度接触性传染病。自30年代在美国爆发以来,现已成为世界各地流行的重要禽病。IBV是单股RNA病毒,该病毒基因可因点突变和重组而发生变异,故传染性支气管炎病毒血清型较多。已知的血清型有以侵害呼吸道为主的Conn、Iowa97、JMK、Florida、Arkansas99等和以侵害肾脏为主的M41、Holte、Gray、Australia“T”等30余种。 Gough等(Gough et al,1992)报道了英国一种新的793/B血清型的IBV,该毒株和其它血清型传支毒株之间无交叉血清学关系;其免疫原基因S1的序列与欧洲17个传支毒株之间差异高达21-25%。目前,该血清型IBV在西班牙、德国、荷兰、意大利、泰国等国均有发生和流行,对养鸡业危害较大。 近年来,山东省各地蛋鸡饲养场不断发生以蛋鸡产蛋下降为主要症状的疾病,给全省的养鸡业造成严重的损失,为查明引发该类疾病的确切病因,找出确切的病原,2002年我们对山东省泰安市某发病典型的蛋鸡场疑似传染性支气管炎病例展开了病原的实验室分离和鉴定工作。 无菌采集发病鸡的气管、肺脏、盲肠扁桃体、肾脏等组织,按照常规试验方法接种SPF鸡胚增殖病毒,传三代后收获尿囊液,用鸡的1%红细胞进行血凝试验检验是否含有新城疫病毒、流感病毒。结果表明,尿囊液无直接血凝活性。 对超速离心纯化的尿囊液电境下观察可发现圆形表面有纤突的直径90nm左右的典型冠状病毒粒子,随即采用Ⅰ型磷酸酯酶C处理纯化后的病毒,再次验证尿囊液的血凝活性,结果显示处理后的尿囊液具有血凝性,初步试验表明分离到的病毒可能为鸡传染性支气管炎病毒(IBV)。 进一步经致鸡胚矮小化试验、新城疫病毒B1株干扰试验和动物回归试验印证了分离到的病原为鸡传染性支气管炎病毒。更多还原

【Abstract】 Infectious Bronchitis(IB) is a severe acute respiratory disease of pouLtry, caused by Infectious bronchitis virus (IBV)。 Since broke out in USA in the 1930s, IB have been an important disease of poultry around the world. IBV is a sRNA virus, the gene of the virus always mutated owing to it’s point-mutation and gene recombination, so the virus have many serotypes. The known serotypes can be classified into respiratory symptomatic serotypes such as Conn, Iowa97, JMK, Florida, Arkansas99 and nephritic symptomatic serotypes such as M41, Holte, Gray, Australia ’T’.Gough reported a new serotype of IBV named 793/B (also known as 4/91 and CR88) in UK in 1992, 793/B isolates have not any or a little serological relation with other serotypes of IBV, the whole S1 gene of 793/B isolates differed by 21% to 25% from those of other 17 European isolates. In recent years, the 793/B serotype of IBV, harmfuL to the healthy development of pouLtry breeding, prevailed and spread in Spain, Germany, Holland, Italy and Thailand, too.At the beginning of 2002, egg drop disease appeared in many layer flocks in Shandong province of China. We isolated and identified a IBV strain from the suffering pouLtry through Electron Microscopic Observation, Hamagglutination (HA) test and animal Regressive Research. The resuLts show that the isolate is a strain of IBV.Trachea, kidney and caecal tonsils samples collected from suspect IB outbreaks were submitted for virological investigation. Clarified suspensions (10% w/v) of tissue homogenates were inocuLated into the allantoic cavity of eggs of 9- to 11 -day-old embryonated specific pathogen free (SPF) chicken eggs., the virus was passaged three times with this method for the study. To know whether the allantoic fluid have NDV or AIV, HA test was detected and the resuLt showed that the allantoic fluid have not hemagglutination tochiken’s 1% red blood cells.The allantoic fluid was centrifuged by hypervelocity centrifuge, Sediment was collected and examined by negative contrast electron microscopy for the presence of Coronavirus particles, the sediment can be observed typical Coronavirus particles which viral spikes surround. Then, we had dealed the allantoic fluid by I type of phosphoesterase C for HA test, the resuLt showed that the allantoic fluid have hemagglutination. So we can drew a primary conclusion that the virus we isolated is a strain of IBV.The following research including chiken embryo dwarfed test, NDV-B1 interference test and animal regressive research confirmed the presence of IBV in the allantoic fluid.The biologically character was determined in after research. Serum VN tests showed that that isolated strain is a mutation strain of IBV. gived fewer or no cross-reactions to the familiar serotype of IBV. For further research, the whole sequence of S1 gene was cloned and analyzed by RT-PCR using specific primer which can differentiate between 793/B IBV and other serotypes of IBV. the strain’s serotype can be deduced as 793/B. the presence of 793/B was confirmed in China.Identity of the S1 gene sequence between the isolated strain and other serotypes of published IBV sequences is <79%. The S1 amino acid sequenced differed by 21 to 25% or more. Our analysis has shown that type 793/B which may came from foreign country has become a component of the IBV population in regions of China.更多还原