【作者】 高文明；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 鸡传染性支气管炎(Avian Infectious Bronchitis, IB)是由冠状病毒科、冠状病毒属的鸡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。上世纪30年代在美国爆发以来,现在已经成为世界各地流行的主要禽病。IBV是单股RNA病毒,该病毒基因可由于点突变和重组而发生变异,因此IBV的血清型较多,已达30余种之多,常见的有M41、Conn、Iowa97、Gray、Hotle、JMK、Clark333、SE17、Florida、Arkanass99和Australian T等,不同血清型毒株之间仅有部分或无交叉保护作用,从而给本病的防治带来困难。我国目前虽然已广泛使用IB疫苗,但由于IBV的易变异型,IB仍然呈高度流行趋势,尤其是在已免疫鸡群中也常常爆发IB,造成严重的经济损失。近年来,河南各地养鸡场在进行了IB的常规免疫程序后,仍爆发了严重的疑似IB的传染性疾病,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,我们对河南某发病鸡场疑似IB的典型病例展开了病原的实验室分离和鉴定工作,并对其生物学特性进行了研究。无菌采集发病鸡的气管、肺脏、肾脏等组织,采用鸡胚盲传观察病毒对鸡胚的致病作用,病毒盲传至第2代后鸡胚出现死亡和侏儒胚;尿囊液经磷酸酯酶C处理后能够凝集1%的鸡红细胞;病毒纯化后经外源病毒检测,无外源病毒污染;将病毒盲传至E8代时,病毒的EID50逐渐增强到106.5/0.1 mL;病毒能够干扰NDV在鸡胚中的增殖;病毒可致SPF雏鸡发病,引起肾脏肿大、花斑肾、尿酸盐沉积等肾脏病变;电镜下可见到直径90～105 nm的冠状病毒粒子;理化特性测定表明,该毒株对乙醚和氯仿敏感,耐酸不耐碱,对热敏感。初步证实分离的病毒为鸡传染性支气管炎病毒,命名为HN99株。对HN99株的Sl基因进行克隆与测序,使用DNAStar软件进行序列分析。结果显示,HN99株与常见的M41、Gray、Holte、H120、H52、Conn等毒株的Sl基因同源性不高,分别为79.4%、72.7%、77.4%、79.1%、78.3%,而与T株的同源性高达99.6%,遗传进化关系也揭示HN99株与T株的亲缘关系较近,而与其它毒株的关系较远。证实HN99株为一新的IBV变异株。利用病毒中和试验和HI试验对HN99株进行了血清分型,结果表明HN99株不属于常见的M41、Conn、Gray等血清型,而HB、JS、SD等地方分离株则属于HN99株血清型,表明HN99株血清型具有一定的地域覆盖性;交叉保护试验证实了血清分型的结果;用HN99株和M41株制备的二价疫苗免疫试验鸡后,不仅产生了较高的抗体水平,而且能够同时抵御HN99株血清型和M41株血清型毒株的攻击,表明HN99株有较好的免疫原性,为利用地方分离株进行IB的防制奠定了基础。更多还原

【Abstract】 Infectious Bronchitis(IB) is an acute and highly infectious respiratory disease,caused by the avian infectious bronchitis virus(IBV). Since broke out in USA in the 1930s,IB have become an important disease of poultry around the world. IBV is a single-stranded RNA virus,the gene of the virus always mutated owing to it’s point-mutation and gene recombination,So the serotype of IBV has more than 30 categories. The known serotypes such as M41、Conn、Iowa97、Gray、Holte、JMK、Clark333、SE17、Florida、Ark99 and Australian T widely exist.Different serotypes have lower or no protection. Currently,IB has often been found in China. Even if in the farm where had vaccinate IB vaccine,the incidence is still widely. Which result in serious economic losses to the poultry industry.There is a serious outbreak of avian infectious bronchitis disease in Henan province recently. In order to identify the exact causes,we isolated and identificated a IBV strain from the suffering farm and researched its biological characteristics. Trachea、kindey、lung samples collected from suspect IB outbreaks were made to suspensions which were inoculated into the allantoic of SPF embryo. The second passage virus produced gross lesions to chicken embryos. The virus was multiplied and purified by chick embryo kidney cell (CEKC),and the SPAFAS Jinan key laboratory certified it was pure and germfree. It’s EID50 was 106.5/0.1 mL and could hemagglutinate chicken red blood cells after treatment with phosphoesterase. Replication of NDV was effected by inoculating in the same chicken embryo after inoculation with the isolated virus. Moreover,the gross lesions in kidney of SPF chickens caused by the virus were distinct. The virus showed typical shape shared by members of Coronaviridae under electron-microscope.In addition,physiochemical properties of the virus shared the typical characteristics of most strains of IBV. All the result showed that the vius we isolated was a strain of IBV,and it was tentatively named HN99.S1 gene of HN99 strain was amplified by RT-PCR and sequenced by automatic sequencing system. The sequence consisted of 1 739 bp. Identity of the S1 gene sequence between the isolated strain and other serotypes of published IBV sequence is <80%.Evolution analysis indicated that the isolated virus was far related to the other strains of IBV.Virus neutralization (VN) and HI test revealed that the serotype of HN99 strain did not belong to common serotypes such as M41、Conn、Hotle、Gray,but HB、JS、SD strains were similar to HN99 serotype. The cross-protection test certified the result of serum test. The inactivated vaccine of HN99 and M41 strains were prepared,moreover,chickens were immunized with the vaccine can supply sufficient protection when challenged use different strains which belonged to M41 or HN99 serotypes. The results indicated that the IBV HN99 strain had fine Immunological Properties.更多还原