【作者】 李新苍；

【导师】 朱瑞良；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 鸡群中常见免疫抑制性病毒主要有5种,分别是IBDV、MDV、ALV、REV及CIAV;除上述5种病毒外,某些毒株的呼肠孤病毒(Reovirus)、新城疫病毒、传染性喉气管炎病病毒等也可以造成免疫抑制(Immunosuppressive)。这些病毒除不同程度的引起机体发生原发感染甚至死亡之外,更严重的是以亚临床感染的形式出现,致使感染禽的免疫系统受到损害,引起机体免疫功能下降,导致疫苗免疫效果降低、并发或继发感染其它病原。根据近年的报道,这几种免疫抑制病在我国鸡群中存在较为普遍,且常常出现多重感染,给养鸡业造成了巨大的经济损失。针对免疫抑制性病毒在我国部分地区广泛流行,且往往呈现混合感染的特点,传统的从病料中直接分离病毒和血清学方法都很难满足临床诊断和做大规模流行病学调查的需要。目前单重PCR、多重PCR已经应用于这几种病毒的诊断,并且取得了较好效果。为了更好的应用PCR方法检测免疫抑制性病毒,本研究对目前使用的PCR检测方法进行了优化,并用优化后PCR检测方法对山东主要养禽地区不同鸡群的鸡只进行了免疫抑制性病毒的调查,进一步了解了几种主要免疫抑制性疾病在山东的流行特点。针对山东某规模化种鸡场的某些分厂出现的不同日龄种鸡及所产商品肉鸡NDV免疫后抗体水平明显偏低,并且所产商品鸡饲养到30日龄左右时生长缓慢、大肠杆菌、球虫及新城疫病毒等感染率明显升高,对该种鸡场的不同日龄种鸡及商品鸡进行了免疫抑制病毒检测和NDV抗体水平的检测,表明免疫抑制性病毒的感染对该鸡场的发病起了重要的作用。本研究分2个部分:1.鸡免疫抑制病毒PCR检测方法的优化及应用为进一步优化免疫抑制性疾病PCR检测方法,在确保与MDV、REV、ALV及CIAV的PCR检测方法反应程序一样的条件下,参照已发表IBDV毒株序列,在结构蛋白VP2编码基因的高变区(VP2 variable domain,vVP2)两端外侧的保守序列内设计合成了2对寡核苷酸引物,对包括疫苗株在内的10株参考毒株进行逆转录酶—聚合酶链式反应(RT-PCR)检测。结果10个参考毒株均能扩增出约492bp的目的片段,而对作为阴性对照的常见5种鸡病病原NDV、MDV、CIAV、B.avium、E.coli均没有扩增出任何片段;同时经临床检验、序列分析表明,该检测技术可用于临床病料、细胞毒及鸡胚毒的检测。优化后的PCR检测技术能同时一次性检测5种病原,大大方便了临床病例的检测和大规模流行病学调查。运用优化后的PCR检测方法,对来自山东主要养禽地区收集、送检的18个鸡群278羽鸡进行免疫抑制性病毒的检测。结果五中免疫抑制病毒的感染率分别为CIAV(46.04%)、MDV(14.03%)、IBDV(12.95%)、REV(2.51%)及ALV(2.16%),其中被检鸡混合感染的总阳性率为21.58%。2.山东某规模化鸡场免疫抑制性病毒分子流行病学调查及鸡群ND疫苗免疫效果分析为了解AA肉种鸡群中免疫抑制性病毒的存在状况及其对NDV疫苗免疫效果和对商品鸡生长的影响,采用多重PCR、RT-PCR和血清学方法对山东某规模化大型AA父母代肉种鸡场的28日龄、180日龄左右种鸡及其所生产的1日龄、30日龄左右商品鸡进行IBDV、MDV、ALV、REV、CIAV及REOV感染状况调查,并同时对鸡群NDV免疫后抗体水平进行跟踪监测,结果表明种鸡和商品鸡均存在CIAV的感染,并不同程度的存在CIAV与MDV、IBDV等共感染;发病鸡群NDV抗体水平明显偏低的原因是由于鸡群感染免疫抑制性病毒所致;种鸡群感染CIAV并垂直传播给商品鸡,间接引起了商品鸡发生免疫抑制性病毒的共感染,导致了商品鸡群30日龄左右生长受阻、免疫能力降低、疾病爆发。因此,免疫抑制性疾病是目前养鸡业的潜在威胁。更多还原

【Abstract】 IBDV(Infectious bursal disease virus)、MDV (Marek’s disease virus)、ALV (Avian leukosis virus)、REV (Reticuloendotheliosis viruses) and CIAV (Chicken infectious anemia virus) were the five major immunosuppressive viruses in current chicken flocks. Besides these five immunosuppressive viruses, REOV(Reovirus)、NDV(Newcastle disease)and ILT (Infectious larygotracheitis)are also immunosuppressive viruses. These immunosuppressive viruses can cause some chichens dead. Even more, these virus can make chicken body immunosupperssive, and then make chichen immunity descend、the effect of vaccine fall and other pathogeny easily infected. Current reports showed that these five immuno suppressive viruses existed widely in chicken flocks in our country. The infection of these five virus in chicken flocks bring mass economic loss for poultry industry at present.Because these five immunosuppressive viruses existed widely and often co-infected in many chicken flocks, it was difficulty to satisfy the need of epidemiological investigations of immunosuppressive viruses for isolating viruses from tissues and the detection of serology. Now PCR were used to detect these five major immunosuppressive virus and the results were good. In order to detect these viruses effectively by PCR, In our research a new technique were established to detect these five immunosuppressive viruses at the same time. Then molecular epidemiological investigations of immunosuppressive viruses were carried out in chicken flocks in shandong using this technique. We knew how these five immunosuppressive virus existed in shandong further. In order to investigate the infections of immunosuppressive viruses in AA broiler chicken flocks and further study how the viruses influence the antbody level of NDV vaccines of different age and the growth of AA broiler chicken flocks. The antibody level of NDV were detected at the same time. The results show that CIAV and several other immunosuppressive viruses were the potential causation.The research divide two parts: 1. The Development of Rapid Diagnostic Technique for Chicken Immunosuppressive Viruses and Epidemiological Surveys on the Immunosuppressive Diseases in Chickens in shandongIn order to optimize the detection technique of immunosuppressive virus, Two sets of primers, flanking the hyper-variable region of VP2 gene, were designed to run a reverse transcription polymerase chain reaction(RT-PCR). Both of these assays could amplify all of 10 reference strains, but not the 5 negative reference pathogens of chicken. 278 chickens from 18 flocks in shandong province were detected using a optimizing PCR technique, which was developed for the rapid differential diagnosis of immunosuppressive diseases. The results showed that the positive rates of CIAV(46.04%)、MDV(14.03%)、IBDV(12.95%)、REV(2.51%)及ALV(2.16%). Dual and multiple infections of the pathogens accounted for 21.58% of all the birds detected..2. Molecular Epidemiological Investigations of Immunosuppressive Viruses in AA Broilerchicken flocks and Analysis of Their Influence on the Function of NDV VaccinesIn order to investigate the infections of immunosuppressive viruses in AA broiler chicken flocks and further study how the viruses influence the function of NDV vaccines and the growth of AA broiler chicken flocks. We select the chickens suspected to be infected by immunosuppressive viruses from one large-scale AA broiler breeder farm and their commercial chicken farms in Shandong province. DNA and RNA were extracted for the multiplex PCR to detect CIAV, MDV, REV, ALV and the RT-PCR to detect IBDV. Also antibody to REOV and NDV were tested. The results showed that CIAV existed in the investigated chicken flocks and Co-infections were serious. The infections of immunosuppressive viruses were the main causation for descent or failure of the function of the NDV vaccines. The breeder flocks were infected by CIAV and then the 1d chicken flocks were infected vertically by CIAV which made the death rate of 30d commercial chicken flocks increase. So infections of immunosuppressive viruses were the potential menace to poultry industry at present.更多还原