【作者】 杨世兴；

【导师】 魏建忠； 范伟兴；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 猪圆环病毒病是由猪圆环病毒II(PCV2)引起的猪的一种多个系统功能障碍的病毒性传染病。临床上以新生仔猪先天性脑震颤和断奶仔猪多系统衰竭综合征等为主要特征。猪断奶后多系统衰竭综合征(postweaning multisystemic wasting syndrome.PMWS)的病原体现已经被确认为猪圆环病毒(PCV2),除了引发PMWS外,PCV2还与猪皮炎肾病综合征( porcine dermatitis and nephropathy syndrome.PDNS)、新生仔猪先天性脑震颤(Congenital tremors.CT)、增生性坏死性肺炎(proliferative and necrotizing pneumonia.PNP)以及怀孕母猪的繁殖障碍(reproductive disorders)等病的发生有关,它已给世界养猪业造成了巨大的经济损失,因此了解目前国内猪圆环病毒的感染情况,对于我们进行预防、治疗,乃至疾病的研究至关重要。本研究根据安徽省的地理分布和养殖状况,从安徽不同地区采集猪组织器官样品进行流行病学调查。1.根据Genbank上已经发表的PCV2基因序列,选取其保守性高的ORF2在其内部设计一对引物(可扩增片段630bp的序列),对两株PCV2参考毒株进行PCR的检测,均能扩增出630bp的目的基因片段,而对PCV1、猪瘟病毒、鸡传染性贫血病毒均没有扩增出特异性目的条带。将PCV2参考毒株作不同倍数稀释后分别进行PCR扩增,当稀释至100倍,即5fg病毒的DNA含量时,仍可以扩增出特异性片段。应用本试验方法对采集于宿州、怀宁、阜阳、合肥四个地区的屠宰场生猪的脾、肺、肾、淋巴结各30份以及采集于华杰、舒城长风、巢湖雄业3个猪场的发病猪的脾、肺、肾、淋巴结各10份进行检测,结果表明:(1)不同地区屠宰场的生猪均有猪圆环病毒II感染,其中,宿州、合肥检出率为6.1%,阜阳的检出率为10%,而怀宁的检出率为23.3%。(2)对3个不同猪场发病猪的调查表明:舒城长风种猪场和巢湖雄业食品有限公司种猪场病猪的检出率均为20%,而华杰良种畜禽有限公司的检出率为70%。2.为了了解病毒的感染组织器官的带毒状况;分别对上述已检测为阳性的样品进行不同部位的检测;检测的部位包括脾、肺、肾、淋巴结等。结果显示不同部位均有阳性目的片段的检出,但不同部位的检出率不同。其中在检测的20份样品中,脾和淋巴结检出率均为100%,肺检出率为80%,肾的检出率为40%。从而表明病毒主要侵害免疫器官,这可能是造成猪免疫机能降低的原因。3.为了比较安徽地区检出的PCV2与国内外其它毒株的亲缘关系,对所检出的部分阳性样品进行PCR扩增,并对其进行序列测定和序列比较分析。通过序列测定表明本试验中的四个样品PCR产物的序列与GenBank上登陆的PCV2序列的同源性达90%以上,证明是PCV2。从系统发育树上可以看出;四个阳性样品之间的亲缘关系密切,它们组成的分支和一株英国毒株AY484416,共同组成另一个细小分支;同时这一分支又和DQ195679及AY556476、AY556473三个毒株关系较近,而与一株美国株(AY325495)、一株福建毒株(AY556474)共同组成的一个分支相距较远。更多还原

【Abstract】 Pig circovirus disease as one of newly identified diseases is associated with postweaning multisystemic wasting syndrome (PMWS). The patients represent the congenital tremors and PMWS in clinic. PCV2 is considered as the pathogen of PMWS. It dose not only induce PMWS, but also is associated with dermatitis and nephropathy syndrome,congenital tremors,proliferative and necrotizing pneumonia and reproductive disorders. PCV2 brought on tremendous economic losses for swine production, so the related investigation is important for us to prevent and treat PCV2.Firstly, according to the published ORF2 genomic sequence of porcine PCV2, we designed a pair of specific primers. The assay of all 2 PCV2 referential strains can amplify the products of 630bp, but not PCV1, CCV and HCV. The sensitive experiment indicated that PCV2 5fg DNA that was diluted to as low as 1/100 was still detected by the PCR. Simultaneously, 120 samples, which were collected from slaughterhouses of Suzhou, Huaining, Fuyang and Hefei, and 30 samples, which were collected from 3 different hogpens of Shucheng, Chaohu and Huajie, were detected by using developed PCR. The results indicated that the PCV2 infection abroad existed in Anhui areas. Thereinto, the positive ratio of Suzhou slaughterhouse was 6.1%, the same as Hefei, Fuyang was 10%, but Huaining was 23.3%. To 3 different hogpens detected, the results indicated that positive ratio of Shucheng hogpen was 20%, Chaohu was 20% while Huajie was 70%.Secondly, for knowing tissue status of infected PCV2 from pathogen, we separately detected the different tissue of detected positive samples. It included spleen, lung, kidney, lymph, and so on. The results indicated that the different tissues were detected PCV2 positive, but the positive ratio was different. The positive ratio of spleen and lymph reached 100%, and it reduced immunity of swine; the positive ratio of lung was 80%, the positive ratio of kidney was about 40%.Thirdly,through amplifying positive samples by PCR and analyzing molecular characteristics of ORF2 region and constructing their phylogenic trees, the results indicated four positive strains connected to AY484416 referential strain from UK. They consisted of an embranchment, and the embranchment was close to three strains of DQ795679, AY556476 and AY556473 in phylogenic tree. But the distance was far away from AY325495 strain and AY556474 strain in phylogenic tree.更多还原