【作者】 杨金龙；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 鹅细小病毒,又称小鹅瘟病毒(Goose Parvovirus,GPV),与人细小病毒HPV、牛细小病毒BPV、犬细小病毒CPV以及猪细小病毒PPV等同属于具有单股线状DNA的细小病毒属病毒的成员。目前世界许多饲养鹅及番鸭地区都有本病的发生,特别是在我国的一些省(市、自治区)的流行、传播速度快,发病率和死亡率较高,给养鹅业造成严重危害,带来重大的经济损失,是目前危害养鹅业的主要传染病之一。虽然已建立了针对GPV特异、敏感的PCR诊断方法,但传统PCR无法对病毒核酸进行准确的定量分析,而对病毒基因组的定量研究在病毒学中有着非常重要的意义,是研究病毒的组织细胞嗜性、致病机理及筛选有效抗病毒药物的有力工具。肠道菌群结构的变化对鹅细小病毒病的发生、发展和转归的影响是一个不容忽视的问题。本文采用不同的研究方法、从不同的角度对健康及感染小鹅瘟病毒雏鹅的消化道菌群结构进行分子解析,获得如下结果:1.本试验以小鹅瘟病毒CH_V强毒株为致病原、以7日龄雏鹅为实验动物,建立细小病毒病动物模型,在国内外首次采用TagMan MGB探针实时荧光定量PCR技术(FQ-PCR)扩增GPV强毒CH_V株的VP3基因,对不同感染途径病毒在体内的分布规律进行实时定量检测,了解其在鹅体内分布规律与鹅临床表现及病程发展的关系,为临床防治提供理论依据。结果表明:(1)优化的FQ-PCR方法可在2 h内实现对GPV的快速定量检测,最低检测限为28 copies/μL,是普通PCR灵敏度的1000倍。(2)口服4 h后雏鹅的食道既可检出GPV DNA,滴鼻感染4 h后雏鹅的气管可检出GPV DNA;注射感染4 h后除雏鹅的肠道、食管、气管、脑、肺外,其它组织均可检测出GPV DNA;12 h后各组织器官中都能检测到GPV DNA,2-9天逐渐达到高峰,之后逐渐减少;GPV DNA含量最多的是肝脏、脾、脑、哈德氏腺等器官。2.采用DGGE技术,对不同途径感染GPV的小鹅肠道菌群结构进行动态的分析,以期揭示肠道菌群结构在感染肠道病毒后的总体变化规律。结果表明:临床健康对照鹅肠道菌群结构相对稳定,PCR-DGGE条带数量以盲肠最多(13条),其次是直肠(12条)、空肠(11条)和回肠(11条),十二指肠最少(8条)。皮下感染鹅各肠段随着感染时间的延长,其PCR-DGGE条带数呈逐渐减少再慢慢恢复正常的趋势;口服感染鹅各肠段PCR-DGGE扩增条带数随着感染时间的延长,呈现先逐渐减少,再慢慢恢复正常的趋势:滴鼻感染鹅各肠道中菌群结构变化与其它两种感染途径引起的变化相似,只是时间延后;死亡鹅各肠段中PCR-DGGE条带数最少。表明鹅细小病毒感染鹅的途径与其致病力密切相关、与感染鹅肠道菌群结构我破坏程度密切相关。3.建立了鹅粪细菌的16S rDNA文库,从鹅粪的16S rDNA文库中获得了5个优势条带进行测序分析。BLAST分析结果显示,在鹅粪的5个优势菌群中,其中4种菌分别与大肠杆菌属、芽孢杆菌、乳酸杆菌、肠球菌成员有96%以上的同源性,而感染GPV后肠道优势菌群则增加了沙门氏杆菌,说明感染GPV后鹅肠道菌群发生了根本性的改变。4.ERIC-PCR是近年研究肠道菌群结构及种群变化的最新分子生物学技术之一,该方法直接根据样品ERIC条带的分布、数量和亮度等信息,分析肠道样品菌群的结构和种群的变化,具有快速、简洁,不依赖纯培养的特点。与其他方法相比,其灵敏度、可重复性和可靠性更好,且省时省力,我们采用ERIC-PCR技术,对不同途径感染GPV的小鹅肠道菌群结构进行动态的分析,以期揭示肠道菌群结构在感染肠道病毒后的总体变化规律。与DGGE检测结果相似:临床健康对照鹅肠道菌群结构相对稳定,ERIC-PCR条带数量以盲肠最多,其次是直肠、空肠和回肠,十二指肠最少。皮下感染鹅各肠段随着感染时间的延长,其ERIC-PCR条带数呈逐渐减少的趋势;口服感染鹅各肠段ERIC-PCR扩增条带数随着感染时间的延长,呈现先逐渐减少,再慢慢恢复正常的趋势;滴鼻感染鹅各肠道中菌群结构变化与口服组相似,只是时间延后;死亡鹅各肠段中ERIC-PCR条带数最少。表明鹅细小病毒感染鹅的途径与其致病力密切相关、与感染鹅肠道菌群的失调程度密切相关。5.关于鹅消化道正常菌群的动态分布、变化及鹅感染小鹅瘟病毒后其体内的正常菌群的动力学变化目前尚无系统性的研究资料,而且建立快速、准确、能够定量检测机体内一些正常菌群的检测方法对于辅助治疗小鹅瘟也是非常必要的。虽然ERIC-PCR、DGGE-PCR等指纹图谱技术能很好地分析消化道的菌群结构,但却不能将细菌的数量进行精确的测定。鉴于此,我们采用Taqman-MGB探针实时荧光定量PCR(FQ-PCR)检测技术,建立大肠杆菌属、双歧杆菌属、芽孢杆菌属和乳酸杆菌属的实时荧光定量PCR检测方法,对经感染小鹅瘟病毒鹅的消化道内正常菌群大肠杆菌、双歧杆菌、芽孢杆菌属及乳酸杆菌等鹅体内常见的特定菌群的数量变化规律进行研究,系统地分析鹅消化道这四种菌群的动态分布、变化,为正常菌群与机体的关系及对小鹅瘟发病机制的影响提供实验依据。结果表明:与正常对照鹅相比,不同途径感染鹅细小病毒CH_V强毒株鹅消化道内检测的四种细菌的基因组拷贝数均不同程度的发生了变化。其中大肠杆菌属细菌数量出现峰值的时间顺序为皮下组＞口服组＞滴鼻组;乳酸杆菌细菌整体含量高低顺序为滴鼻组＞口服组＞皮下组;其他二种细菌三个攻毒途径的变化趋势相近,但波动大小顺序为皮下组＞口服组＞滴鼻组。表明细菌失调程度与鹅细小病毒感染途径密切相关。更多还原

【Abstract】 Goose Parvovirus,which also known as Gosling Plague Virus(Goose Parvovirus,GPV),is the causative agent of Gosling plague(GP),an acute,contagious,and fatal disease, variously known as Derzsy’s disease.The virus was classified as an autonomous parvovirus belonging to the family parvovirdae and it was more closely related to the human parvovirus HPV,bovine parvovirus BPV,canine parvovirus CPV and porcine parvovirus PPV.While qualitative PCR was useful for laboratory diagnosis of GPV infection,it still had some problems,it had the processes of electrophoresis and dyeing which had the shortcomings such as time consuming and easy to contaminate,poor quantification and unsuitable for large-scale investigations.Moreover,the amount of virus in different tissues and cells,was very useful for exploring the nosogenesis,virus replication,host-virus interactions,tropism,and effective for screening anti-viral drug,it couldn’t be determined by qualitative PCRIt should not be ignored that the occurrence and development and turnover of GP with the changes in the structure of intestinal flora of goose.The following results were obtained by using different research methods and from different perspectives on health and infected GPV1.In this study,7-day-old goslings were chosen as animal model of the virus disease to research the pathogens of GPV CHv strain.To provide the theoretical basis of prevention and treatment of the disease and understanding the relationgship between the distribution of virus in vivo and the clinical manifestations and the development of the disease,the TagMan MGB probes were the first time used for real-time fluorescence quantitative-PCR (FQ - PCR) to amplificate the VP3 gene of GPV whth different routes of infection of the virus.The results showed that:(1) Optimize the FQ-PCR method can be achieved in 2 h for the rapid quantitative detection of GPV,the minimum detection limit was 28 copies /μL,are ordinary PCR sensitivity of 1000 times.(2) 4 h after oral administration of the esophagus can be detected gosling CH_V strain,4 h after intranasal infection of the trachea gosling CHV strain could be detected;injection 4 h after infection of the intestine in addition to Gosling,esophagus,trachea,brain,lung,other organizations can detect CHV strain;12 h after the different organs and tissue can be detected CHV strain,2 - 9 days gradually reached a peak,followed by gradually reduced;viral load Most of the liver, spleen,brain,Harderian gland and other organs.It is suggested that it should be based on different time of infection with different organizations which containning more virus when prepare or detect the antigen,it should be preferred with liver,spleen,brain,Harderian gland when do not know the exact time of infection.2.To reveal the overall regularity of the structure of intestinal flora in the gastro-intestinal virus and to analyze the dynamic of intestinal flora by the variation infection ways with GPV,DGGE technology was used based on the success of the quantitative detection of GPV.The results showed that:the structure of intestinal microflora was relatively stable in healthy control goslings,it is at most of the number of PCR-DGGE band with cecum, followed with the rectum,jejunum and ileum,and it is at least in duodenum.PCR-DGGE bands of hypodermically inoculated goslings gradually decreased and then gradually increased to normal with the extension of time after inoculation.PCR-DGGE bands of orally inoculated goslings decreased after inoculation and then gradually increased to normal.The changes of microbial community of nasally infected goslings were the same as other two inoculated way only delay the time.It was the least in dead goslings.The maladjusted degree of intestinal microbial community was correlative with the infected routes of GPV. 3.The 16S rDNA library of goose fecal bacterial was set up based on successful analysis the overall situation of intestinal flora.Five major bands from 16S rDNA library of gosling feces were sequenced.It was revealed that four major bands which has more than 96% homology with Bacillus,Lactobacillus,Enterococcus members and coliform respectively by BLAST analysis,the dominant microorganisms in intestinal flora was increased with Salmonella after infected GPV and had fundamental changed.4.ERIC-PCR is one of the molecular biology techniques on study the change of structure and flora species in recent years.It has characteristic such as fast and simple and not rely on pure culture with the change of structure and the species of intestinal microflora in the sample by analysis directly from the information such as distribution and quantity and brightness of ERIC strip.Compared with other methods,it has the advantege such as sensitivity,better repeatability and reliability,and saving time and labor.ERIC-PCR was used to reveal the overall variation of the structure of intestinal bacteria flora in the gastro-intestinal virus with analysis of intestinal bacteria flora dynamic structures by different ways of GPV infection.With the similar to DGGE results:it is relatively stable of bacillus flora of clinical healthy controls goslings,The band numbers formed by ERIC-PCR of duodenum were the least and were the most of cecal,followed by the rectum, jejunum and ileum.ERIC-PCR bands of hypodermically inoculated goslings gradually decreased and then gradually increased to normal with the extension of time after inoculation.ERIC-PCR bands of orally inoculated goslings decreased after inoculation and then gradually increased to normal.The changes of microbial community of nasally infected goslings were the same as other two inoculated way only delay the time.It was the least in dead goslings.The maladjusted degree of intestinal microbial community was correlative with the infected routes of GPV and the study plays an important role to elucidate the pathogenesis of GPV infection.5.There is no systematic research data on the dynamics distribution and changes of normal flora in vivo of healthy goslings and infected with GPV.It is very necessary to set up the detection method which with the advantage of fast,accurate and quantitative detection to some normal flora for the adjuvant treatment of GP.ERIC-PCR or DGGE-PCR was a good fingerprint technique to analysis the structure of the flora but with the accurate number of bacteria.In view of this,Taqman-MGB probe real-time fluorescence quantitative PCR(FQ-PCR) was adopted for detect E.coli,Bifidobacterium genus Bacillus and Lactobacillus in normal gosling flora infected with GPV.The results indicated that the four kind of bacteria of all infected goslings with GPV were varying degrees of changing. The chronologicaol order of Escherichia coli’s quantity crest value is hypodermically inoculated goslings＞orally inoculated goslings＞nasally inoculated goslings.The quantity of Lactobacillus is nasally inoculated goslings＞orally inoculated goslings＞hypodermically inoculated goslings.The dynamic changes of the other two kind of bacterium are also hypodermically inoculated goslings＞orally inoculated goslings＞nasally inoculated goslings.So the maladjusted degree of microbial community in respiratory tract was correlative with the infected routes.This study will provide valuable insight into fully understand the pathogenesis of GPV infection.更多还原