【作者】 史鸿飞；

【导师】 薛飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 牛呼吸道传染病多表现为牛呼吸道疾病综合征，对养牛业危害严重。牛腺病毒3型与牛副流感病毒3型都是已知的牛呼吸道疾病综合征的重要病原。2009年国内首次报道从黑龙江的分离到一株BAV-3，命名为HLJ0955株。而在2008年本实验室从山东省牛群中首次在国内分离到4株BPIV3，为一新的基因型。由于牛群中BAV-3和BPIV3自然感染非常普遍，导致在进行实验感染时获取合适的实验用牛非常困难。因此，SPF实验动物也许是研究BAV-3和BPIV3致病性的有效动物模型。本研究应用病毒分离、荧光定量PCR和免疫组化检测BAV-3在实验感染BALB/c小鼠和豚鼠中的组织分布。通过滴鼻接毒（104.5TCID50）小鼠以及豚鼠（105.2TCID50），在接毒后不同时间采集肺脏等组织进行相关试验。结果表明BAV-3能够在2周龄小鼠肺脏中有效地复制。IHC结果表明在接毒后第1到第11天能够在肺脏内皮细胞检测到BAV-3抗原。荧光定量PCR能够在接毒小鼠第1到第7天的肺脏中检测到病毒DNA。BAV-3接毒小鼠会造成出血、肺泡内皮细胞增生等病理变化。而在接毒4周龄小鼠体内，肺脏病变程度较轻。所有接毒的豚鼠在接种后体温升高,剖检可见肺脏实变和出血；病毒分离和免疫组化在感染后24小时检测到病毒复制；荧光定量PCR在感染后第1天到第11天的肺脏中检测到病毒DNA。病毒中和试验表明在接毒后第11天产生中和抗体。豚鼠病理组织学变化与小鼠变化相似。本研究还利用BPIV3SD0835株接毒16只豚鼠(3.55×106TCID50)。接毒豚鼠表现出呼吸道相关的临床症状，且有2只豚鼠死亡；解剖学可见肺脏暗红、实变。组织病理学变化包括肺泡间隔增宽和纤维素性肺炎。利用病毒分离滴定、Real-time RT-PCR和免疫组化在接毒24小时呼吸道组织中检测到了病毒复制。同时，IHC染色揭示了肺脏和气管是SD0835株主要复制的组织部位。在接毒后期能够检测到病毒特异性的中和抗体。综上所述，由研究结果可知BALB/c小鼠作为一种研究BAV-3感染的动物模型是可行的，且2周龄的小鼠相对于4周龄小鼠对HLJ0955株更加敏感,但在实验感染BALB/c小鼠中未观察到临床症状及解剖学变化。相反，BAV-3HLJ0955株能够在豚鼠肺部复制，且能引起豚鼠体温升高以及肉眼和组织学的病理变化。这些结果表明豚鼠感染模型相对于BALB/c小鼠感染模型更为理想，可以用作检测BAV-3HLJ0955株感染过程和致病性的系统。BPIV3SD0835对豚鼠具有致病性，能够导致豚鼠产生临床可见的症状以及肺脏的组织病理学变化。因此，豚鼠是一种理想的研究BPIV3感染的动物模型，为今后BPIV3基因C型感染过程、组织嗜性、致病性的深入研究提供了新的思路。更多还原

【Abstract】 Bovine respiratory disease complex is the major clinical manifestation of bovine respiratoryinfectious disease, which causes serious economic losses for the global cattle industry annually, andbovine adenovirus type3and bovine parainfluenza type3are the most important pathogen. A ChineseBAV-3strain HLJ0955was first isolated in2009in Heilongjiang Province, China.4BPIV3strains werefirst isolated in Shandong Province by our laboratory in2008, the4BPIV3strains were different fromthe previously reported genotypes. Natural infections with BAV-3or BPIV3in calves are very popularand result in difficulties of acquiring the suitable calves for experimental infection. Therefore specificpathogen free animals might be a useful animal infection model for studies on the pathogenesis ofBAV-3and BPIV3isolates.The present study used virus isolation, real-time PCR and immunohistochemistry staining to detectbovine adenovirus type3in tissues from experimentally infected BALB/c mice and guinea pigs. As afirst step, the infected lungs and other organs of2week old and4week old female BALB/c mice werecollected on different time post inoculation (105.2TCID50) for subsequent tests. BAV-3replicates wellin the lungs of2week old BALB/c mice, no virus could be isolated by day5PI, but IHC results showedthat the antigen of BAV-3in the alveolar epithelial cells can be detected from1to11days PI. ViralDNAs were detectable in the lungs of experimentally infected BALB/c mice during7days ofobservation by real-time PCR. Infection of BALB/c mice causes an interstitial pneumonia characterizedby hemorrhage, alveoli septa thickening, alveolar epithelial cell proliferation. On the other hand, thehistological lesions in the lungs of4week old BALB/c mice inoculated with BAV-3were less severeand their lasting time was shorter. All of the infected guinea pigs had apparently elevated rectaltemperatures (39.2℃–39.9℃), consolidation and petechial hemorrhage were observed in guinea pigsexperimentally infected with the HLJ0955. Viral replication was detectable by virus isolation andtitration and IHC in the lungs of guinea pigs as early as24hr PI. Viral DNA was detectable in the lungsof infected guinea pigs during11days of observation by real-time PCR. Virus neutralization antibodiesagainst BAV-3were detectable from11days PI. Histopathological changes were the same as thechanges in mice.An experimental infection of SPF guinea pigs with the Chinese BPIV3strain SD0835of thegenotype C was performed. Sixteen guinea pigs were intranasally inoculated with the suspension ofSD0835(3.55×106TCID50). The virus-inoculated guinea pigs displayed a few observable clinical signsthat were related to the respiratory tract disease and two experimentally infected guinea pigs died, thegross pneumonic lesions in guinea pigs inoculated with SD0835consisted of dark red, consolidationand atelectasis. Histopathological changes including alveoli septa thickening and focal cellulosepneumonia were also observed in the lungs of guinea pigs experimentally infected with SD0835. Viralreplication was detectable by virus isolation and titration, real-time RT-PCR and immunohistochemistry(IHC) staining in the respiratory tissues of guinea pigs as early as24hours after intranasal inoculation with SD0835. As well, the results of IHC staining implicated that the lungs and tracheas were the majortissues in which SD0835replicated. Virus-specific serum neutralizing antibodies against BPIV3weredetected in virus-inoculated guinea pigs.In conclusion, it is eligible to the BALB/c mice as the animal model for the BAV-3strain HLJ0955infection, and the2week old mice were more sensitive than the4week old mice to HLJ0955infection.However, no clinical sign and gross lesion was observed in BALB/c mice experimentally infected withBAV-3strain HLJ0955. On the contrary, the HLJ0955could replicate in lungs of guinea pigs and causefever and gross and histological lesions in infected guinea pigs. Therefore the guinea pig infectionmodel of BAV-3would serve as a more useful system than BALB/c mice for monitoring the infectionprocess and pathogenesis of the Chinese BAV-3strain HLJ0955. BPIV3strain SD0835of the genotypeC was pathogenic to guinea pigs and could cause a few observable clinical signs, and gross andhistologic lesions in virus-inoculated guinea pigs. Thus guinea pig is an ideal laboratory animalinfection model for BPIV3and would cast more light on the genotype C of BPIV3infection process, invivo tropism and pathogenesis.更多还原