【作者】 胡守萍；

【导师】 吴东来；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2009， 博士

【摘要】 2006年,在我国爆发了高致病性猪繁殖与呼吸综合征(High pathogenic porcine reproductive and respiratory syndrome,HPPRRS)。南方的很多地区从规模化大猪场到农村的散养户都遭到了不同程度影响。表现为不同年龄和体重的猪都能感染,感染猪群表现为高热(41-42℃),昏睡,呼吸困难。在仔猪发病率达100%,死亡率可达50%以上,母猪流产率可达30%以上,育肥猪也可发病死亡。病原主要为美洲型经典毒株PRRSV在ORF1a处的部分Nsp2基因缺失所产生的变异株。HPPRRSV潜伏期2-7天,主要表现高热,怀孕各阶段的母猪流产、死胎,及后备猪发病后死亡,且哺乳仔猪及生长育肥猪也有较高的死亡率。为评价HPPRRS病毒(HPPRRS virus,HPPRRSV)的致弱效果,筛选弱毒疫苗株,将HPPRRSV HUN4株(以下略为强毒株)和其致弱株HUN4-Fxx(以下略为弱毒株)分别接种54头40-42天的仔猪。接种后,分别于1、3、5、7、10、14、21、28天剖杀,其间观察临床症状及病理剖检变化,取心脏、肝脏、脾脏、肺脏、肾脏、淋巴结、大脑、小肠、胃、扁桃体等器官用4%多聚甲醛固定后,HE染色,进行组织病理学观察。结果表明在接种强毒株后的5-21天可出现一些仔猪死亡,剖检表现为全身淋巴结出血,脾脏边缘有梗死灶,肝脏出现灰白色结节,肺脏出现肉样变及间隔增宽,脑有轻度水肿等病变。病理组织学观察发现大部分接种强毒的猪肺脏出现明显的间质性肺炎变化;淋巴结中淋巴细胞明显缺失,伴有不同程度的嗜酸性粒细胞浸润;脾脏白髓淋巴细胞明显坏死减少;扁桃体淋巴小结内淋巴细胞有显著减少,伴有少量嗜酸性粒细胞浸润;大脑分子层有血管套形成。而接种弱毒株的仔猪未见明显的临床和病理剖检变化,病理组织学观察则仅见淋巴器官内淋巴细胞减少。应用间接免疫荧光( indirect immunofluorescence assay, IFA )、免疫组织化学(immunohistochemical procedrues,IHC)和原位杂交(in situ hybrisization,ISH)方法检测了强弱毒株病毒抗原和病毒核酸在接种猪体内的分布,三种方法在强、弱毒株接种猪的检出率分别为肺脏33.33%,0%;75%,37.5%;95.8%,62.5%。扁桃体87.5%,62.5%;91.7%,87.5%;95.8%,87.5 %。脾脏45.8%,37.5%;83.33%,50%;66.7%,56.25%。颌下淋巴结91.6% ,75% ;100%,100% ;100%,87.5%。肠系膜淋巴结87.5% ,68.75% ;95.8%,87.5% ;100%,87.5%。腹股沟淋巴结95.8%,81.25% ;100%,81.25%;100%,87.5%。大脑4.16%,0%;12.5%,0% ;16.7%,0%。小肠54.16%,37.5% ;83.33%,56.25% ;87.5%,56.25%。肾脏37.5% ,18.75% ;58.33%,25%;0%,0%。心肌、肝脏和胃均未检出阳性细胞。IFA检测强毒组在接种后的10-14天各组织的荧光阳性反应达到最强,而弱毒组无明显荧光反应; IHC检测强毒组各组织阳性细胞的峰值出现在接种后的3-21天,在此期间弱毒组无明显峰值出现,强弱毒组差异显著; ISH检测强毒组各组织阳性细胞的峰值出现在接种后的5-21天,强弱毒组阳性细胞数具有显著的统计学差异。IFA和IHC检测肾小球呈阳性反应,但ISH检测呈阴性,PCR检测弱毒组及强毒组的多数肾脏呈阴性,据以上结果推测阳性反应物大部分可能是抗原抗体复合物。研究结果表明,虽然弱毒株在淋巴器官的分布与强毒株差异不十分明显,但病毒含量明显降低,对淋巴器官的损伤不明显,并且不侵害脑,在其靶器官肺的检出率也明显低于强毒株;同时也未导致任何高致病性猪繁殖与呼吸综合征临床症状和病理学变化,说明弱毒株致病力已明显降低,有进一步作为高致病性猪繁殖与呼吸综合征弱毒疫苗研究的价值。更多还原

【Abstract】 In 2006, the highly pathogenic porcine reproductive and respiratory syndrome were outbreak in mainland of China. A lot of areas from the large-scale farms to the rural households had been attacked in varying degrees in the south. Different ages and weights swine can be aggressed and the performance of infected swines were high fever (41-42℃), drowsiness, difficulty in breathing. The infected rate was 100% and the mortality rate can reach to 50% in pigs. The abortion of sow was highly up to 30%. Foster pigs can be attacked to death. Pathogen were the variant which were American classical- PRRSV in part Nsp2 Department ORF1a gene deletion mutant. The incubation period of HPPRRSV can last 2-7 days, infected pigs mainly showed high fever, pregnant sows at various stages were miscarriage, stillbirth and back-up pigs can be attacked to death. Suckling piglets and foster pigs were also infected and had higher mortality rates.In order to evaluation the attenuated effect of high pathogenic porcine reproductive and respiratory syndrome virus (HPPRRSV) and screen the attenuated vaccine candidate, fifty-two 40-42 days old pigs were inoculated with HPPRRSV HUN4 strain and its attenuated strain HUN4-Fxx respectively. Pigs from these groups were necropsied at 1, 3, 5, 7, 10, 14, 21 and 28 days postinoculation. We took heat, liver, spleen, lung, kidney, submandibular lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, cerebrums, intestines, stomach and tonsil and fixed with 4% paraformaldehyde for pathology. Gross lesions shown pigs demonstrated death after incoculated with HUN4 frome 5 to 21 days . Lymphoid node were hemorrhage, the edges of spleen necrosis,gray-white nodule were seen in liver, lung had hypertrophy and hyperplasia and multifocal septal thickening, Brain had mild edema. Miscroscopic lesions were as follows. The multifocal interstitial pneumonia was prestent. Lymphocytes necrosis and decreased and foci of eosinophile granulocyte cell infiltrates were seen in the paracortical zone and medullary region. Necrosis and decrease of lymphocytes were also detected in spleen and tonsil. Eosinophile granulocyte cell infiltrated present in the tonsil. Lymphocytic infiltration around blood vessels in cerebrums. Lymphocytic cuffing around blood vessels in molecular layer. Contrastly, there were no obviously clinical and gross changes and lymphocytes necrosis and decrease were mild in immune organs of inoculated with HUN4-Fxx pigs in pathohistology.We used indirect immunofluorescence assay (IFA), immunohistochemical procedrues (IHC) for PRRSV antigen and in situ hybrisization for PRRSV nucleic acid. The percentage of PRRSV antigen in pigs which inoculated with HUN4 or HUN4-Fxx were different by using three ways . Lungs were 33.33%,0%;75%,37.5%;95.8%,62.5%, respectively. Tonsils were 87.5%,62.5%;91.7%,87.5%;95.8%,87.5 %, respectively. Spleens were 45.8%,37.5%;83.33%,50%;66.7%,56.25%, respectively. Submandibular lymph nodes were 91.6% ,75% ;100%,100% ;100%,87.5%, respectively. Mesenteric lymph nodes were 87.5% ,68.75% ;95.8%,87.5% ;100%,87.5% , respectively. Inguinal lymph nodes were 95.8%,81.25% ;100%,81.25%;100%,87.5% , respectively. Cerebrums were 4.16%,0%;12.5%,0% ;16.7%,0% , respectively. Intestines were 54.16%,37.5% ;83.33%,56.25% ;87.5%,56.25% , respectively. Kidneys were 37.5% ,18.75% ;58.33%,25%;0%,0% , respectively. There were no positive cells in heart, liver and stomach.Positive reaction fluorescence achieved the most strong during 10-14DPI inoculated with HUN4 in IFA, whereas postitive reaction was gentle inoculated with HUN4-Fxx strain. Antigen was most intense from 3-21DPI in HUN4 group of IHC and two groups had significantly distinction. PRRSV nucleic acid was most intense from 3-21DPI in HUN4 group of ISH and the quantity of postive cells of two groups were notable differently. The positive signals were detected in the endothelium of glomerulus by IFA, IHC except ISH. PCR results of kidneys in HUN4-Fxx group and th most of HUN4 group were negative. According to above results, we presumed the postive reactions wer antigen-antibody complex.The research results showed that the distribution of attenuated strain HUN4-Fxx in the immune organs were not different with HUN4, whereas the HUN4-Fxx strain lead to the degree of lesions were obviously decreased. The HUN4-Fxx strain could not be detected in cerebrums and in lung which virus target organ detection rate were significant lower than HUN4 and no obviously clinical and pahtological changes. All above researches indicated the virulence were notable decreased and could be the strain for the attenuated vaccine.更多还原