【作者】 刘雯；

【导师】 靳朝；

【作者基本信息】 吉林大学 ， 临床兽医学， 2011， 硕士

【摘要】 犬瘟热(Canine distemper, CD)由副粘病毒科麻疹病毒属犬瘟热病毒(canine distemper virus, CDV)引起,目前在世界范围内普遍发生。CDV的原始宿主为食肉目犬科动物,但近些年宿主范围扩大到几乎所有陆生食肉动物,甚至海洋食肉动物。许多野生动物群体也频频爆发犬瘟热感染。CDV为泛嗜性病毒,可引发呼吸、消化及神经系统多系统症状,导致间质性肺炎、卡他性肠炎和非化脓性脑炎的发生。曾林等(2008)在疑似犬瘟热病毒感染的实验恒河猴中首次分离到一株犬瘟热病毒,对N蛋白基因的系统进化分析显示该毒株属于我国流行野毒株。为进一步了解该毒株引起的多系统症状和病理变化,及其在不同物种间的差异性,本研究通过1、不同动物感染该CDV毒株的组织病理学比较；2、病毒抗原在不同动物体内分布的比较；3、病毒核酸在不同动物体内分布的比较,进行该犬瘟热病毒在不同动物体内分布的比较病理学研究。首先,采集3只患病死亡恒河猴的肺脏、脾脏、小肠、淋巴结、肝脏等组织,HE染色后进行组织病理学观察。取恒河猴肺脏、脾脏、淋巴结组织制成病毒组织匀浆,将上清液接种于雪貂和犬,观察临床症状及病理剖检变化,采集各动物组织,固定并染色进行组织病理学观察,对不同动物感染该CDV毒侏的病理变化进行比较。结果表明在接种后第7天和第8天时雪貂和犬先后发病，均表现为严重的呼吸系统症状和胃肠炎,个别病例表现轻微神经症状。病理组织学观察发现,恒河猴、犬肺脏均出现明显的肺泡壁增厚,恒河猴表现为间质性肺炎,犬肺脏为明显的卡他性肺炎,部分犬发生严重化脓性病变,雪貂肺脏以出血渗出为主,肺泡腔内有大量红细胞；恒河猴、犬的小肠绒毛损伤严重,雪貂小肠仅见杯状细胞增多,固有层结缔组织增生；脾脏、淋巴结均表现为淋巴细胞大量坏死、凋亡,见核固缩和核碎裂相；犬膀胱上皮细胞内有大量嗜酸性包涵体；部分雪貂可见神经元周围胶质细胞增生；其他脏器无明显病理变化。实验结果显示,该CDV毒株对3种动物的呼吸系统、消化系统和淋巴器官均可造成严重损伤。病理学观察结果表明,间质性肺炎是CDV感染的共同特征,灵长类动物也不例外,雪貂更易表现出出血性病变；雪貂和犬容易形成膀胱包涵体,通过排泄物大量传播病毒。然后,应用免疫组织化学(immunohistochemisty, IHC)和原位杂交in situ hybrisization, ISH)方法检测了该CDV病毒抗原和病毒核酸在自然发病恒河猴和接种雪貂、犬体内的分布。IHC对恒河猴、雪貂、犬组织检测结果为：肺100%、和接种雪貂、犬体内的分布。IHC对恒河猴、雪貂、犬组织检测结果为：肺100%、100%、100%；小肠100%、100%、100%；脾脏100%、100%、100%；淋巴结100%、100%、9/10；膀胱0、1/6、1/2；脑0、5/6、1/5；肾脏0、1/3、1/10；肝脏0、1/3、0。ISH对恒河猴、雪貂、犬组织检测结果为：肺100%、100%、100%；小肠100%、5/6、100%；脾脏1/3、1/3、2/5；淋巴结0、1/3、3/10;脑0、1/3、0。IHC、ISH检测结果显示,三种动物的肺脏、脾脏、淋巴结几乎全部存在阳性细胞。阳性细胞类型主要为肺支气管上皮细胞、肺泡巨噬细胞、脾脏巨噬细胞、淋巴细胞、小肠绒毛上皮细胞和隐窝上皮细胞,说明上皮细胞、巨噬细胞和淋巴细胞是该CDV主要的靶细胞。实验结果表明,该CDV毒株在不同物种体内的分布差异性并不明显,特别是对呼吸系统、消化道和淋巴器官的损伤都比较严重,仅在侵害的个别细胞类型上有轻微差异性。对于中枢神经系统的侵害,雪貂较为严重,犬也会产生脑炎症状。可以推断CDV对非人灵长类动物的感染并非偶然,提示CDV宿主范围和趋向性研究的重要意义,以及判断新的感染动物种群的必要性。更多还原

【Abstract】 Canine distemper (CD) is caused by canine distemper virus (CDV) which belongs to the genus Morbillivirus within the family Paramyxoviridae, it was occurrence all over the world.The natural host for CDV was canine carnivores, but it has expanded to predominantly carnivores during the past two decades. Recently many wildlife populations were infected with canine distemper virus. CDV belongs to pan-tropic virus, infects different cell types and induce interstitial pneumonia, catarrhal enteritis and non-suppurative encephalitis. Lin Zeng et al (2008) isolated CDV from Rhesus monkeys in the quarantine time. Phylogenetic analysis indicated the CDV in the Rhesus monkeys belonged to the clade of the epidemic CDV types of China. We designed three experiments to compare the clinical symptom, pathological changes and interspecific difference among three Mammals infected with CDV.First, the lung, spleen, small intestine, lymph nodes, brain, liver, and other organizations from the Rhesus monkeys were fixed in 10% neutral buffered formalin and then embedded in paraffin. Serial sections were cut and stained with hematoxylin and eosin (HE) for histopathological examination. Made tissue homogenate and inoculate ferrets and dogs for supernatant solution. We observed the changes in clinical symptoms and pathological autopsy, fixed the tissues and stained for histopathological observation. The results showed that on the 7th day and 8th day after inoculation, ferrets and dogs were both sick and made severe respiratory symptoms and gastroenteritis, several cases showed mild neurological symptoms. Necropsy visible:lung of ferrets were severe congestion and hemorrhage. Lung of dogs were purulent lesions in large area and hemorrhage in the edge. Intestinal tract was catarrhal enteritis and hemorrhage in varying degrees and spleen was minor swelling. Part of ferrets and dogs had showed mild cerebral edema lesions. Rhesus monkeys, ferrets and dogs show obvious interstitial pneumonia by pathology observing. However, lung of ferrets were serious hemorrhage and alveolar space filled with a large number of red blood cells. The lung of dogs showed a severe suppurative pneumonia, pulmonary parenchyma was replaced by connective tissue and inflammatory cells. The intestinal villi of Rhesus monkeys and dogs were injured seriously. Lymphocytes, macrophages and eosinophils were infiltrated in the intestine lamina propria. Ferrets showed catarrhal enteritis. Lesions in the spleen and lymph nodes consisted of focal or diffuse lymphoid depletion and lympholysis were spleen and lymph nodes consisted of focal or diffuse lymphoid depletion and lympholysis were observed. Canine bladder epithelial cells emerged a large number of eosinophilic inclusion bodies. Some ferrets can be seen gliosis. Other tissues. including the cerebrum and cerebellum, were apparently normal. Analyzing the experimental results. this CDV strain can cause serious damage on the respiratory system, digestive system and lymphoid organs of three different animals. Histopathology results proofed that interstitial pneumonia was the common feature of CDV infected containing primates. Ferret exhibited hemorrhagic lesions and dogs easy to form inclusion bodies.Then Immunohistochemistry (IHC) and In Situ Hybridization (ISH) were used to detect CDV virus antigen and virus nucleic acid distribution in the natural incidence of Rhesus monkeys and experimental inoculated animal. We detected tissues of Rhesus monkeys, ferrets and dogs by IHC. The results showed that positive rate are 100%,100%.100%of lung; 100%,100%,100% of small intestine:100%.100%,100% of spleen:100%,100%,9/10 of lymph nodes; 0%,1/6,1/2 of bladder; 0,5/6,1/5 of brain:0,1/3,1/10 of kidney; 0.1/3.0 of liver. The results of ISH detected showed: 100%,100%,100% of lung; 100%,5/6.100% of small intestine:1/3,1/3,2/5 of spleen; 0,1/3, 3/10 of lymph nodes:0,1/3,0 of brain. The results showed that the lung, spleen and lymph nodes in all animals almost positive.In this study, the variability of CDV distribution in different species was not obvious, the respiratory system, digestive tract and lymphoid organ were damaged seriously. However, the disoperation of central nervous system in ferrets was much more serious than Rhesus monkeys. Anyway, it suggested that nonhuman primates infecting with CDV was not occasional, and it was very important and necessary to research the evolutionary basis of host switching of CDV and forecast new animal populations which could infected with CDV.更多还原