【作者】 罗玲；

【导师】 程国富；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 4周龄健康海兰灰蛋鸡100羽随机分成两组：试验组50羽，对照组50羽。试验组经滴鼻-点眼的方式接种H₉N₂亚型禽流感病毒尿囊液0.4ml／羽(含毒量为10⁷-10⁹EID₅₀)，复制禽流感病理模型。对所建立的病理模型进行临床症状、病理组织学变化的检查，在接种后1d、3d、7d、14d、21d测定感染蛋鸡血清生化指标的变化，用微量平板红细胞凝集抑制试验（HI试验）测定不同日龄鸡抗体水平的变化，应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（简称TUNEL）法对感染蛋鸡脾脏、胸腺、法氏囊、盲肠扁桃体进行细胞凋亡的动态变化研究。 临床症状为：接种后第3天，感染蛋鸡精神萎靡，食欲下降，饮水量增加。剖检可见，感染蛋鸡肝脏肿大、出血、淤血，密布针尖大小灰白色或灰黄色坏死灶；肾脏肿大，淤血；肠道粘膜水肿、出血。 分批剖杀接种蛋鸡，组织学检查结果表明：该病毒可引起多种组织细胞出现变性、坏死，尤以肝脏、肾脏的损害最重，且在不同时间内其损伤程度不同。肝：出血、淤血，肝细胞变性、坏死。肾：轻度淤血，肾小管上皮细胞发生严重变性、坏死。心脏：心肌出血、变性。肠道：粘膜上皮细胞变性、坏死，脱落。肺：出血。气管：粘膜层脱落，内有浆液性渗出物。脾、胸腺、法氏囊：淋巴细胞大量减少，部分细胞发生变性、坏死。肝和肾组织进行Mann亚甲蓝伊红染色表明胞质内有嗜酸性包涵体。 电镜观察结果表明：感染蛋鸡胸腺、脾脏、法氏囊、盲肠扁桃体等免疫器官部分细胞出现细胞凋亡的形态学特征，细胞染色质固缩并凝结成块，聚集在核膜周边呈新月状或环状小体。肝、肾组织多数细胞发生坏死，血管内皮细胞发生肿胀、变形。 血清生化指标测定结果表明：接种后第3天，血清中天门冬氨酸氨基转移酶（AST／GOT）、丙氨酸氨基转移酶（ALT／GPT）、磷（P）含量升高。钙（Ca）含量在接种后第7天下降。总蛋白（TP）、白蛋白（ALB）、球蛋白（GLB）、尿酸（UA）和肌酐（CRE）、胆碱酯酶（ChE）无明显变化。HgNZ亚型禽流感病毒人工感染蛋鸡的病理学研究感染蛋鸡Hl抗体水平测一定结果表明:鸡接种后第3天检测出H工抗体，随后持续升高，14天升至最高值1:256，21天下降到1:64。免疫器官细胞凋亡研究结果表明:感染蛋鸡脾脏、胸腺、法氏囊的细胞凋亡率升高，表明禽流感病毒感染诱导了免疫器官的细胞凋亡。 由此作者认为，在蛋鸡感染禽流感病毒的发生发展过程中，病毒通过呼吸道等途径侵入动物机体，随血液循环到达体内特定部位增殖。血管内皮细胞发生变性、坏死和脱落，造成微循环障碍。肝肾组织变性坏死较严重，免疫器官细胞凋亡率显著升高。另一方面，机体的抗体水平升高。这些表明禽流感病毒在使机体致病的同时，也刺激机体的免疫应答。更多还原

【Abstract】 100 4-day old healthy layers were divided randomly into 2 groups, 50 for the tested group and 50 for the control group. The layers of the tested group were inoculated 0.4ml(107-109EID50) H9N2 subtype virus allantoic fluid of poultery embryo. In a chicken model of layers inoculated with H9N2 subtype avian influenza virus, clinical signs and histopathology were observed and transmissional electron microscope were carried out on the tissues of inoculated layers. At the same time, apoptosis of the immune organ in layers infected with avian influenza virus was demostrated by the nick end-labelling of DNA in situ (TUNEL). Meantime, the serum biochemical value and antibody level were determined after inoculation by intranasal-intraocular.The results showed that layers were dispirited, began to eat little and drank more on 3 day post inoculation. Liver had a change of swelling, and appeared white and yellow necrosed focus. Swelling and petechchiae appeared in kidney and intestines.Histopathological changes showed that liver had disseminated hemorrhage, congestion, Hepatocytes showed degeneration, necrosis. Heavy degeneration and necrosis occurred in the renal tubular epithelial cells. The pathological changes of lung and heart showed hemorrhage. Swelling and hemorrhage occurred in the mucosa of intestines, and there were degeneration, necrosis in the mucosa epithelial cells of intestines. Mucosa were deprived from trachea. The number of lymphocyte reduced sharply in spleen, thytnus and bursa of Fabricius. Eosinophilic inclusion bodies were found in the cytoplasm of hepatocytes and the renal tubular epithelial cells by Mann staining methods for liver and kidney.Transmissional electron microscope showed that some cells had the morphological characteristics of siginificant apoptosis in the spleen, thymus, bursa of Fabricius and caecum tonsil, and morphological changes were condensation of nuclear chromatin into dense masses, followed by itsmarginating against the nuclear envelope, and concentration of cytoplasm association with lots of cytoplasmic vacuolization. Cytoplasm was swollen, cell nuclear was dissolved in the hepatocytes and the renal tubular and epithelial cells. The swelling and distortion appeared in the intravascular cells.The results of the serum biochemical value showed that the level of serum AST, ALT and P began to go up on 3 PID. The value of Ca went down on 7 PID. The level of TP, ALB, GLB, UA, CRE or ChE had no significant differences within the whole period.Antibody against H9N2 subtype AIV was tested with the hemagglutinin inhibition(HI)and the findings indicated that HI antibody began to rise on 3 PID and rose to maximum (1:256) on 14 PID. HI antibody reduced to 1:64 on PID later.The apoptosis levels of immune organs were dynamically observed in the experimental inoculation layers. The result showed that apoptosis levels of spleen, thymus and bursa of Fabricius rose distinctively. The results showed AIV could induce apoptosis of the immune organs in vivo.So we could see that virus followed into the tissues of layers and reproduced in sensitive organs or tissues during the infection. Heavy degeneration and necrosis occurred in liver and kidney. Meantime, apoptosis levels increased sharply in the immune organ. On the other hand, HI antibody levels rose distinctively. The results showed AIV could induce apoptosis and immune reaction in vivo.更多还原