【作者】 王彬；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2003， 硕士

【摘要】 本研究以养殖场、临床病料、采集雏鹅或成年鹅的泄殖腔分泌物或肠道、肝脏、脾脏作为分离样品，通过离心和抗生素处理灭菌后，接种鸭胚，以血球凝集(HA)和血球凝集抑制(HI)的试验方法检测病毒。试验结果表明，50份样品中有2份样本分离到腺病毒，分离率为4％。将分离到的两株分离物病毒分别命名为H5J24和N8J8。HA和HI试验结果表明，该2株分离物病毒能够凝集鸡、鸭、鹅的红细胞，且这种凝集作用可被抗Y81G4株鹅腺病毒多抗血清所抑制，但抗新城疫(ND)病毒的单克隆抗体(单抗)、抗H5和H9亚型禽流感病毒(AIV)的单抗对其无抑制作用。透射电镜进行经形态学观察，结果发现大小约为78nm、球形、无囊膜的病毒粒子。理化特性测定结果表明，分离到的病毒血凝素蛋白对50％氯仿、0.25％胰酶、pH3.0和pH9.0处理1小时以及50℃ 30分钟表现稳定，而60℃ 45分钟和100℃ 1分钟可使其失活。根据Ⅲ群腺病毒EDS标准毒株基因组序列，设计并合成一对可扩增出E3区的PCR引物，实验证明可以扩增出特异性核酸片段。这些结果证明该两株分离物病毒为Ⅲ群禽腺病毒。 腺病毒E3区两端分别是保守性较强的pⅧ蛋白基因和纤维蛋白基因。通过特定的引物，用PCR方法从2株鹅腺病毒基因组中扩增出E3区并进行了序列测定。结果表明，这2株病毒分离物E3区的长度较短，全长约为240扬州大学硕士论文个核昔酸，不具有明显的编码框。与EDS一76以及其他腺病毒的E3区比较结果提示，此2株鹅腺病毒应归类于m群禽腺病毒。 为了弄清楚这2株病毒的病原性，我们通过肌肉注射感然8日龄肉鹅，进行人工致病性实验.结果发现，实验鹅在感染病毒后，未出现任何明显临床症状，提示两株病毒的病原性较弱。对这些人工感染鹅进一步研究，通过在不同时间段的排毒、带毒情况及血清中抗体水平检测，证明病毒能够在实验鹅体内存在并持续排毒，同时实验鹅血清中循环抗体能够较长时间维持在2”一2”的较高水平.组织病理学切片观察结果表明，病毒可以在内脏组织器官引起一定的病理变化.如肾小管上皮细胞颗粒变性，有些细胞出现脂肪变性，有些发生充血或轻微出血;肺三级支气管上皮细胞坏死脱落，管腔内有脱落的上皮细胞及淋巴细胞，肺房有大量红细胞及少盘的淋巴细胞:脾脏有轻度充血和出血;小肠部分猫膜层与肌层剥离，肠猫膜上皮细胞坏死、脱落，小肠官腔内有坏死脱落的上皮细胞，有的肠绒毛脱落，固有层肠腺体上皮细胞发生水泡性变性，固有层、猫膜下层有淋巴细胞浸润，肌层血管有充血;肝细胞水泡变性、坏死.这些结果提示，我们分离到的两株分离物病毒对小鹅没有临床致病作用，但仍有一定的毒力。更多还原

【Abstract】 Cloacae secretion of healthy geese, livers, spleens and guts of clinical young or adult geese were collected for viruses isolation. These samples were sterilized by centrifuge and antibiotics and then inoculated into duck embryos. Two virus strains named H5J24 and N8J8 respectively were isolated from 50 samples and identified by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The isolation rate is 4%. Both viruses could agglutinate 0.8% red blood cells (RBC) of chicken, duck or goose. The agglutination activity could be inhibited by the serum against virus Y81G4 isolated from goose, but not by the monoclonal antibodies to Newcastle disease virus, Avian influenza virus subtype H5 or H9. The virus particles are non-enveloped, 78nm diameter , and have icosahedrad symmetry visible in the transmission electron microscope(TEM) by negative stain. Based on the morphology, physical and chemical properties of the viruses and PCR analysis, the viruses were located at group III Adenoviruses.Adenoviruse E3 region locates between the conservative genes pVIII and Fiber. We designed a pair of primers to amplify the fragement from two isolates by PCR. The results showed that E3 regions of the 2 virus strains was about240bp in size. There was no distinct opening reading frame (ORF) in sequence analysis with DNAstar software. The 2 isolate strains from geese were further classified as Group III Avianadenovirus compared with the E3 region sequences of EDS-76 and other adenovirus.The 2 isolate virus strains were experimentally inoculated into 10-day-old goslings in order to understand their pathogenicity. During different infection phases, the experimental geese did not exhibit any clinical syndrome. This result suggested that their pathogenicity were very weak for geese. Following detection made it clear that the 2 virus strains could long exist in geese and be expelled into environment for long time. Meanwhile the serum antibodies were maintained at a high level. The tilers of HI of the serum were about 211~212. Based on the tissue pathology, the 2 virus strains still could induce pathological changes in some organs, such as kidney, spleen, small intestine and lung.更多还原