【作者】 孟琼华；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 本研究以实验病理学方法对人工感染鹅源1型鸭疫里默氏杆菌（RA）的21日龄的雏鹅进行了系统病理组织学的研究，建立了RA感染雏鹅的病理模型。感染30小时后，急性死鹅1只，其他鹅出现典型的临床症状；感染46到72小后，半数鹅死亡，达到死亡高峰；感染9天后，20％鹅耐过。大体解剖病变为全身各组织器官的浆膜面广泛的纤维素性渗出；肝脏、胸腺肿大；脾脏、法氏囊感染前期肿大，感染后期萎缩；肠道出血，盲肠前期出血，后期畸变；皮下充血出血，并有胶冻样浸润，脑充血。病理组织学变化为纤维素性坏死性心肌炎、肝周炎、脑膜炎；急性肾小管坏死性肾炎、胰腺炎、肠炎；出血性胸腺炎、肺炎、脾炎等；盲肠扁桃体、脾脏、胸腺淋巴滤泡中淋巴细胞减少；除胰腺外，所有检测的组织中都有大量嗜中性白细胞出现。 本研究建立了间接免疫组化（Indirect Immunoperoxidase Staining,IIS）检测RA抗原的方法，该法的最适条件为：以PLP为固定液，以APES为粘片剂，以0.3％的甲醇-H₂O₂在湿盒中室温下孵育20分钟消除组织中的过氧化物酶及假过氧化物酶活性，兔抗RAIgG与羊抗兔—HRP的稀释比例均为1：25，以4℃过夜作兔抗RAIgG的孵育条件，以10％白蛋白0.05％Tween-20pH7.4的PBS湿盒内室温孵育60分钟处理组织中非特异性染色因素；以TBST为洗涤液，空气振荡器70转/分钟摇洗两次，每次10分钟洗涤切片；以0.5％甲苯胺蓝作为复染液。该方法用于检测感染雏鹅组织器官中的RA抗原具有特异、敏感、直观的优点。 本研究应用建立的间接免疫组化法对RA人工感染21日龄雏鹅后RA抗原在雏鹅各组织器官的分布规律检测结果表明，鹅人工感染RA24小时后，心脏、肝脏、脾脏、肾脏、回肠和直肠RA抗原呈强阳性分布，其次在肺脏、十二指肠、空肠和盲肠RA抗原呈弱阳性分布，胰腺、胸腺、法氏囊和大脑为阴性。在感染46小时后，所有检测器官中都呈阳性，大部分RA抗原阳性组织（肾脏、肺脏、胸腺、十二指肠、回肠、大脑）中都出现嗜甲苯胺蓝物质。在感染58小时后，各个器官RA抗原呈强阳性分布。感染第5天后肝细胞和肺房上皮细胞RA抗原分布呈阳性，感染9天后，肝、肾、胸腺的阳性强度没有下降，其他器官的阳性强度都下降。整个感染过程中，空肠和胰腺RA抗原阳性强度分布较低，大脑RA抗原阳性分布最低。各组织器官中RA抗原阳性分布呈现从间质到实质、从血管及血管周围到组织细胞规律分布的特点。RA感染对鹅的实质器官、肠道、免疫器官都有严重的损伤；RA抗原的分布与RA感染途径及其机体的抵抗力有关；RA对雏鹅机体组织器官的损伤程度与RA抗原的分布阳性强度及其嗜中性白细胞的浸润呈正相关。更多还原

【Abstract】 Geese were artificially infected with Remerella anatipestifer serotype 1 at 21 days old to study the morphological pathology and to establish indirect immunohistochemistry methods to detect RA antigen and RA antigen distribution in infected geese. 30 hours post infected(PI) ,one goose was died and most of the others demonstrated clinical signs which were similar to that of duckings infected with RA; 46 to 72 hours PI, 50% of the goose showed highest mortality; 22 days PI20% of the geese survived. The gross pathological changes were as follows: fibrins on the seromembrane of all the organs, especially on the heart, liver, air sac and meningitis; swelling of liver and thymus ,swelling of spleen and bursal of Fabricius in early period and atrophy in the later stage, hyperemia and hemorrhage of the intestine, aberration of cecum in the later stage. Many parenchyma organs of infected geese showed fibrins and necrosis inflammation. Intestine showed hemorrhage inflammation. Few lymphocyte was found in spleen, thymus, bursa of fabricius and cecal tosil. Neutrophils infiltration could be seen in most of the organs.The best conditions for indirect immunochemistry were as follows: Fixing specimens with PLP, processing slides with APES, eliminating the activity of catalase and its similar enzyme with 0.3% formaldehyde-H2O2, blocking the tissue section with 10% albumin-tween-20 in humidity, restaining with toluidine blue. It was found that this method showed great specificity and sensitivity.The result of indirect immunochemistry demonstrated that: 24 hours PI, heart, liver, spleen, kidney, ileum, rectum, lung, duodenum and cecum were positive; 46 hours PI, all the organs showed positive results and some of the organs showed heterophile substances which could be stained by toluidine blue; 58 hours PI, the positive results were most obvious in all organs; 5 days PI, alveloar endothelial cells and hepatocytes showed positive in immunochemistry detection; 9 days PI, liver, kidney and thymus still showed strong results in immunohistochemistry detection while that of other organs began to decline; jejunum and pancreas showed less obvious results compared with other organs listed above in Immunohistochemistry detectionand brain was the least obvious.In summary, RA infection in geese could cause severe damages to parenchyma organs, intestines and the immune organs; pathological lesions were correlated with the distribution of RA antigen and neutrophils.更多还原