鸭出血性大肠杆菌O46分离株实验病理模型的建立

Establishment of an Experimental Pathological Model by Using EHEC O46 Isolate in Duck

【目的】为了建立鸭源出血性大肠杆菌(Enterohaemorrhagic E.coli,EHEC)O46分离株的实验病理模型并观察其在雏鸭体内的动态分布及组织病理学和超微病理学变化。【方法】本研究以O46分离株通过口服、肌肉和皮下注射3种途径感染10日龄健康雏鸭,感染剂量均为0.5 mL/只(2×108CFU·mL-1),感染后2、4、6、12、24h剖杀、取样,以后每隔12h剖杀、取样并对剖解变化进行详细观察,在每个安排的时间点平行采集2只雏鸭的心、肝、脾、肺、肾、脑、食道、胸腺、十二指肠、空肠、盲肠、直肠、法氏囊、胰腺和气管等组织制备组织切片和超薄切片,通过H.E染色、醋酸铀、柠檬酸铅染色和免疫组化染色,对感染雏鸭的组织病理学变化及超微病理变化、细菌抗原定位进行观察。【结果】实验病理模型能复制出与自然感染相同的病例,除气管未检测到细菌抗原外,其余的组织均检测到细菌抗原,心、肺、脾、肾和肠道是感染的主要靶器官,抗原主要存在其感染细胞的细胞质中,阳性信号最早出现于心脏。尸体剖解、组织病理学及超微病理学观察表明,雏鸭人工感染鸭源EHEC O46分离株后主要病理学损害为浆膜广泛性纤维素性炎症,心、肝、脾、肺、肾、法氏囊、小肠、胰腺、脑等器官充血、出血、炎性细胞浸润,肾小管上皮细胞、肝细胞、心肌细胞、肠上皮细胞等实质细胞变性、坏死或凋亡,法氏囊淋巴细胞减少。超薄切片电镜观察可在心、肺、脾、肝和小肠中观察到大肠杆菌,其侵害的主要靶细胞包括淋巴细胞、巨噬细胞、肠道上皮细胞、心肌纤维细胞。【结论】鸭源EHEC O46分离株能使雏鸭发病和死亡,实验病理模型能复制出EHEC引起的出血性肠炎和溶血性尿毒症等病理变化,该分离株能在鸭体内进行大面积的侵嗜,心脏、肺脏、脾脏、肾脏和肠道是O46分离株感染的主要靶器官。

[ Objective ] This experiment was conducted to build an experimental pathological model of EHEC 046 isolate and observe its dynamic distribution, and the dynamic changes of histopathology and ultra-structural pathology in ekperimentally infected ducklings. [ Method ] The 10-day-old ducklings were infected with 0.5 mL of 046 isolate （2 x 108CFU-mLI ） through oral, intramuscular and subcutaneous injection separately. Tissue samples of heart, liver, spleen, lung, kidney, cerebra, esophagus, thymus, duodenum, jejunum, cecum, rectum, bursa of fabricius, pancreas and trachea were taken from the infected ducklings after being infected for 2, 4, 6, 12, and 24 h, and ever since for every 12 hours to make tissue biopsies and ultrathin sections, which were then stained with haematoxylin and eosin （H.E）, as well as uranyl acetate, lead citrate and immunohistochemistry to observe the changes of histopathology, ultra-structural pathology and the bacterial antigen localization in experimentally infected ducklings. [ Result] Same clinical symptoms as with naturally infected cases were duplicated in experimental pathological model. Bacterial antigens were detected in different tissues except trachea. The target tissues were hearts, lungs, spleens, kidneys and intestinal tracts where bacterial antigen located in cytoplasm and the positive signals were found in heart firstly. Necropsy, histopathological changes and ultra-structural pathological changes showed that main pathological damages of EHEC 046 isolate in ducklings were as follows： serous generalized fibrinous infammation could be seen; hyperemia, hemorrhage and inflammatory cell infiltration were presented in heart, liver, spleen, lung, kidney, bursa of fabricius, intestinum tenue, pancreas and cerebrum; histopathological changes of necrosis and apoptosis were displayed in parenchyma cell such as renal tubular epithelial cell, hepatocyte, cardiomyocyte and enterocyte; reduction of lymphocytes could be seen in bursa of fabricius. E.coli was observed in heart, lung, spleen, liver, and intestinum tenue by SEM. The target tissue cells where isolate located were lymphocytes, macrophages, enterocytes and cardiac fibroblasts. [Conclusion] EHEC 046 isolate in duck showed high pathogenicity in ducklings. Pathologcial changes such as hemorrhagic enteritis and hemolytic uremic syndrome caused by EHEC were duplicated in experimental pathological model. It was concluded that 046 isolate was the pantropic bacteria injuring most tissues and cells in ducklings, and the principal target organs were heart, lung, spleen, kidney and intestine.