鸡流行性腹泻病原的分离鉴定及特性研究

Isolation and identification and characteristics of the pathogen of avian epidemic diarrhea

用胰蛋白酶处理发病鸡的粪便和肠内容物 ,接种Marc14 5细胞 ,盲传数代后 ,从山东不同地区发生流行性腹泻的鸡群中分离到轮状病毒 ,并对分离的轮状病毒的生物学特性和理化特性进行了部分研究。结果表明病毒粒子的形态呈车轮状、大小为 70～ 80nm ;其病毒基因组的电泳图谱为 5∶1∶3∶2 ;病毒在Marc14 5细胞上传到第 9代时的TCID50 为10 _4.62 /0 .1mL ;该分离株病毒对氯仿、乙醚有抵抗力 ;对pH3.0处理 6 0min稳定 ;5 0℃ 30min能使其感染力下降 10 2 ;1mol/LMgCl2 不能增强其对 5 0℃ 6 0min的抵抗力。动物回归试验中接种两周龄SPF鸡 ,2 4h后陆续发病 ,表现为持续性水样腹泻 ,与自然发病相同 ;剖检可见病鸡脱水、小肠内有大量的液体和气泡、肠粘膜变薄 ;组织学变化为肠绒毛上皮坏死、脱落 ,绒毛平均长度减少而隐窝深度增加 ,固有层中淋巴细胞浸润。其临床症状及病理组织学变化与自然发病相同。因此确定发生在山东鸡流行性腹泻的病原为轮状病毒。

Avian rotavirus was isolated from chicken farms with natural diarrthoea in different districts of Shandong province.Isolation and culture of the rotavirus was attemped by the inoculating on monolayers of Marc145 cells with a suspension of faecal material containing the rotavirus.Marc145 cells were found to be suitable for primary isolation and propagation .While trypsin appeared to be essential for the enhancement of infectivity and the occurance of cytopathic effect(CPE).Serum neutralization (SN),electron microscopy(EM) and analysis of genomic RNA were done to identify and confirm the identity of the rotavirus. The rotavirus isolated from the intestinal contents of commercial chickens was determined to be group A rotavirus based on RNA electropherotype and neuralization test.The average size for the isolated rotavirus was 80 nm for double-shelled particles and 70 nm for single-shelled particles. Further characterization revealed that the buoyant density in saccharose was 1.35 g/cm_3.The TCID50 was 10^(-4.62) /ml when avian rotavirus was propagated for nine passages.The rotavirus was resistant to chloroform ,ether and pH3.0 for 60 min.The infectivity was decreased when processed at 50℃ for 30 min;MgCl_2 can not enhance the resistant to 50 ℃ for 60 min. The clinical symptoms and histology of chickens with epidemic diarrhea in natural and artificial infection were studied. Specific Pathogen-free (SPF) chickens developed diarrhea 1 day after inoculation with avian rotavirus. The characteristic changes at necropsy were dilated ceca, frothy gaseous intestinal contents and shedding of intestinal mucous membrane.The histologic lesions were characterized by the remarkable atrophy of intestinal villi, basal vacuolation of enterocytes; the depth of crypt increased and the villus-to-crypt ratio had significantly decrease,compared with that of noninoculated control chickens.In addition,intestinal villus become short and flat,or cuboid,and nccropic.Many villous enterocytes were detached from the lamina propria and the lamina propria contained a diffuse infiltrate of lymphocytes ,plasma cells and macrophages.The characteristic ultrastructural changes observed by electronmicroscope were swelled mitochondria and dilated endoplastic reticulum.Ribosome on the surface of endoplastic reticulum fell off and the number of ribosomes within the cell decreased.