禽戊型肝炎病毒与马立克病毒混合感染的鉴定及禽戊肝病原学调查

发布时间：2018-05-12 01:00

本文选题：禽戊肝病毒 + 马立克病毒　；参考：《山东农业大学》2017年硕士论文

【摘要】：禽戊型肝炎病毒(Hepatitis E virus,HEV)是鸡大肝大脾病(Big liver and spleen disease,BLS)或肝脾肿大综合征(Hepatitis-Splenomegaly,HS)的主要病原,通常造成30~72周龄的蛋鸡和肉种鸡产蛋率下降以及死淘率升高。马立克病毒(Marek’s Disease virus,MDV)是常见的禽肿瘤病病毒之一,引发鸡的高度接触性恶性淋巴肿瘤性的马立克病(Marek’s Disease),感染鸡群造成鸡的免疫抑制给家禽养殖业造成很大的经济损失。目前国内鸡群感染禽HEV的报道较少,还没有发现蛋鸡群中存在禽HEV。MDV与REV、ALV等双重或多重混合感染的病例报道显示出,MDV与其他病毒的共感染比单独MDV感染造成的危害更大。截至目前没有禽HEV与MDV共感染的报道。本研究从患有大肝大脾病的海兰褐蛋鸡群采集发病鸡25只和表观健康鸡5只。鸡只进行剖检并制备病理组织切片HE染色。剖检发现肝脾肿大,并有肿瘤存在,病理组织学观察发现肝细胞水泡变性普遍,个别小血管周围有数量不多的、大小不一的淋巴细胞聚集,肝窦内出现零星嗜异白细胞,表现出典型MD症状并伴有炎症反应。无菌采集30只鸡的抗凝血,离心取其靠近白细胞层的血浆接种CEF,培养7~9d后,细胞上清用于ELISA试剂盒检测ALV-P27抗原,细胞用于间接免疫荧光试验(IFA)。ELISA试验结果表明,所有样品均为ALV-P27抗原阴性。IFA试验结果表明,用MDV的单抗H19检测到阳性细胞,而用ALV、REV的单抗JE9和11B118未检测到阳性信号。该结果表明培养的细胞中存在MDV野毒。提取接种的CEF DNA和30只鸡肝脏DNA,用特异性的5对引物PCR扩增检测MDV、ALV、REV。PCR扩增试验结果显示,MDV阳性率为46.7%(14/30),而ALV、REV全部为阴性。对部分MDV阳性样品进行克隆测序并分析所得序列。结果表明MDV分离株meq基因序列与经典的MDV参考株的同源性为98.7%~100%。分析碱基序列发现MDV分离株为强毒株特征,其575-577位点的碱基为CAC;在meq基因的多脯氨酸功能区EELCAQLCSTPPPPI重复序列中没有脯氨酸(P)的缺失。综合分析结果可以判定分离株来源于野毒。提取送检鸡肝脏样品RNA,反转录为cDNA,通过套式PCR方法扩增禽HEV ORF2部分基因序列并对阳性样品进行克隆测序,根据得到的禽HEV ORF2部分基因序列,比较其与国内外参考株的同源性以及确定基因型。RT-PCR检测结果显示,发病鸡肝脏样品中禽HEV检出率为56%(14/25),表观健康鸡禽HEV检出率为80%(4/5)。测序结果显示样品禽HEV ORF2基因片段与国内外经典参考株的同源性为77.3%~98.3%,属于禽HEV基因3型。取禽HEV RNA阳性鸡的肝脏与PBS缓冲液混合研磨并过滤,肝脏悬液作为病毒,翅静脉注射到4只两周龄SPF鸡,对照组SPF鸡注射生理盐水。饲养至14天采集肝脏,提取RNA并反转录,进行HEV RNA的鉴定,对阳性结果克隆测序以确定病毒来源。采集的肝脏样品,套式PCR方法检测到攻毒组有250bp大小的目的条带,所得序列与攻毒肝脏悬液序列同源性为100%,而对照组未检测到禽HEV RNA。该检测结果表明,用于攻毒的脏悬液中禽HEV具有完整的生物活性可以再次传染给鸡。为了解禽HEV在肿瘤病发病的种鸡场中的感染率,对七个肿瘤病发病的种鸡场,进行禽HEV病原学调查。从该禽HEV阳性海兰褐蛋鸡的父母代种鸡群采集粪便40份和抗凝血20份,另外从其他6个发生肿瘤病的种鸡场采集粪便和鸡只等样品,共165份样品。提取样品RNA,反转录后进行禽HEV的套式PCR检测。结果显示,来自该海兰褐父母代种鸡群的粪便样品中禽HEV检出率为67.5%(27/40),血浆样品中禽HEV检出率为20%(4/20),而从其他6个种鸡场采集的样品中没有检测到禽HEV。随机挑选两个样品克隆测序。序列比对结果显示,与分离自该海兰褐蛋鸡的3个禽HEV序列同源性为98.3%~100%,进化树分析显示在同属于基因3型,并在同一分支。为调查市场活禽和野生鸟类禽HEV感染的状态,给禽HEV的防控和野生鸟类疾病监测提供数据参考。东亚-澳大利亚迁徙路线经过我国南北两点:黄河三角洲自然保护区和江苏盐城湿地珍禽自然保护区。2014年~2016年共采集野生鸟类和活禽市场粪便样品2691份,提取RNA并反转录后,荧光定量PCR方法检测禽HEV RNA。结果显示,未检测到阳性样品,可疑样品7份。通过套式PCR将可疑样品进行验证,结果发现全部没有扩增出目的条带。本次调查未发现采集的野生鸟类和市场活禽样品中存在禽HEV。
[Abstract]:Hepatitis E virus (HEV) is the main pathogen of chicken liver splenomegaly (Big liver and spleen disease, BLS) or hepatomegaly syndrome (Hepatitis-Splenomegaly, HS). It usually causes the decline of egg production rate and the increase of the rate of death in the laying hens and broilers. The Marek virus is a common cause. One of the avian tumor virus (Marek "s Disease) caused by the chicken's highly contagious malignant lymphatic tumor (Marek 's), the infection of chickens caused by chicken group caused great economic loss to poultry breeding industry. At present, there are few reports on avian HEV in chickens in domestic chickens, and there is no avian HEV.MDV and REV, ALV in laying hens. The case reports of double or multiple mixed infection showed that the co infection of MDV and other viruses was more harmful than that of individual MDV infection. There were no reports of coinfection between avian HEV and MDV. This study collected 25 hens and 5 epigenetic chickens from the group of hens with great hepatomegaly. The liver splenomegaly was found in the liver and splenomegaly, and the tumor was found in the liver and splenomegaly. The histopathological observation found that the liver cell was denatured, the number of the small blood vessels around the small vessels was not much, the size of the lymphocytes gathered, the hepatic sinusoids appeared in the hepatic sinusoids, and the typical MD symptoms were accompanied by inflammatory reaction. 30 chickens were collected asepically. 30 chickens were collected asepically. The anticoagulant, centrifugally taken the plasma near the white cell layer, was inoculated with CEF, and after the culture of 7~9d, the cell supernatant was used to detect the ALV-P27 antigen by the ELISA kit. The cells were used for the indirect immunofluorescence test (IFA).ELISA test. All the samples were found to be ALV-P27 antigen negative.IFA test results, and the positive cells were detected by MDV McAb H19, and the results of the test of ALV-P27 antigen negative.IFA test showed that the positive cells were detected by MDV McAb H19. The positive signals were not detected by the monoclonal antibodies JE9 and 11B118 of ALV, REV. The results showed that there were MDV wild poison in the cultured cells. The CEF DNA and 30 chicken liver DNA were extracted, and the specific 5 pairs of primers were used to detect MDV, ALV, and REV.PCR amplification test results showed that the positive rate was 46.7%. The sexual samples were cloned and sequenced and analyzed. The results showed that the homology of the meq gene sequence of the MDV isolate and the classic MDV reference strain was that the MDV isolate was the strong strain of the 98.7%~100%. analysis base sequence. The base of the 575-577 site was CAC, and there was no in the EELCAQLCSTPPPPI repeat sequence of the meq gene in the function region of the proproline function. The comprehensive analysis results can determine the origin of the isolated strain from wild virus. RNA of chicken liver samples was extracted and sent back to cDNA. The sequence of partial gene sequence of avian HEV ORF2 was amplified by nested PCR method and the positive samples were cloned and sequenced. According to the sequence of partial gene of avian HEV ORF2, it was compared with the domestic and foreign reference strains. The results of origin and determination of genotype.RT-PCR showed that the detection rate of avian HEV in chicken liver samples was 56% (14/25), and the detection rate of HEV in apparent healthy chickens was 80% (4/5). The sequencing results showed that the homology of the HEV ORF2 gene fragment from the domestic and foreign classical reference strains was 77.3%~ 98.3%, which belonged to the avian HEV gene 3. The avian HEV RNA positive chicken was taken. The liver and PBS buffer were mixed and filtered, the liver suspension was used as a virus, 4 two weeks old SPF chickens were injected into the wing vein, and the control group SPF chickens were injected with physiological saline. The liver was collected for 14 days, and RNA was extracted and reverse transcribed. The identification of HEV RNA was carried out. The positive results were cloned and sequenced to determine the source of the virus. The liver samples collected and the set PCR method were collected. It was detected that the target band of the attack group had 250bp size, and the homology of the obtained sequence and the sequence of the liver suspension was 100%, while the control group did not detect the avian HEV RNA.. The results showed that the bird HEV in the dirty suspension used for the attack could be transmitted to the chicken again. The infection rate, the avian HEV pathogen investigation was carried out on the chicken farm of seven oncological diseases. 40 feces and 20 anticoagulants were collected from the parents of the HEV positive hens of the fowl, and 165 samples were collected from the other 6 other kinds of oncology chicken farms. The samples were extracted and RNA was extracted, and the avian HEV was carried out after reverse transcription. The results showed that the detection rate of avian HEV was 67.5% (27/40) and 20% (4/20) in the plasma samples from the faecal samples from the broiler chick group, and two samples from 6 other chicken farms were not detected randomly and two samples were cloned and sequenced. The sequence alignment results showed that it was isolated and isolated from the samples. The 3 avian HEV sequence homology of the hens was 98.3%~100%. The phylogenetic tree analysis showed the same belongs to the gene 3, and in the same branch. It provides data reference for the prevention and control of avian HEV and the surveillance of wild bird disease. The East Asia Australia migration route passes through two of the north and south of our country, in order to investigate the state of HEV infection of live birds and wild birds in the market. Points: 2691 fecal samples of wild birds and live birds were collected in the Yellow River Delta Nature Reserve and Jiangsu Yancheng Wetland Nature Reserve in ~2016 year.2014. After extracting RNA and reverse transcription, fluorescence quantitative PCR method was used to detect the results of avian HEV RNA.. The positive samples were not detected and 7 suspected samples were not detected. The suspicious samples were entered through nested PCR. The results showed that no target bands were found. No avian HEV. was found in the wild birds and live poultry samples collected from the survey.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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