本文選題：禽戊肝病毒 + 馬立克病毒�。� 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：禽戊型肝炎病毒(Hepatitis E virus,HEV)是雞大肝大脾病(Big liver and spleen disease,BLS)或肝脾腫大綜合征(Hepatitis-Splenomegaly,HS)的主要病原,通常造成30~72周齡的蛋雞和肉種雞產(chǎn)蛋率下降以及死淘率升高。馬立克病毒(Marek’s Disease virus,MDV)是常見的禽腫瘤病病毒之一,引發(fā)雞的高度接觸性惡性淋巴腫瘤性的馬立克病(Marek’s Disease),感染雞群造成雞的免疫抑制給家禽養(yǎng)殖業(yè)造成很大的經(jīng)濟(jì)損失。目前國內(nèi)雞群感染禽HEV的報(bào)道較少,還沒有發(fā)現(xiàn)蛋雞群中存在禽HEV。MDV與REV、ALV等雙重或多重混合感染的病例報(bào)道顯示出,MDV與其他病毒的共感染比單獨(dú)MDV感染造成的危害更大。截至目前沒有禽HEV與MDV共感染的報(bào)道。本研究從患有大肝大脾病的海蘭褐蛋雞群采集發(fā)病雞25只和表觀健康雞5只。雞只進(jìn)行剖檢并制備病理組織切片HE染色。剖檢發(fā)現(xiàn)肝脾腫大,并有腫瘤存在,病理組織學(xué)觀察發(fā)現(xiàn)肝細(xì)胞水泡變性普遍,個(gè)別小血管周圍有數(shù)量不多的、大小不一的淋巴細(xì)胞聚集,肝竇內(nèi)出現(xiàn)零星嗜異白細(xì)胞,表現(xiàn)出典型MD癥狀并伴有炎癥反應(yīng)。無菌采集30只雞的抗凝血,離心取其靠近白細(xì)胞層的血漿接種CEF,培養(yǎng)7~9d后,細(xì)胞上清用于ELISA試劑盒檢測ALV-P27抗原,細(xì)胞用于間接免疫熒光試驗(yàn)(IFA)。ELISA試驗(yàn)結(jié)果表明,所有樣品均為ALV-P27抗原陰性。IFA試驗(yàn)結(jié)果表明,用MDV的單抗H19檢測到陽性細(xì)胞,而用ALV、REV的單抗JE9和11B118未檢測到陽性信號。該結(jié)果表明培養(yǎng)的細(xì)胞中存在MDV野毒。提取接種的CEF DNA和30只雞肝臟DNA,用特異性的5對引物PCR擴(kuò)增檢測MDV、ALV、REV。PCR擴(kuò)增試驗(yàn)結(jié)果顯示,MDV陽性率為46.7%(14/30),而ALV、REV全部為陰性。對部分MDV陽性樣品進(jìn)行克隆測序并分析所得序列。結(jié)果表明MDV分離株meq基因序列與經(jīng)典的MDV參考株的同源性為98.7%~100%。分析堿基序列發(fā)現(xiàn)MDV分離株為強(qiáng)毒株特征,其575-577位點(diǎn)的堿基為CAC;在meq基因的多脯氨酸功能區(qū)EELCAQLCSTPPPPI重復(fù)序列中沒有脯氨酸(P)的缺失。綜合分析結(jié)果可以判定分離株來源于野毒。提取送檢雞肝臟樣品RNA,反轉(zhuǎn)錄為cDNA,通過套式PCR方法擴(kuò)增禽HEV ORF2部分基因序列并對陽性樣品進(jìn)行克隆測序,根據(jù)得到的禽HEV ORF2部分基因序列,比較其與國內(nèi)外參考株的同源性以及確定基因型。RT-PCR檢測結(jié)果顯示,發(fā)病雞肝臟樣品中禽HEV檢出率為56%(14/25),表觀健康雞禽HEV檢出率為80%(4/5)。測序結(jié)果顯示樣品禽HEV ORF2基因片段與國內(nèi)外經(jīng)典參考株的同源性為77.3%~98.3%,屬于禽HEV基因3型。取禽HEV RNA陽性雞的肝臟與PBS緩沖液混合研磨并過濾,肝臟懸液作為病毒,翅靜脈注射到4只兩周齡SPF雞,對照組SPF雞注射生理鹽水。飼養(yǎng)至14天采集肝臟,提取RNA并反轉(zhuǎn)錄,進(jìn)行HEV RNA的鑒定,對陽性結(jié)果克隆測序以確定病毒來源。采集的肝臟樣品,套式PCR方法檢測到攻毒組有250bp大小的目的條帶,所得序列與攻毒肝臟懸液序列同源性為100%,而對照組未檢測到禽HEV RNA。該檢測結(jié)果表明,用于攻毒的臟懸液中禽HEV具有完整的生物活性可以再次傳染給雞。為了解禽HEV在腫瘤病發(fā)病的種雞場中的感染率,對七個(gè)腫瘤病發(fā)病的種雞場,進(jìn)行禽HEV病原學(xué)調(diào)查。從該禽HEV陽性海蘭褐蛋雞的父母代種雞群采集糞便40份和抗凝血20份,另外從其他6個(gè)發(fā)生腫瘤病的種雞場采集糞便和雞只等樣品,共165份樣品。提取樣品RNA,反轉(zhuǎn)錄后進(jìn)行禽HEV的套式PCR檢測。結(jié)果顯示,來自該海蘭褐父母代種雞群的糞便樣品中禽HEV檢出率為67.5%(27/40),血漿樣品中禽HEV檢出率為20%(4/20),而從其他6個(gè)種雞場采集的樣品中沒有檢測到禽HEV。隨機(jī)挑選兩個(gè)樣品克隆測序。序列比對結(jié)果顯示,與分離自該海蘭褐蛋雞的3個(gè)禽HEV序列同源性為98.3%~100%,進(jìn)化樹分析顯示在同屬于基因3型,并在同一分支。為調(diào)查市場活禽和野生鳥類禽HEV感染的狀態(tài),給禽HEV的防控和野生鳥類疾病監(jiān)測提供數(shù)據(jù)參考。東亞-澳大利亞遷徙路線經(jīng)過我國南北兩點(diǎn):黃河三角洲自然保護(hù)區(qū)和江蘇鹽城濕地珍禽自然保護(hù)區(qū)。2014年~2016年共采集野生鳥類和活禽市場糞便樣品2691份,提取RNA并反轉(zhuǎn)錄后,熒光定量PCR方法檢測禽HEV RNA。結(jié)果顯示,未檢測到陽性樣品,可疑樣品7份。通過套式PCR將可疑樣品進(jìn)行驗(yàn)證,結(jié)果發(fā)現(xiàn)全部沒有擴(kuò)增出目的條帶。本次調(diào)查未發(fā)現(xiàn)采集的野生鳥類和市場活禽樣品中存在禽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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.31

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