血清4型Ⅰ群禽腺病毒的分離鑒定及流行病學(xué)調(diào)查
發(fā)布時(shí)間：2018-09-10 18:06
【摘要】：心包積水-肝炎綜合征(Hydropericardium hepatitis syndrome,HHS)是由血清4型I群禽腺病毒(Fowl Adenovirus serotype 4,FAV4)引起的一種國(guó)內(nèi)新發(fā)家禽傳染性疾病。該病一經(jīng)發(fā)生,便迅速向周圍地區(qū)蔓延,迄今在我國(guó)多個(gè)省市均有該病的報(bào)道,目前已確定該病毒可以感染多個(gè)品種的雞和鴨�；疾〖仪葜饕憩F(xiàn)為無明顯先兆而突然倒地死亡,發(fā)病家禽多見于3~5周齡的商品家禽,種雞和種鴨也可以感染。其特征性癥狀為3~5周齡的肉雞突然死亡,剖檢主要變化為心包積水和出血性肝炎。該病感染率可高達(dá)90%以上,死亡率在20%~75%,最高可達(dá)80%,嚴(yán)重危害著我國(guó)養(yǎng)禽業(yè)的健康發(fā)展。本研究從疑似患病的商品肉雞肝臟組織中分離到FAV4,并對(duì)其進(jìn)行生物學(xué)特性作了研究,對(duì)分離到的病毒進(jìn)行鑒定,以及對(duì)山東地區(qū)血清4型I群禽腺病毒的臨床感染情況檢測(cè)及結(jié)果統(tǒng)計(jì)分析,旨在為今后更好的闡明FAV4的流行規(guī)律以及防制提供理論依據(jù)。1、血清4型I群禽腺病毒的分離鑒定本研究采用雞胚卵黃囊方式接種的方法從山東某地區(qū)以心包積液為主要特征的疑似FAV4感染的商品雞肝臟組織中分離到病毒,并對(duì)分離的毒株進(jìn)行PCR擴(kuò)增、血凝試驗(yàn)、ELD50測(cè)定、Hexon基因序列測(cè)定與分析以及對(duì)人工感染試驗(yàn)進(jìn)行組織病理學(xué)觀察、抗體水平檢測(cè)等一系列試驗(yàn)。結(jié)果顯示,分離的毒株不能凝集雞和鴨的紅細(xì)胞,但能夠凝集大鼠的紅細(xì)胞;ELD50為10-3.33/0.2 m L。組織學(xué)變化顯示肝細(xì)胞纖維化和脂肪變性,腎小管上皮細(xì)胞腫脹,心肌纖維斷裂、顆粒變性。對(duì)臨床采集的血清檢測(cè)結(jié)果顯示發(fā)病雞群抗體水平為陽性,發(fā)病雞群中的健康雞陽性率為40%(40/100),表明雞群中存在隱性感染。將分離株的Hexon基因組與已發(fā)表的血清4型I群禽腺病毒基因進(jìn)行同源性比較,發(fā)現(xiàn)該分離株與印度株在同一個(gè)分支上,同源性在99.6%以上,而與韓國(guó)、歐洲、美洲株同源性較遠(yuǎn)。用分離的毒株感染10日齡的雛雞能引起心包積液、肝臟出血、腎臟腫大等病變。上述結(jié)果表明該分離株為血清4型I群禽腺病毒。2、血清4型I群禽腺病毒Taqman熒光定量PCR方法的建立本試驗(yàn)針對(duì)Genbank上已發(fā)表的I群禽腺病毒的12個(gè)血清型的Hexon基因,建立了特異性檢測(cè)血清4型I群禽腺病毒的Taqman熒光定量PCR方法。將PCR擴(kuò)增的片段連接到pMD18-T載體上構(gòu)建重組質(zhì)粒,經(jīng)序列分析正確后,測(cè)定重組質(zhì)粒濃度,將標(biāo)準(zhǔn)品10倍梯度稀釋后用于構(gòu)建實(shí)時(shí)熒光定量PCR標(biāo)準(zhǔn)曲線,并進(jìn)行反應(yīng)的靈敏性、特異性和重復(fù)性試驗(yàn)。結(jié)果表明,標(biāo)準(zhǔn)曲線Ct值與模板濃度呈良好的線性關(guān)系,標(biāo)準(zhǔn)曲線方程為y=-3.1077x+38.305,相關(guān)系數(shù)R2為0.9951,此方法可以檢測(cè)出最低量為40 copies,靈敏性是普通PCR的100倍;重復(fù)性試驗(yàn)結(jié)果顯示批內(nèi)變異系數(shù)CV均小于0.40%,批間變異系數(shù)CV均小于0.65%;對(duì)臨床采集的679份疑似感染FAV4的病料檢測(cè)表明,Taqman熒光定量PCR和普通PCR檢測(cè)陽性率分別為76.29%和65.24%,兩者符合率為84.71%。用該方法檢測(cè)I群禽腺病毒的其他11個(gè)血清型病毒,結(jié)果均為陰性,無交叉反應(yīng)。研究結(jié)果表明,該方法敏感性高、特異性強(qiáng)、重復(fù)性好,可應(yīng)用于FAV4的臨床診斷和流行病學(xué)調(diào)查。3、山東地區(qū)血清4型I群禽腺病毒的流行病學(xué)調(diào)查對(duì)山東地區(qū)養(yǎng)殖場(chǎng)采集的679份病料進(jìn)行檢測(cè),共檢測(cè)出FAV4陽性樣品443份,陽性率為65.2%;其中商品雞的檢出率為71.9%,蛋(種)雞的檢出率為34.2%,商品雞的檢出率明顯的高于種雞的檢出率,表明該病毒主要感染商品雞;而蛋(種)鴨的檢出率為68.6%,與商品鴨的檢出率(79.2%)相差不大,但蛋(種)鴨的檢出率明顯的高于種雞的檢出率。對(duì)臨床采集的172份種雞病料進(jìn)行H9N2、CIAV和IBDV等免疫抑制性病原的檢測(cè),H9N2和CIAV的檢出率較高,感染率在30%左右。FAV4與H9N2和CIAV的混合感染比率相對(duì)較高,表明家禽發(fā)生免疫抑制性疾病時(shí)能夠促進(jìn)FAV4的發(fā)生。而對(duì)149份商品雞樣品進(jìn)行H9N2、CIAV和IBDV等免疫抑制性病原的檢測(cè),商品肉雞感染FAV4與H9N2和IBDV的感染呈一定的相關(guān)性。對(duì)358份鴨源樣品進(jìn)行檢測(cè),FAV4陽性率為77.1%,其中蛋(種)鴨的FAV4陽性率為68.6%,商品鴨的FAV4陽性率為79.2%。對(duì)檢測(cè)出FAV4的276份樣品進(jìn)行H9N2和IBDV的檢測(cè),48份陽性蛋(種)鴨樣本中H9N2檢測(cè)出20份,陽性率為41.67%,228份陽性商品鴨樣本中H9N2檢測(cè)出88份,IBDV檢測(cè)出61份。蛋(種)鴨同時(shí)感染FAV4和H9N2的比率高達(dá)40%,而在檢測(cè)出FAV4的商品鴨中,H9N2和IBDV的比率都較高。統(tǒng)計(jì)結(jié)果表明雞、鴨的FAV4檢出率均高于70%,種鴨的檢出率明顯高于種雞,商品家禽的感染率明顯高于種禽,鴨子的檢出率(77.10%)明顯高于雞的51.20%;FAV4與H9N2和CIAV的混合感染比率相對(duì)較高,表明家禽發(fā)生免疫抑制性疾病時(shí)能夠促進(jìn)FAV4的發(fā)生,為臨床上防制FAV4提供一定的流行病學(xué)資料。
[Abstract]:Hydropericardium hepatitis syndrome (HHS) is a new domestic poultry infectious disease caused by serum fowl Adenovirus serotype 4 (FAV4). Once the disease occurs, it spreads rapidly to the surrounding areas. So far, the disease has been reported in many provinces and cities in China and has been confirmed. The virus can infect many breeds of chickens and ducks. The disease is characterized by sudden death of poultry without obvious precursors. The disease occurs mostly in commercial poultry aged 3-5 weeks. Breeding and breeding ducks can also be infected. The characteristic symptoms are sudden death of broilers aged 3-5 weeks. The main change of the disease is hydrocardia and hemorrhagic hepatitis. The infection rate can be as high as 90%. The mortality rate can be as high as 20%~75%. The highest mortality rate can be as high as 80%. It seriously endangers the healthy development of poultry industry in China. The purpose of this study is to provide theoretical basis for further elucidation of the epidemic regularity and prevention and control of FAV4. 1. Isolation and identification of serotype 4 avian adenovirus I. In this study, chicken embryo yolk sac inoculation method was used to inoculate suspected FAV4 infections from pericardial effusion in Shandong province. The virus was isolated from chicken liver tissues and tested by PCR amplification, hemagglutination test, ELD50 assay, Hexon gene sequencing and analysis, histopathological observation and antibody level detection. ELD50 was 10-3.33/0.2 m L. Histological changes showed hepatocyte fibrosis and steatosis, renal tubular epithelial cells swelling, myocardial fibers rupture, granular degeneration. Serum test results showed that antibody level was positive in infected chickens, and the positive rate of healthy chickens was 40% (40/100) in infected chickens. It was found that the isolate was more than 99.6% homologous to the Indian strain in the same branch, but far more homologous to the Korean, European and American strains. The results showed that the isolate was serotype 4 avian adenovirus type I. 2 and serotype 4 avian adenovirus type I. Taqman fluorescence quantitative PCR was used to detect the Hexon gene of 12 serotypes of avian adenovirus group I published in Genbank. The recombinant plasmid was constructed by connecting PCR amplified fragment to pMD18-T vector. The recombinant plasmid concentration was determined after the sequence analysis was correct. The standard sample was diluted 10 times and used to construct a real-time fluorescence quantitative PCR standard curve. The sensitivity, specificity and repeatability of the reaction were tested. The correlation coefficient R2 was 0.9951. This method could detect the lowest amount of 40 copies, and the sensitivity was 100 times higher than that of ordinary PCR. The repeatability test showed that CV in batch was less than 0.40%, CV between batches was less than 0.65%. The positive rates of Taqman fluorescence quantitative PCR and conventional PCR were 76.29% and 65.24%, respectively, and the coincidence rate was 84.71%. The results of the detection of other 11 serotypes of avian adenovirus group I by this method were negative and no cross reaction was found. The epidemiological investigation of serotype 4 avian adenovirus group I in Shandong area detected 679 samples collected from farms in Shandong area, and 443 positive samples of FAV4 were detected, with a positive rate of 65.2%. The detection rate of commercial chickens was 71.9%, and that of eggs (chickens) was 65.2%. The detection rate of commercial chickens was 34.2%. The detection rate of commercial chickens was significantly higher than that of breeding chickens, indicating that the virus was mainly infected with commercial chickens. The detection rate of egg (breed) ducks was 68.6%, which was similar to that of commercial ducks (79.2%), but the detection rate of egg (breed) ducks was significantly higher than that of breeding chickens. Immunosuppressive pathogens such as CIAV and IBDV were detected, and the detection rate of H9N2 and CIAV was high, and the infection rate was about 30%. The positive rate of FAV4 in 358 duck samples was 77.1%. The positive rate of FAV4 in egg (breed) duck was 68.6%. The positive rate of FAV4 in commercial duck was 79.2%. H9N2 and IBDV were detected in 276 samples of FAV4 and H9N2 in 48 duck samples. Among 228 positive commercial ducks, 88 were detected for H9N2 and 61 were detected for IBDV. The rate of simultaneous infection of FAV4 and H9N2 in egg (breed) ducks was as high as 40%. However, the ratio of H9N2 and IBDV in commercial ducks detected for FAV4 was higher. The statistical results showed that the detection rate of FAV4 in chickens and ducks was higher than 70%, and the detection rate of FAV4 in breed ducks was significantly higher than that in chickens and ducks. The infection rate of commercial poultry was significantly higher than that of breeding poultry, and the detection rate of duck (77.10%) was significantly higher than that of chicken (51.20%).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65;S858.3
本文編號(hào)：2235209
[Abstract]:Hydropericardium hepatitis syndrome (HHS) is a new domestic poultry infectious disease caused by serum fowl Adenovirus serotype 4 (FAV4). Once the disease occurs, it spreads rapidly to the surrounding areas. So far, the disease has been reported in many provinces and cities in China and has been confirmed. The virus can infect many breeds of chickens and ducks. The disease is characterized by sudden death of poultry without obvious precursors. The disease occurs mostly in commercial poultry aged 3-5 weeks. Breeding and breeding ducks can also be infected. The characteristic symptoms are sudden death of broilers aged 3-5 weeks. The main change of the disease is hydrocardia and hemorrhagic hepatitis. The infection rate can be as high as 90%. The mortality rate can be as high as 20%~75%. The highest mortality rate can be as high as 80%. It seriously endangers the healthy development of poultry industry in China. The purpose of this study is to provide theoretical basis for further elucidation of the epidemic regularity and prevention and control of FAV4. 1. Isolation and identification of serotype 4 avian adenovirus I. In this study, chicken embryo yolk sac inoculation method was used to inoculate suspected FAV4 infections from pericardial effusion in Shandong province. The virus was isolated from chicken liver tissues and tested by PCR amplification, hemagglutination test, ELD50 assay, Hexon gene sequencing and analysis, histopathological observation and antibody level detection. ELD50 was 10-3.33/0.2 m L. Histological changes showed hepatocyte fibrosis and steatosis, renal tubular epithelial cells swelling, myocardial fibers rupture, granular degeneration. Serum test results showed that antibody level was positive in infected chickens, and the positive rate of healthy chickens was 40% (40/100) in infected chickens. It was found that the isolate was more than 99.6% homologous to the Indian strain in the same branch, but far more homologous to the Korean, European and American strains. The results showed that the isolate was serotype 4 avian adenovirus type I. 2 and serotype 4 avian adenovirus type I. Taqman fluorescence quantitative PCR was used to detect the Hexon gene of 12 serotypes of avian adenovirus group I published in Genbank. The recombinant plasmid was constructed by connecting PCR amplified fragment to pMD18-T vector. The recombinant plasmid concentration was determined after the sequence analysis was correct. The standard sample was diluted 10 times and used to construct a real-time fluorescence quantitative PCR standard curve. The sensitivity, specificity and repeatability of the reaction were tested. The correlation coefficient R2 was 0.9951. This method could detect the lowest amount of 40 copies, and the sensitivity was 100 times higher than that of ordinary PCR. The repeatability test showed that CV in batch was less than 0.40%, CV between batches was less than 0.65%. The positive rates of Taqman fluorescence quantitative PCR and conventional PCR were 76.29% and 65.24%, respectively, and the coincidence rate was 84.71%. The results of the detection of other 11 serotypes of avian adenovirus group I by this method were negative and no cross reaction was found. The epidemiological investigation of serotype 4 avian adenovirus group I in Shandong area detected 679 samples collected from farms in Shandong area, and 443 positive samples of FAV4 were detected, with a positive rate of 65.2%. The detection rate of commercial chickens was 71.9%, and that of eggs (chickens) was 65.2%. The detection rate of commercial chickens was 34.2%. The detection rate of commercial chickens was significantly higher than that of breeding chickens, indicating that the virus was mainly infected with commercial chickens. The detection rate of egg (breed) ducks was 68.6%, which was similar to that of commercial ducks (79.2%), but the detection rate of egg (breed) ducks was significantly higher than that of breeding chickens. Immunosuppressive pathogens such as CIAV and IBDV were detected, and the detection rate of H9N2 and CIAV was high, and the infection rate was about 30%. The positive rate of FAV4 in 358 duck samples was 77.1%. The positive rate of FAV4 in egg (breed) duck was 68.6%. The positive rate of FAV4 in commercial duck was 79.2%. H9N2 and IBDV were detected in 276 samples of FAV4 and H9N2 in 48 duck samples. Among 228 positive commercial ducks, 88 were detected for H9N2 and 61 were detected for IBDV. The rate of simultaneous infection of FAV4 and H9N2 in egg (breed) ducks was as high as 40%. However, the ratio of H9N2 and IBDV in commercial ducks detected for FAV4 was higher. The statistical results showed that the detection rate of FAV4 in chickens and ducks was higher than 70%, and the detection rate of FAV4 in breed ducks was significantly higher than that in chickens and ducks. The infection rate of commercial poultry was significantly higher than that of breeding poultry, and the detection rate of duck (77.10%) was significantly higher than that of chicken (51.20%).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65;S858.3
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