血清4型Ⅰ群禽腺病毒的分离鉴定及流行病学调查

发布时间：2018-09-10 18:06

【摘要】：心包积水-肝炎综合征(Hydropericardium hepatitis syndrome,HHS)是由血清4型I群禽腺病毒(Fowl Adenovirus serotype 4,FAV4)引起的一种国内新发家禽传染性疾病。该病一经发生,便迅速向周围地区蔓延,迄今在我国多个省市均有该病的报道,目前已确定该病毒可以感染多个品种的鸡和鸭。患病家禽主要表现为无明显先兆而突然倒地死亡,发病家禽多见于3~5周龄的商品家禽,种鸡和种鸭也可以感染。其特征性症状为3~5周龄的肉鸡突然死亡,剖检主要变化为心包积水和出血性肝炎。该病感染率可高达90%以上,死亡率在20%~75%,最高可达80%,严重危害着我国养禽业的健康发展。本研究从疑似患病的商品肉鸡肝脏组织中分离到FAV4,并对其进行生物学特性作了研究,对分离到的病毒进行鉴定,以及对山东地区血清4型I群禽腺病毒的临床感染情况检测及结果统计分析,旨在为今后更好的阐明FAV4的流行规律以及防制提供理论依据。1、血清4型I群禽腺病毒的分离鉴定本研究采用鸡胚卵黄囊方式接种的方法从山东某地区以心包积液为主要特征的疑似FAV4感染的商品鸡肝脏组织中分离到病毒,并对分离的毒株进行PCR扩增、血凝试验、ELD50测定、Hexon基因序列测定与分析以及对人工感染试验进行组织病理学观察、抗体水平检测等一系列试验。结果显示,分离的毒株不能凝集鸡和鸭的红细胞,但能够凝集大鼠的红细胞;ELD50为10-3.33/0.2 m L。组织学变化显示肝细胞纤维化和脂肪变性,肾小管上皮细胞肿胀,心肌纤维断裂、颗粒变性。对临床采集的血清检测结果显示发病鸡群抗体水平为阳性,发病鸡群中的健康鸡阳性率为40%(40/100),表明鸡群中存在隐性感染。将分离株的Hexon基因组与已发表的血清4型I群禽腺病毒基因进行同源性比较,发现该分离株与印度株在同一个分支上,同源性在99.6%以上,而与韩国、欧洲、美洲株同源性较远。用分离的毒株感染10日龄的雏鸡能引起心包积液、肝脏出血、肾脏肿大等病变。上述结果表明该分离株为血清4型I群禽腺病毒。2、血清4型I群禽腺病毒Taqman荧光定量PCR方法的建立本试验针对Genbank上已发表的I群禽腺病毒的12个血清型的Hexon基因,建立了特异性检测血清4型I群禽腺病毒的Taqman荧光定量PCR方法。将PCR扩增的片段连接到pMD18-T载体上构建重组质粒,经序列分析正确后,测定重组质粒浓度,将标准品10倍梯度稀释后用于构建实时荧光定量PCR标准曲线,并进行反应的灵敏性、特异性和重复性试验。结果表明,标准曲线Ct值与模板浓度呈良好的线性关系,标准曲线方程为y=-3.1077x+38.305,相关系数R2为0.9951,此方法可以检测出最低量为40 copies,灵敏性是普通PCR的100倍;重复性试验结果显示批内变异系数CV均小于0.40%,批间变异系数CV均小于0.65%;对临床采集的679份疑似感染FAV4的病料检测表明,Taqman荧光定量PCR和普通PCR检测阳性率分别为76.29%和65.24%,两者符合率为84.71%。用该方法检测I群禽腺病毒的其他11个血清型病毒,结果均为阴性,无交叉反应。研究结果表明,该方法敏感性高、特异性强、重复性好,可应用于FAV4的临床诊断和流行病学调查。3、山东地区血清4型I群禽腺病毒的流行病学调查对山东地区养殖场采集的679份病料进行检测,共检测出FAV4阳性样品443份,阳性率为65.2%;其中商品鸡的检出率为71.9%,蛋(种)鸡的检出率为34.2%,商品鸡的检出率明显的高于种鸡的检出率,表明该病毒主要感染商品鸡;而蛋(种)鸭的检出率为68.6%,与商品鸭的检出率(79.2%)相差不大,但蛋(种)鸭的检出率明显的高于种鸡的检出率。对临床采集的172份种鸡病料进行H9N2、CIAV和IBDV等免疫抑制性病原的检测,H9N2和CIAV的检出率较高,感染率在30%左右。FAV4与H9N2和CIAV的混合感染比率相对较高,表明家禽发生免疫抑制性疾病时能够促进FAV4的发生。而对149份商品鸡样品进行H9N2、CIAV和IBDV等免疫抑制性病原的检测,商品肉鸡感染FAV4与H9N2和IBDV的感染呈一定的相关性。对358份鸭源样品进行检测,FAV4阳性率为77.1%,其中蛋(种)鸭的FAV4阳性率为68.6%,商品鸭的FAV4阳性率为79.2%。对检测出FAV4的276份样品进行H9N2和IBDV的检测,48份阳性蛋(种)鸭样本中H9N2检测出20份,阳性率为41.67%,228份阳性商品鸭样本中H9N2检测出88份,IBDV检测出61份。蛋(种)鸭同时感染FAV4和H9N2的比率高达40%,而在检测出FAV4的商品鸭中,H9N2和IBDV的比率都较高。统计结果表明鸡、鸭的FAV4检出率均高于70%,种鸭的检出率明显高于种鸡,商品家禽的感染率明显高于种禽,鸭子的检出率(77.10%)明显高于鸡的51.20%;FAV4与H9N2和CIAV的混合感染比率相对较高,表明家禽发生免疫抑制性疾病时能够促进FAV4的发生,为临床上防制FAV4提供一定的流行病学资料。
[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%).
【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】：S852.65;S858.3

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