湖北地区Ⅰ群禽腺病毒的分离鉴定及其致病性研究

发布时间：2018-06-13 00:24

本文选题：Ⅰ群禽腺病毒 + 分离鉴定　；参考：《长江大学》2017年硕士论文

【摘要】：鸡心包积液-肝炎综合征(HHS)是由I群禽腺病毒(Fowl adenovirus I,FAV-I)血清4型引起的一种传染病。本病典型症状为心包积水、坏死性肝炎,主要以3~6周龄肉仔鸡最易感,死亡率高达80%。在印度、加拿大、日本、俄罗斯等许多国家均有报道该病的发生。近几年,在我国江苏、河南、山东和福建等地陆续在817、三黄鸡及地方品种的青年鸡群,发生了这种流行病。由于I群禽腺病毒作为原发性病原的作用至今仍不清楚,因此免疫途径也一直未能确定。鸡群在感染病毒后,会造成鸡体全身免疫功能下降,使用疫苗后达不到免疫效果,给我国养禽业发展带来严重的影响。本课题利用PCR方法从湖北省养禽地区送检的病料中检测禽腺病毒,进行病毒分离和hexon基因测序,了解湖北地区I群禽腺病毒血清型的分布和感染状况。为进一步认识湖北地区分离毒株的病原特性,选择一株血清4型分离毒株进行人工感染试验,通过免疫组化方法检测FAV-I,了解湖北地区禽腺病毒在鸡体内的分布规律。取得的主要结果如下:1.湖北地区I群禽腺病毒的分离鉴定从湖北省养禽地区送检的12份心包积水-肝炎综合征发病鸡中,利用PCR方法扩增出hexon基因,经序列比对,12株分离株与Genbank中I群禽腺病毒12个不同血清型序列之间核苷酸同源性为31.1%~98.3%,与血清4型和血清10型的禽腺病毒同源性最接近,与血清4型的同源性最高达到98.1%~98.3%。遗传进化树分析显示与I群禽腺病毒血清4型均在同一分支上,说明12株分离株属于I群禽腺病毒血清4型。病料上清接种鸡胚肝细胞(CEL),发现细胞收缩变圆,细胞膜萎缩,界限模糊不清,折光性增强。出现的细胞病变与I群禽腺病毒在细胞上的病变过程一致。并测定得到12株分离毒株的TCID50分别在104.75-107.5之间。2.FAVI/HBtuanfeng/151023分离株的致病性研究本研究选择血清4型FAVI/HBtuanfeng/151023分离株人工感染33日龄的尼克白羽蛋鸡。将试验分为3组:肌注组、口服组和对照组,同时肌注组和口服组每组放10只不接种鸡为同居。肌注组通过皮下注射,口服组通过口服途径接种0.2mL?(0.2×107.5TCID50)羽的病毒液,对照组接种0.2mL?羽灭菌生理盐水,每日观察记录鸡只发病情况。感染后第3d、5d、7d对试验组和对照组的鸡进行剖杀,采集组织器官,制作病理切片。结果表明该分离毒株具有较强的致病性,感染率为100%,能引起鸡精神沉郁、食欲减退、羽毛松乱、嗜睡等症状。剖检发病鸡可见胸腔内有明显的心包积液,肝脏表面布满了大大小小的出血斑点,边缘有白色的坏死,肺水肿充血。组织病理学变化可见肝组织内有淤血,细胞核浓缩,肝脂肪变性坏死,肝细胞核内见形状不规则的核内包涵体。3.利用免疫组化方法检测FAV-I通过蔗糖浓缩方法将FAVI/HBtuanfeng/151023分离毒株进行浓缩,经甲醛灭活后免疫家兔,制备FAV-I参考毒株高免血清,琼扩抗体效价高达1:64。应用免疫组化技术对人工感染鸡不同分组的组织器官进行检测,结果表明:不同试验组的鸡感染后第3d,肝脏、心脏、肾脏、脾脏、胸腺、十二指肠中都检测到阳性信号,FAV-I侵害鸡的器官为肝脏、脾脏、肾脏和肠道。随着时间的推移,检测到靶器官阳性信号逐渐增强,免疫器官的阳性信号则呈现出强弱交替的情况。综上所述:本研究从湖北养禽地分离到的12株分离株属于I群禽腺病毒血清4型。选取FAVI/HBtuanfeng/151023株经口服、肌肉注射途径感染试验鸡均可导致发病后死亡,具有较强的致病性。利用免疫组化方法检测FAV-I,结果表明肝脏、脾脏、肾脏和肠道是禽腺病毒主要侵害的靶器官。该方法具有良好的特异性,可用于FAV-I感染鸡组织的亚细胞定位与FAV-I感染死亡鸡的诊断,为阐明FAV-I的致病机理提供有用的科学数据。
[Abstract]:Chicken cardiac pericardium hepatitis syndrome (HHS) is an infectious disease caused by Fowl adenovirus I (Fowl adenovirus I, FAV-I) serotype 4. The typical symptom of this disease is pericardial water and necrotic hepatitis. It is the most susceptible to 3~6 weeks old broilers. The mortality rate is as high as 80%. in India, Canada, Japan, Russia and many other countries. In recent years, this epidemic has occurred in 817, three yellow chickens and young chickens of local varieties in Jiangsu, Henan, Shandong and Fujian. The role of the I avian adenovirus as a primary pathogen is still unclear, so the immune pathway has not been confirmed. The chicken group will cause the whole body immunity after the virus infection. The function decreased, the immune effect was not reached after the use of the vaccine, which had a serious effect on the development of poultry industry in China. The PCR method was used to detect the avian adenovirus in the sick materials from the Hubei breeding poultry area, and the virus isolation and hexon gene sequencing were carried out to understand the distribution and infection status of the I group of avian adenovirus in the Hubei region. To understand the pathogenic characteristics of isolated strains in Hubei, a serotype 4 isolate was selected for artificial infection test, and FAV-I was detected by immunohistochemical method to understand the distribution of avian adenovirus in Hubei area. The main results were as follows: 1. the isolation and identification of avian adenovirus in the group of I in Hubei region from Hubei Province In 12 pericardial hydronephrosis syndrome chickens, the hexon gene was amplified by PCR method. By sequence alignment, the nucleotide homology between the 12 isolates and the 12 different serotype sequences of Genbank I avian adenovirus was 31.1%~98.3%, which was closest to the serotype 4 and serotype 10 of avian adenovirus, and was homologous with the serotype 4. The analysis of the highest 98.1%~98.3%. genetic evolution tree showed that the I group of avian adenovirus 4 was on the same branch, indicating that 12 isolates belonged to the I group of avian adenovirus type 4. The inoculated chicken embryo liver cells (CEL) in the supernatant, found the cell contraction, the cell membrane atrophy, the limit blurred and the refraction enhancement. The appearance of the cytopathic and I The pathological process of the avian adenovirus on the cell was consistent. The pathogenicity of the TCID50 isolated between 12 isolates and the.2.FAVI/HBtuanfeng/151023 isolates between 104.75-107.5 isolates was studied. The study selected the nick feathered laying hens of 33 days of age for artificial infection of type 4 FAVI/HBtuanfeng/151023 isolates. The oral group and the control group were given 10 non inoculated chickens in the injection group and the oral group. By subcutaneous injection, the oral group inoculated the 0.2mL? (0.2 * 107.5TCID50) feathers by oral administration. The control group was inoculated with 0.2mL? Sterilized physiological saline, and the daily observation of the chickens was recorded. The test group was 3D, 5D, 7d. The chickens of the control group were killed, the tissues and organs were collected and the pathological sections were collected. The results showed that the isolated strain had a strong pathogenicity, the infection rate was 100%, which could cause the symptoms of chicken spirit depression, anorexia, feathers loosening, drowsiness and so on. The infected chicken can see the obvious pericardial effusion in the chest, and the liver surface is full of large and small out of the liver. Blood spots, white necrosis on the edge and hyperemia of the pulmonary edema. Histopathological changes can be seen in the liver tissue with congestion, nuclear concentration, liver fatty degeneration and necrosis, and the irregularly shaped inclusions of the inclusion body.3. in the nucleus of the liver detect the concentration of FAV-I by the sucrose concentration method to isolate FAVI/HBtuanfeng/151023 strains by sucrose concentration method. After being inactivated by formaldehyde, the rabbit was immunized with the FAV-I reference strain of high serum free serum, and the titer of agar expanded antibody was detected by 1:64. application immunization technique to detect the tissues and organs of different groupings of artificial infected chickens. The results showed that the liver, the liver, the heart, the kidney, the spleen, the thymus, the duodenum and the duodenum were positive after the infection of the chickens in the different test groups. The signal that FAV-I violates chicken's organs is liver, spleen, kidney and intestines. As time goes on, the positive signals of target organs are gradually enhanced and the positive signals of immune organs show the alternation of strength and weakness. In summary, 12 strains isolated from Hubei breeding poultry land belong to the I group of avian adenovirus type 4. Select FAVI/ HBtuanfeng/151023 strains can cause death after oral administration by intramuscular injection. It has strong pathogenicity. FAV-I is detected by immunohistochemical method. The results show that liver, spleen, kidney and intestines are the main target organs of avian adenovirus. This method has good specificity and can be used in FAV-I infection of chicken tissues. Subcellular localization and the diagnosis of FAV-I infected dead chickens provide useful scientific data for elucidating the pathogenesis of FAV-I.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

【相似文献】