本文選題：Ⅰ群禽腺病毒 + 分離鑒定��； 參考：《長(zhǎng)江大學(xué)》2017年碩士論文

【摘要】：雞心包積液-肝炎綜合征(HHS)是由I群禽腺病毒(Fowl adenovirus I,FAV-I)血清4型引起的一種傳染病。本病典型癥狀為心包積水、壞死性肝炎,主要以3~6周齡肉仔雞最易感,死亡率高達(dá)80%。在印度、加拿大、日本、俄羅斯等許多國(guó)家均有報(bào)道該病的發(fā)生。近幾年,在我國(guó)江蘇、河南、山東和福建等地陸續(xù)在817、三黃雞及地方品種的青年雞群,發(fā)生了這種流行病。由于I群禽腺病毒作為原發(fā)性病原的作用至今仍不清楚,因此免疫途徑也一直未能確定。雞群在感染病毒后,會(huì)造成雞體全身免疫功能下降,使用疫苗后達(dá)不到免疫效果,給我國(guó)養(yǎng)禽業(yè)發(fā)展帶來(lái)嚴(yán)重的影響。本課題利用PCR方法從湖北省養(yǎng)禽地區(qū)送檢的病料中檢測(cè)禽腺病毒,進(jìn)行病毒分離和hexon基因測(cè)序,了解湖北地區(qū)I群禽腺病毒血清型的分布和感染狀況。為進(jìn)一步認(rèn)識(shí)湖北地區(qū)分離毒株的病原特性,選擇一株血清4型分離毒株進(jìn)行人工感染試驗(yàn),通過免疫組化方法檢測(cè)FAV-I,了解湖北地區(qū)禽腺病毒在雞體內(nèi)的分布規(guī)律。取得的主要結(jié)果如下:1.湖北地區(qū)I群禽腺病毒的分離鑒定從湖北省養(yǎng)禽地區(qū)送檢的12份心包積水-肝炎綜合征發(fā)病雞中,利用PCR方法擴(kuò)增出hexon基因,經(jīng)序列比對(duì),12株分離株與Genbank中I群禽腺病毒12個(gè)不同血清型序列之間核苷酸同源性為31.1%~98.3%,與血清4型和血清10型的禽腺病毒同源性最接近,與血清4型的同源性最高達(dá)到98.1%~98.3%。遺傳進(jìn)化樹分析顯示與I群禽腺病毒血清4型均在同一分支上,說(shuō)明12株分離株屬于I群禽腺病毒血清4型。病料上清接種雞胚肝細(xì)胞(CEL),發(fā)現(xiàn)細(xì)胞收縮變圓,細(xì)胞膜萎縮,界限模糊不清,折光性增強(qiáng)。出現(xiàn)的細(xì)胞病變與I群禽腺病毒在細(xì)胞上的病變過程一致。并測(cè)定得到12株分離毒株的TCID50分別在104.75-107.5之間。2.FAVI/HBtuanfeng/151023分離株的致病性研究本研究選擇血清4型FAVI/HBtuanfeng/151023分離株人工感染33日齡的尼克白羽蛋雞。將試驗(yàn)分為3組:肌注組、口服組和對(duì)照組,同時(shí)肌注組和口服組每組放10只不接種雞為同居。肌注組通過皮下注射,口服組通過口服途徑接種0.2mL?(0.2×107.5TCID50)羽的病毒液,對(duì)照組接種0.2mL?羽滅菌生理鹽水,每日觀察記錄雞只發(fā)病情況。感染后第3d、5d、7d對(duì)試驗(yàn)組和對(duì)照組的雞進(jìn)行剖殺,采集組織器官,制作病理切片。結(jié)果表明該分離毒株具有較強(qiáng)的致病性,感染率為100%,能引起雞精神沉郁、食欲減退、羽毛松亂、嗜睡等癥狀。剖檢發(fā)病雞可見胸腔內(nèi)有明顯的心包積液,肝臟表面布滿了大大小小的出血斑點(diǎn),邊緣有白色的壞死,肺水腫充血。組織病理學(xué)變化可見肝組織內(nèi)有淤血,細(xì)胞核濃縮,肝脂肪變性壞死,肝細(xì)胞核內(nèi)見形狀不規(guī)則的核內(nèi)包涵體。3.利用免疫組化方法檢測(cè)FAV-I通過蔗糖濃縮方法將FAVI/HBtuanfeng/151023分離毒株進(jìn)行濃縮,經(jīng)甲醛滅活后免疫家兔,制備FAV-I參考毒株高免血清,瓊擴(kuò)抗體效價(jià)高達(dá)1:64。應(yīng)用免疫組化技術(shù)對(duì)人工感染雞不同分組的組織器官進(jìn)行檢測(cè),結(jié)果表明:不同試驗(yàn)組的雞感染后第3d,肝臟、心臟、腎臟、脾臟、胸腺、十二指腸中都檢測(cè)到陽(yáng)性信號(hào),FAV-I侵害雞的器官為肝臟、脾臟、腎臟和腸道。隨著時(shí)間的推移,檢測(cè)到靶器官陽(yáng)性信號(hào)逐漸增強(qiáng),免疫器官的陽(yáng)性信號(hào)則呈現(xiàn)出強(qiáng)弱交替的情況。綜上所述:本研究從湖北養(yǎng)禽地分離到的12株分離株屬于I群禽腺病毒血清4型。選取FAVI/HBtuanfeng/151023株經(jīng)口服、肌肉注射途徑感染試驗(yàn)雞均可導(dǎo)致發(fā)病后死亡,具有較強(qiáng)的致病性。利用免疫組化方法檢測(cè)FAV-I,結(jié)果表明肝臟、脾臟、腎臟和腸道是禽腺病毒主要侵害的靶器官。該方法具有良好的特異性,可用于FAV-I感染雞組織的亞細(xì)胞定位與FAV-I感染死亡雞的診斷,為闡明FAV-I的致病機(jī)理提供有用的科學(xué)數(shù)據(jù)。
[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.
【學(xué)位授予單位】：長(zhǎng)江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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