本文選題：雞傳染性喉氣管炎病毒 + 池州分離株��； 參考：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：雞傳染性喉氣管炎(Infectious Laryngotracheitis,ILT)是由雞傳染性喉氣管炎病毒(Infectious Laryngotracheitis Virus,ILTV)引起的雞病毒性呼吸道疾病,可引起雞死亡或產(chǎn)蛋下降而造成嚴(yán)重的經(jīng)濟(jì)損失。2015年安徽省池州某雞場120日齡蛋雞發(fā)生疑似雞傳染性喉氣管炎,該場存欄5000只,臨床癥狀為呼吸困難,發(fā)病率為60%,死亡率為60%,為了對(duì)該場發(fā)病情況進(jìn)行確診,本研究采集病雞喉頭氣管組織及其分泌物,剪碎加入3倍體積無菌0.2MPBS充分研磨,反復(fù)凍融三次后離心取上清加入青-鏈霉素(500U/mL)4℃孵育過夜,置于-80℃冰箱備用。提取病雞喉頭氣管及其分泌物總DNA并根據(jù)GenBank發(fā)表的雞傳染性喉氣管炎病毒gD基因設(shè)計(jì)合成一對(duì)特異性引物,從發(fā)病雞喉頭及其分泌物中擴(kuò)增出1134bp大小片段,.制備的組織液上清經(jīng)雞胚絨毛尿囊膜(chorioallantoic membrane,CAM)途徑接種10日齡SPF雞胚,120h后打開雞胚,發(fā)現(xiàn)在雞胚CAM上發(fā)現(xiàn)典型痘斑,收集病變雞胚痘斑和尿囊液,充分研磨,反復(fù)凍融并離心取上清按照相同途徑連續(xù)接種雞胚三次,取第4代雞胚分離毒作為種毒,保存于-80℃;將種毒通過滴鼻、點(diǎn)眼的方式接種5日齡健康麻羽肉雞,每只102EID50作為試驗(yàn)組,并使用同等劑量的無菌PBS設(shè)置陰性對(duì)照,連續(xù)觀察,結(jié)果發(fā)現(xiàn):攻毒3d后試驗(yàn)組出現(xiàn)采食量下降,呼吸困難,眼結(jié)膜壞死等癥狀,第4～5d癥狀加重并出現(xiàn)兩只雛雞死亡,第9d開始預(yù)后;而同時(shí)對(duì)照組無任何癥狀;剖檢病死雞發(fā)現(xiàn)其喉頭氣管出血,盲腸扁桃體腫大,并可從病死雞喉頭氣管及其分泌物中檢測到病毒。結(jié)果證實(shí)分離到一株雞傳染性喉氣管炎病毒,命名為CZ-ILTV16株。以第4代雞胚分離毒為材料提取病毒DNA,并擴(kuò)增gD基因,膠回收純化后克隆至pMD18-T載體,轉(zhuǎn)化入DH5α感受態(tài)細(xì)胞,挑取單克隆鑒定正確后進(jìn)行測序,結(jié)果發(fā)現(xiàn)與GenBank上已發(fā)表gD基因序列同源性在99%以上,基因進(jìn)化樹分析結(jié)果發(fā)現(xiàn),分離毒處于獨(dú)立分支,且與Serva株的親緣性較近;
[Abstract]:Infectious laryngotrachetitis (ILT) is a viral respiratory disease caused by infectious laryngotracheitis virus (ILTV).In 2015, a 120-day-old laying hens in Chizhou, Anhui Province, was suspected to have infectious laryngotracheitis. 5000 chickens were stored in the farm, with clinical symptoms of dyspnea.The incidence rate was 60 and the mortality rate was 60. In order to confirm the incidence of the disease, the trachea tissue and its secretion were collected, and three times the volume of aseptic 0.2MPBS were shredded.After repeated freezing and thawing for three times, the supernatant was centrifuged and incubated overnight at 4 鈩，

本文編號(hào)：1773735