Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13642
標題: 新城雞瘟活毒疫苗投予後雞隻體內病毒分布與消長之探討
Studies on the distribution and vicissitude of Newcastle disease virus in chickens vaccinated with live vaccine
作者: 曾啟文
Tseng, Chi-Wen
關鍵字: NDV
新城雞瘟病毒
administration route
viral distribution
antibody response
RT-PCR
投予方式
病毒分布
抗體反應
反轉錄-聚合連鎖反應
出版社: 獸醫學系
摘要: 為了解新城雞瘟活毒疫苗免疫後,病毒在雞隻體內的分布及消長情形, 採用聚合連鎖反應 (PCR) 來進行病毒核酸的偵測,欲借重該項技術的 高敏感性,以期提高檢測的正確性。過去應用 PCR 來增幅 RNA 必須經過 兩個實驗步驟:首先 RNA 模板經過反轉錄作用合成第一股 cDNA,再以第 一股 cDNA 作為模板來進行特定核酸片段的增幅反應。本實驗使用針對已 發表的新城雞瘟病毒 B1 株核酸序列所設計的特異性引子,在確定其特異 性後,結合反轉錄作用與聚合連鎖反應,將之簡化於一個反應管中進行 ,來偵測新城雞瘟病毒 (NDV) 在免疫後於雞隻體內的分布及消長。結果 顯示,SPF 雞隻以活毒疫苗免疫後,可偵測到病毒核酸的時間較帶有移行 抗體的雞隻為長;就所能偵測到的分布區域而言,則明顯與疫苗投予的方 式有關。在點眼、口服、噴霧三種疫苗投予方式中,以點眼方式免疫的雞 隻,病毒在體內分布的部位皆為三種方式中最為平均而且持續時間最久的 ,其中又以免疫組織器官所能檢測到病毒核酸的時間最長。以口服方式投 予,病毒分布局限於上消化道,能檢出病毒核酸的時間則為三種方式之末 。至於噴霧免疫的方式,病毒同樣呈現局部分布的情形,以上呼吸道為主 ,檢出病毒核酸的時間則有些許的差異。以上述實驗結果為基礎所進行的 雞隻免疫抗體力價測定實驗,採行點眼、口服、噴霧、皮下注射等方式投 予新城雞瘟活毒疫苗。在基礎免疫後,各組抗體力價的變化情形差異很小 ,但經過油劑疫苗補強免疫之後,以點眼方式進行基礎免疫的雞隻抗體上 升的速度最快,程度最高且均勻度最好;噴霧免疫次之,皮下注射方式則 其高抗體力價持續時間最長,而以口服免疫效果最差。另外,本實驗亦進 行包括疫苗株與野外分離株在內的 RT-PCR,結果均能出現特異長度的產 物片段。至此已成功發展出利用單管 RT-PCR (single-tube RT-PCR) 來 檢測新城雞瘟病毒的技術,其快速、高敏感性及特異性的優點,可應用於 臨床新城雞瘟病毒的診斷。
To evaluate the distribution and vicissitude of Newcastle disease virus (NDV) in chicken vaccinated by live vaccines, we detected the presence of viral RNA by polymerase chain reaction (PCR). This approach took advantage of the high sensitivity and accuracy of PCR. Conventional protocols for RNA-polymerase chain reaction (RT-PCR) requires two separated steps: reverse transcription (RT) and polymerase chain reaction. This study developed a simplified, single-tube RT-PCR for the detection of NDV RNA. Primers designed for this RT-PCR were selected from a region within the F gene of NDV B1 strain. The result showed that, after vaccination, the presence of NDV RNA in SPF chickens was longer than that in maternal antibody-containing chickens. In addition, the location of viral RNA obviously depended on the route of vaccination. The eye-drop administration not only generated the widest range of viral distribution but also gave the longest period of presence of viral RNA. Moreover, after the eye-drop administration of vaccination, NDV appeared to present in the lymphoid tissues longer than in any other tissues. On the other hand, oral administration gave a viral distribution only limited to the upper respiratory tract; moreover, the period of presence of viral RNA was short. Aerosol administration gave a similar result to that of oral vaccination, but the period of presence of detectable viral RNA was digferent. In primary immunization, the titer of antibody generated by different administration routes ( eye-drop, oral, aerosol and subcutaneous injection) exhibited no significant difference. However, after the boost immunization by of oil- emulsified vaccine, the eye-drop administration showed the best increasing rate of antibody titer, followed by the aerosol administration. On the other hand, the subcutaneous injection gave the longest period of high antibody titer. The oral administration showed the poorest result of vaccination. The RT- PCR we developed was able to detect both the vaccine strain and field isolates of NDV; specific PCR product was visible in all of the NDV strains tested. This single-tube RT-PCR procedure present here therefore provided a rapid, highly specific and sensitive method for the clinical diagnosis of ND.
URI: http://hdl.handle.net/11455/13642
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