摘 要
本研究之目的在證明雞傳染性貧血病毒（chicken infectious anemia virus; CIAV）可經由雞柔嫩艾美球蟲（Eimeria tenella）傳播的可能性。將CIAV接種於一日齡的SPF雞，待雞隻三週齡時再接種無CIAV污染的球蟲卵囊（oocyst），過一週後再剖檢，病雞盲腸以免疫螢光染色法可發現球蟲上具有CIAV陽性的螢光反應。收集卵囊並以浮游法純化之。為避免污染可能存在於宿主細胞中的病毒，將純化而得的卵囊先以SDS、Proteinase K、DNase及氯仿處理後再離心收集。將此蟲卵所萃取而得的DNA以CIAV特異性的引子對（primers）進行聚合酶鏈反應（PCR），結果得到一預期的 420 bp PCR產物。將此PCR產物定序，其基因序列與接種雞的CIAV序列完全吻合，可說明球蟲卵囊所攜帶的CIAV來自於接種的病雞。將含有病毒的卵囊接種於 MSB1 細胞，可導致CPE的現象。進一步以PCR分析鑑定結果也呈現CIAV 陽性反應。由以上的結果可以證明當雞感染CIAV並在體內增殖時，可將病毒散佈至盲腸內的E. tenella球蟲，至於這些帶有病毒的球蟲對宿主的影響，以及病毒是否會造成球蟲代謝反應甚或致病力的改變則需更進一步的研究來證明。

Abstract


The purpose of this study is to investigate the probability of Eimeria tenella as a carrier for the transmission of chicken infectious anemia virus（CIAV）. One-day-old specific pathogen free（SPF）chickens were inoculated with CIAV, and then infected 20 days later with E. tenella. CIAV proteins were detected by immunofluorescent test in schizont, gametocytes and zygotes of E. tenella. Oocysts were purified from infected chicken by NaNO3 gradient centrifugation. Oocyst DNA was extracted by the treatments of SDS, proteinase K, DNase and chloroform. PCR template preparated from oocyst was used for amplification of CIAV DNA which is a 420 bp DNA fragment. The sequence of the PCR product from oocyst is similar to that of CIAV DNA from blood of infected chicken. The oocysts of E. tenella containing CIAV could also be suggested by inducing cytopathic effect after inoculated into cultured MSB1 cells. Our results indicated aprobability that E. tenella oocysts contain CIAV proteins and nucleic acids and thus E. tenella might paly an important role in the epizootiology.

目 次

中文摘要………………………………………………………..Ⅰ
英文摘要……………………………………………………….Ⅱ
目 次……………………………………………………….Ⅲ
表 次………………………………………………………..Ⅴ
圖 次……………………………………………………… Ⅵ
第一章 前言…………………………………………………..1
第二章 材料與方法…………………………………………..8
第一節 病毒株及陽性血清的製備……………………… 8
第二節 柔嫩艾美球蟲的純化與收集…………………….9
第三節 病毒與球蟲接種試驗…………………………… 12
第四節 細胞培養與病毒接種…………………………….17
第五節 雞隻接種試驗…………………………………….18
第六節 電子顯微鏡之CIAV的偵測……………………..19
第三章 結果…………………………………………………….21
第一節 病毒株及陽性血清的製備….…………………… 21
第二節 柔嫩艾美球蟲的純化與收集……………………. 21



第三節 病毒與球蟲接種試驗……………………………. 22
第四節 細胞培養與病毒接種……………………………. 24
第五節 雞隻接種試驗……………………………………. 25
第六節 電子顯微鏡之CIAV的偵測…………………….. 25
第四章 討 論………………………………………………….26
第五章 參考文獻……………………………………………….32

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