臺灣博碩士論文加值系統

犬瘟熱 (canine distemper,簡稱CD) 的疾病。其病原為犬瘟熱病毒，隸屬於副黏液病毒科，麻疹病毒屬之病毒。該疾病所感染的對象以肉食目為主，包括犬科、貓科、鬣狗科、鼬科、浣熊科、熊科、靈貓科及海豹科。在國內野生動物方面，目前已知台灣南投郊外已有一隻豹貓血中帶有CDV的抗體，由此可知台灣的野生動物也對CDV具感受性。因此本研究係嘗試以酵素結合免疫吸附法 (ELISA)、形態病理學及反轉錄聚合酶鏈反應 (RT-PCR) 的技術，應用於偵測動物園內野生動物CDV感染情況。以ELISA的技術偵測動物CDV抗體，另以RT-PCR偵測CDV的核酸。藉以評估動物園的野生動物感染是否已遭受CDV感染及其感染程度、症狀及具感受性的動物種別等。本研究材料來自台北市立動物園所飼養的動物合計1998年至2002年庫存196個血清分別屬於131隻動物，以ELISA檢測有有40隻動物有CDV陽性抗體 (30.5%)。調查動物園肉食獸的對外展示區，包括台灣鄉土動物區、非洲動物區、亞洲熱帶雨林區、溫帶動物區、夜行館皆出現CDV抗體的動物。就動物園外的地區調查，野柳海洋世界及私人居家飼養的貓科動物也有CDV抗體出現。病理學檢查有2例發現類似CDV感染的病變，以CDV免疫組織化學染色，在肺臟的融合細胞及第二型肺泡細胞，胃黏膜的融合細胞和腎臟的腎盂部上皮出現CDV陽性訊號而確診；RT-PCR檢測41隻動物的體液或組織，有16隻動物 (39%, 16/41) 呈現陽性反應，其中有10隻存活，6隻死亡。將RT-PCR所檢測之16隻陽性動物中，有8隻也曾做過ELISA試驗：其中有3隻動物ELISA為陽性；另有5隻動物的ELISA檢測為陰性。在RT-PCR產物之核酸定序分析中，山獅No.3的PCR產物比對200 bp的CDV的磷酸蛋白基因，證實為CDV感染所致。其基因相似度與日本的Yanaka 和 Hamamatsu株比較為100%，並與目前本土性的犬隻感染全身型的CDV磷酸蛋白序列相同。本研究利用大規模血清學的監測，得知國內許多動物種別如前人文獻所述對CDV均具感受性。此外，本實驗結合血清學、病理學及RT-PCR確認國內動物園的圈養動物已有CDV感染的病例。

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores, including canids, felids, hyaenids, mustelids, procyonids, ursids, and viverrids. Taiwan is an endemic area of CDV infection in dogs. A seroepidemiological survey of CDV infection revealed that the prevalence of antibodies was found in domestic cats and a leopard cat in Taiwan. It is speculated that wildlife in Taiwan is also susceptible to CDV. To investigate current status of CDV infection in wildlife in Taiwan, ELISA and pathology combined with RT-PCR were conducted in the study. All samples were collected from the routine health check and necropsy materials of carnivores at Taipei zoo. A total of 196 sera obtained from 131 carnivores were used in serological investigation. CDV-positive was detected in 72 sera (72/196, 36.7%) from 40 carnivores (40/131, 32%). Antibodies against CDV were detected at display areas including Formosan Animal Area, African Animal Area, Asian Tropical Rainforests Animal Area, Temperate Zone Animal Area, and Nocturnal Animal House. In addition, positive ELISA results were also found in feline species at private and Ocean World. By histopathology and ABC-AP immunohistochemistry against CDV nucleoprotein employed in the lung, stomach, and renal pelvis specimens, 2 cases were diagnosed as CD. Tissues and body fluids obtained from 41 carnivores were examined by RT-PCR and nested PCR. Phosphoprotein gene of canine distemper virus (CDV) was used as the target gene for RT-PCR. By RT-PCR, positive signals were detected in the 16 of 41 (16/41, 39%) animals. Nucleic acid sequence analysis of part of the P genes demonstrated that the virus from Mountain lion No.3 had 100% similarities with Yanaka and Hamamatsu strain of Japan and those confirmed from dog with CD in Taiwan. In conclusion, serological survey provides a general screening data that wildlife in Taipei Zoo has been exposed to CDV which was also confirmed in carnivores by pathology and RT-PCR.

目錄--------------------------------------------------------------------------------------------I
圖次--------------------------------------------------------------------------------------------IV
表次--------------------------------------------------------------------------------------------V
中文摘要--------------------------------------------------------------------------------------VI
英文摘要--------------------------------------------------------------------------------------VIII
第一章 文獻探討與實驗目的------------------------------------------------------------1
第一節 犬瘟熱病毒感染之歷史背景---------------------------------------------1
第二節 犬瘟熱病毒之宿主範圍--------------------------------------------------2
第三節 犬瘟熱病毒之病毒特性--------------------------------------------------- 5
第四節 犬瘟熱病毒之流行病學---------------------------------------------------6
第五節 犬瘟熱病毒感染之致病機轉---------------------------------------------8
第六節 野生動物感染犬瘟熱病毒之臨床症狀---------------------------------9
第七節 犬瘟熱病毒之診斷---------------------------------------------------------11
第八節 犬瘟熱病毒感染之形態病理學變化------------------------------------14
第九節 實驗目的---------------------------------------------------------------------15
第二章 材料與方法------------------------------------------------------------------------16
第一節 實驗設計及流程圖---------------------------------------------------------16
第二節 材料來源---------------------------------------------------------------------17
第三節 間接酵素結合免疫吸附法------------------------------------------------17
2.3.1 實驗材料----------------------------------------------------------------17
2.3.2 實驗方法-----------------------------------------------------------------19
2.3.3 結果判讀----------------------------------------------------------------19
第四節 形態病理學檢查------------------------------------------------------------20
2.4.1 肉眼病理學檢查--------------------------------------------------------20
2.4.2 組織病理學檢查--------------------------------------------------------20
2.4.3 犬瘟熱病理切片的判定標準----------------------------------------22
2.4.4 組織化學染色-----------------------------------------------------------23
2.4.5 免疫組織化學染色-----------------------------------------------------24
第五節 巢式反轉錄聚合酶鏈反應檢測犬瘟熱病毒之感染------------------27
2.5.1 樣材-----------------------------------------------------------------------27
2.5.2 引子之選定--------------------------------------------------------------27
2.5.3 病毒核酸之萃取--------------------------------------------------------28
2.5.4 反轉錄反應--------------------------------------------------------------29
2.5.5 聚合酶鏈反應-----------------------------------------------------------30
2.5.6 聚合酶鏈反應之電泳分析--------------------------------------------31
2.5.7 聚合酶鏈反應產物之定序--------------------------------------------32
2.5.8 聚合酶鏈反應產物之序列分析--------------------------------------32
第三章 結果---------------------------------------------------------------------------------33
第一節 間接酵素結合免疫吸附法之結果---------------------------------------33
3.1.1 判讀結果----------------------------------------------------------------33
3.1.2 ELISA檢測動物的飼養地區-----------------------------------------33
3.1.3陽性動物的血清力價消長--------------------------------------------34
3.1.4未施打疫苗且為ELISA陽性動物之病史回溯-------------------34
3.1.5已施打疫苗且為ELISA陽性動物之病史回溯-------------------34
第二節 形態病理學檢查------------------------------------------------------------35
3.2.1 肉眼病理學檢查--------------------------------------------------------35
3.2.2 組織病理學檢查--------------------------------------------------------35
3.2.3 組織化學染色-----------------------------------------------------------36
3.2.4 免疫組織化學染色-----------------------------------------------------36
第三節 巢式反轉錄聚合酶鏈反應檢測及核酸序列分析--------------------- 36
3.3.1 巢式反轉錄聚合酶鏈反應檢測-------------------------------------36
3.3.2 核酸序列分析-----------------------------------------------------------37
第四章 討論---------------------------------------------------------------------------------38
第五章 參考文獻---------------------------------------------------------------------------49

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