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研究生:傅瀅濱
研究生(外文):Fuh, Yng-Bin
論文名稱:應用聚合�t鏈鎖反應偵測野生動物結核病之感染及動物組織病理變化之比較
論文名稱(外文):The application of polymerase chain reaction(PCR) on the diagnosis of tuberculosis in wildlife animals and comparison of histopathologic changes among animal species
指導教授:龐飛龐飛引用關係鄭謙仁---
指導教授(外文):Victor Fei PangChian-Ran Jeng
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學系研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:118
中文關鍵詞:結核病聚合鏈鎖反應野生動物
外文關鍵詞:tuberculosisPCRwildlife animal
相關次數:
  • 被引用被引用:4
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結核病的病灶一般是以結節狀的肉芽腫炎症反應為主,而此典型的病灶在顯微鏡下的特
徵為中央呈乾酪壞死樣伴隨著鈣化,外圍以淋巴球、類上皮細胞、巨噬細胞、多核巨細胞
等炎症細胞,並由一層莢膜包圍在外;但仔細研究不同野生動物的結核病感染例發現,各
動物種之間的病灶組成結構上仍有差異性存在。本研究的主要目的即欲從病理的角度,配
合分子生物學技術,進行野生動物感染本病的診斷,並比較野生動物間感染同型或不同型
分枝桿菌病灶之異同。以聚合鏈鎖反應進行結核病診斷的技術可直接應用於各種檢體,
使本病診斷的速率大幅提升。本研究以聚合鏈鎖反應,直接由福馬林固定組織檢體,進
行分枝桿菌的偵測及型別的鑑定,並做為野生動物結核病病灶病理學分類之依據。本研究
中共使用三組引動子,首先以第一組引動子 (PCR-TB) 進行聚合鏈鎖反應,可成功地對
分枝桿菌,包括: Mycobacterium tuberculosis, M. bovis, M. avium, M. paratubercu
losis 及M. fortuitum的65 kD表面抗原基因增幅出一383 bp 核酸片段。以第二組引動子
(PCR-MT),成功地對典型分枝桿菌,包括: M. tuberculosis和M. bovis 的38 kD prote
in antigen b 基因增幅出一419 bp核酸片段: 經第一組及第二組引動子作用,在肉眼下
沒有看見產物時,則分別以巢式PCR (Nested PCR)對產物進行再增幅及確認。對於第二組
引動子及其巢式PCR所無法偵測出的病材,則嘗試以第三組引動子 (PCR-JB) 輔助進行診
斷,對於陽性病材,第三組引動子可增幅出一段495 bp 核酸片段。在所收集的90個疑似
結核病感染的野生動物的福馬林固定組織檢體中,抗酸性染色、分枝桿菌及人型分枝桿菌
叢 (M. tuberculosis complex) 的檢出陽性率分別為40/90(44.4%) 、63/90 (70%)、59/
90 (65.56%)。對於無法進行細菌培養鑑定的檢體而言,此 PCR 技術將有助於結核病的檢
出。就所蒐集剖檢疑似結核病感染,並經PCR偵測確定為 M. tuberculosis complex 感染
的不同種別野生動物而言,其組織病理特徵的確有不同之處。以鹿科動物肺臟為例,水鹿
多以大區域融合性的壞死灶為主,病灶與周圍未波及組織間的界限明顯。梅花鹿與山羌的
病灶較相似,病灶與周圍組織間的界限較不明顯,並有不等程度的氣腫與炎症滲出液存在
,多核巨細胞的分佈較水鹿集中,並有形狀奇特、多達30-60個核的多核巨細胞。白鹿在
乾酪壞死區中有小化膿區與小鈣化區。而就猴科動物而言,其病灶以大區域的乾酪壞死配
合輕微的鈣化及炎症反應為特徵。本研究的結果顯示,不同種別野生動物間結核病病灶的
組織病理變化仍有不同的特徵,對於無法進行細菌培養鑑定的疑似結核病感染例,以PCR
技術配合組織病理學的檢查將會是結核病診斷上的一項重要輔助工具。
The typical lesion of tuberculosis is a nodular granulomatous inflammatory
reaction characterized by central caseous necrosis and calcification
accompanied with lymphocytes, epithelioid cells, macrophages, and
multinucleated giant cells in the peripheral region and surrounded by a
fibrous capsule. More recent studies have shown that differences in lesion
composition exist between different animal species. The present study
attempted to diagnose TB-infected wild animals from the aspect of pathology
by using molecular biology technique and to compare the lesion among different
animal species infected by same or different species of Mycobacterium. The
polymerase chain reaction (PCR) technology can be applied to several kind of
tissue samples and makes the diagnosis of TB more efficiently. In our study,
formalin-fixed tissues were used for the detection and typing of Mycobacterium
infection by PCR. The PCR results were then used as the basis for pathological
evaluation of the TB lesions among wild animals. Three sets of primers were
used for PCR. The first one amplified a 383 bp nucleotide fragment that is
encoded for the 65 kDa surface antigen of M. tuberculosis, M. bovis, M. avium,
M. paratuberculosis and M. fortuitum. The second primer set amplified a 419 bp
nucleotide fragment that is encoded for the 38 kDa protein antigen b of M.
tuberculosis and M. bovis. When the PCR product of the first and/or second
sets of primer was invisible by electrophoresis, it was further amplified and
confirmed by nested PCR. The third primer set functioned as an alternative for
the second one since it could amplify a 495 bp nucleotide fragment that
couldn''t be done by the second primer set. Within the 90 formalin-fixed
TB-suspected cases that we collected, the detecting frequency of acid-fast
positive bacteria, Mycobacterium sp. by the 1st primer set, and M. tuberculosis
complex by the 2nd/3rd primers set were 40/90 (44.4%), 64/90 (71.1%), and
58/90 (64.4 %), respectively. Thus, for those tissue samples that bacterial
isolation could not be performed, PCR technique can provide a significant
help in the diagnosis and typing of Mycobacterium infection. When comparing
the lesions observed in those cases that had M. tuberculosis complex infection
confirmed by PCR, it was found that different histopathologic characteristics
did occur between different species of wild animals. Taking the pulmonary
lesions seen in cervid species as an example, large confluent necrotic lesion
with emphysema and inflammatory exudate of varying amounts in the surrounding
tissue was the typical feature of Formosan sambar (Cervus unicolor swinhoei).
The lesions seen in Formosan muntjac (Muntiacus reevesi micrucus) and Formosan
sika deer (Cervus nippon taiouanus) were quite similar and generally well
circumscribed; in these two species, the multinucleated giant cells often
formed aggregates, were variable in shapes, and had up to 30-60 nuclei. Within
the caseous necrotic lesion of fallow deer (Dama dama) were small abscesses
and calcified areas. As for the non-human primate, the inflammatory reaction
was in general not obvious in the affected region. Our result has clearly
demonstrated that the histopathologic changes of TB lesion were different
among several wild animal species. It is concluded that for those TB-suspected
but bacterial isolation unavailable cases, PCR technique accompanied by
histopathological examination will be an useful tool for the diagnosis and
typing of Mycobacterium infection.
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