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研究生:謝金偉
研究生(外文):Jim-Wei Hsieh
論文名稱:台灣中部地區小型哺乳類動物Bartonella屬菌之流行病學研究
論文名稱(外文):Epidemiology of Bartonella spp. in Small Mammals in central Taiwan
指導教授:張照勤張照勤引用關係
指導教授(外文):Chao-Ching Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫公共衛生學研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:63
中文關鍵詞:小型哺乳類流行病學哺乳動物流行病學麥寮港危險性相關性巴東體屬菌
外文關鍵詞:Bartonellasmall mammalscentral Taiwan
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Bartonella spp.為新興人畜共通傳染疾病病原,其自然宿主涵蓋種類廣泛,過去研究顯示小型哺乳類動物為其重要宿主之一。本研究樣本捕捉地點分別位於中部地區兩商業港口(2002年8月起到翌年7月間,台中港與麥寮港),中部地區乳牛牧場與都市地區(2003年8月起到翌年11月間)進行野生小型哺乳類動物捕捉採樣,共計捕獲310隻鼠類,包括六物種:溝鼠(Rattus norvegeicus)169隻,錢鼠(Suncus murinus)126隻,小黃腹鼠(Rattus losea)3隻,家鼷鼠(Mus domesticus)1隻,小月鼠(Mus formosanus)1隻,屋頂鼠(Rattus rattus)10隻。結果顯示,菌血症陽性率為41.0%(127/310)。菌種鑑定則利用16S/23S rRNA基因 ITS片段以及gltA基因片段進行限制片段長度多型性(RFLP)技術組合分析,可將目前已知大部分之菌種區分,其中16S/23S rRNA基因 ITS片段可以快速觀察菌種差異性,RFLP分析可更仔細區別各分離菌株在具有相同PCR增幅基因片段中的差異。進一步將分離株之gltA基因片段進行定序,依親緣樹分析結果發現台灣地區Bartonella屬菌於小型哺乳類動物中相當多樣,並有混合感染之發現;其中B. elizabethae佔25.1%(32/127)、B. grahamii佔7.8%(10/127)、B. taylorii佔2.4%(3/127)與B. tribocorum佔41.7%(53/127),其中有22個血液樣本出現兩種以上基因型別,推測為混合感染(17.3%);此外並發現自錢鼠分離的菌株中有部分菌株為台灣特有的菌種佔30.7%(39/127),目前將其暫命為B. Taiwan-1 株,其種別特性待更深入研究。將全血萃取DNA後針對gltA 基因與16S/23S rRNA基因 ITS片段進行PCR檢測,所得總體陽性率分別為24.7%(75/304)及38.8%(118/304)。血清檢體利用間接螢光抗體法(IFA)針對台灣四種鼠類常見菌種抗原B. elizabethae, B. Taiwan-1, B. grahamii及B. tribocorum進行抗體檢測,陽性率結果分別為0.0%(0/308)、3.2%(10/308)、3.2% (10/308)及5.6%(12/308)。總體樣本分析流行病學資料的結果顯示菌血症的有無與動物種別、性別、年齡與季節有顯著相關(p<0.05);外寄生蟲的暴露有無雖未達顯著相關,但觀察兩港區螨的寄生與菌血症仍具有相關性(p<0.05)。本研究結果得知Bartonella屬菌在台灣野生小型哺乳動物中的生態分佈,而其中為人畜共通致病原的Bartonella屬菌株(B. elizabethae與 B. grahamii)分離結果,值得未來觀察與警戒台灣居民因意外接觸小型哺乳動物而感染Bartonella屬菌疾病的危險性。
Most of Bartonella spp. have been identified to be zoonotic pathogens. Previous studies showed that small mammals are important reservoirs for Bartonellae. This study was conducted in small mammals collected form harbor areas during August 2002 and July 2003, and one dairy cattle herd as well as urban area during August 2003 and November 2004 in central Taiwan. A total of 310 blood specimens were obtained, including specimens of Rattus norvegicus(169), Suncus murinus(126), Rattus losea(3), Mus domesticus(1), Mus formosanus(1) and Rattus rattus(10). It was identified that 41.0%(127/310) of the samples were Bartonella bacteremic. The isolates were further characterized by PCR of the 16S/23S ITS gene, and PCR/RFLP and partial sequencing of the gltA gene. The analysis of 16S/23S ITS gene could be used as a rapid diagnosis at the species level, and RFLP analysis of the gltA gene could further differentiate isolates with similar PCR products. According to the phylogentic analysis, various species of Bartonella were identified in small mammals tested, including B. elizabathae(25.1%, 32/127), B. grahamii(7.8%, 10/127), B. taylorii(2.4%, 3/127) and B. tribocorum(41.7%, 53/127). It was also found that 17.3%(22/127) of the animals were co-infected with different Bartonella species. Of major importance, 30.7%(39/127) of the isolates were with similar results by molecular characterization but determined to be unknown Bartonella species; they were mostly isolated from Suncus murinus. The strain was thus designated as B. Taiwan-1 in this study. Using PCRs of the 16S/23S ITS and gltA genes for DNA extractants from whole blood samples, 24.7% and 38.8% of them were positive. Seroprevalence of B. elizabathae, B. Taiwan-1, B. grahamii and B. tribocorum by IFA test were 0%, 3.2%, 3.2% and 5.6%, respectively. Epidemiologic data analyses further showed that bacteremic status was significantly associated with species of small mammals, sex, age and season of collection (p<0.05). Although the presence of ectoparasites was not associated with Bartonella bacteremia overall, mite infestation was a significant risk factor for Bartonella bacteremia in small mammals from two harbor areas. The results of this study offered valuable information regarding Bartonella infections in small mammals in Taiwan. The identification of zoonotic Bartonella spp. further highlights the possible risk of people acquiring the infections from small mammals in Taiwan.
目次

致謝……………………………………………………………………………….i
中文摘要…………………………………………………………………………ii
英文摘要………………………………………………………………………...iv
第一章 前言…………………………………………………………………......1
第二章 文獻探討……………………………………………………………......2
第一節 Bartonella 屬菌細菌學特性及歷史…………………………….......2
第二節 Bartonella 屬菌所引起之人畜共通傳染疾病與自然傳播途徑.......2
第三節 Bartonella 屬菌於小型哺乳類的研究………………………….......4
第四節 Bartonella 屬菌之基因型分析……………………………………...5
第五節 Bartonella 屬菌演化關係與自然宿主特異性探討………………...6
第三章 材料與方法…………………………………………………………..11
第一節 Bartonella spp.標準菌株來源……………………………………..11
第二節 檢體收集與製備……………………………………………………11
2-1 鼠隻樣本捕捉……………………………………………………….11
2-2 血液樣本製備與保存……………………………………………….11
2-3 全血DNA萃取……………………………………………………...12
第三節 Bartonella spp. 培養、分離與鑑定………………………………..12
3-1 Bartonella spp. 培養與分離………………………………………..12
3-2 Bartonella spp. 菌種鑑定與分型…………………………………..13
第四節Bartonella spp. 間接螢光抗體檢測(Indirect Immunofluorescence antiboby Test, IFA)…………………………………………………….............14
4-1 抗原玻片製作………………………………………………..............15
4-2 間接螢光抗體檢測(Indirect Immunofluorescence antiboby Test, IFA)..16
第五節 各項分析法與使用軟體…………………………………………..16
5-1 統計…………………………………………..……………………...16
5-2 核酸序列分析………………………………..……………………...17
第四章 結果…………………………………………..……………………....18
第一節 樣本採集:鼠類捕捉記錄與地理分佈…..……………………....18
第二節 Bartonella spp.分離與各菌血症陽性率…..……………………....18
第三節 菌種鑑定與基因型分析…. ………………..………………..…....19
第四節 全血直接PCR檢測………………………..……………………....21
第五節 Bartonella spp. 間接螢光抗體檢測(IFA)……………………....21
第五章 討論…………………………………………..……………………....23
第六章 參考文獻……………………………………..……………………....30













圖表索引

圖一. 粗糙型菌落外觀…………………..……………………………...…....38
圖二. 細小型菌落外觀…………………..……………………………...…....38
圖三. 結合16S/23S ITS基因與gltA基因PCR/RFLP分析所得之分型結果.39
圖四. 以最大簡約法(maximum parsimony method)進行親緣樹譜分析結
果(本研究分離株與標準株)…………………………………………40
圖五. 以最大可能性法(maximum Likelihood method)進行親緣樹譜分析
(本研究分離株與標準株)…………………………………………41
圖六. IFA陽性結果…………..……………………………...….....................38
圖七. IFA 陰性結果…………..……………………………...…....................38
圖八. 以最大簡約法(maximum parsimony method)進行親緣樹譜分析
(本研究分離株與世界各地小型哺乳類動物之分離株)…………..42
圖九. 以最大可能性法(maximum Likelihood method)進行親緣樹譜分析
(本研究分離株與世界各地小型哺乳類動物之分離株)…………43
表一.台灣中部地區三處捕捉地點所捕捉之鼠種記錄……………...…..........44
表二. 各鼠種於台灣中部地區各捕捉地點分佈情形……………...…............45
表三. 台灣中部地區Bartonella屬菌於小型哺乳類動物之菌血症盛行率.....46
表四. Bartonella屬菌之菌血症盛行率相關之危險因子分析……...…............47
表五. 三處採樣區內Bartonella屬菌之菌血症盛行率與相關危險因子分析.48
表六. 本研究中各分子型與其相關菌株標號…………………...…..............49
表七. 本研究分離株各型代表之gltA基因核酸序列與標準菌株之gltA基因序列基因相似度…………………...….......…………………...….......50
表八. 各潛在影響因子與各菌種分離株相關性分析.....………………….....51
表九. gltA基因及16S/23S rRNA基因ITS片段之PCR結果與菌血症分離結果比較.52
表十. 利用四種抗原所進行之Bartonella spp.血清抗體盛行率分析結果與各潛在危險因子相關性分析……………………………………………..53
表十一. 利用四種抗原所進行之Bartonella spp.血清抗體力價分佈………54

附錄
表一. 限制酵素作用表(廠商提供)…………………………………………55
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