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研究生:曾宇銓
研究生(外文):Yu-Chuan Tseng
論文名稱:臺灣花蓮地區動物之梨形鞭毛蟲暨隱胞子蟲流行病學調查暨蟲株基因分型研究
論文名稱(外文):Epidemiology and genotyping of Giardia lamblia and Cryptosporidium sp. in animals in Hualien of Taiwan
指導教授:彭士奕
指導教授(外文):Shih-Yi Peng
口試委員:廖建維鄭柏青
口試委員(外文):Chien-Wei LiaoPo-Ching Cheng
口試日期:2013-06-25
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學檢驗生物技術學系醫學生物技術碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:68
中文關鍵詞:梨形鞭毛蟲隱胞子蟲
外文關鍵詞:Giardia lambliaCryptosporidium sp.
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梨形鞭毛蟲和隱胞子蟲皆為人畜共通之原蟲寄生蟲,藉由糞口感染或汙染水源傳播而引起流行性下痢。他們的囊體或卵囊可以抵抗自來水的加氯消毒處理,故容易造成飲用水的污染而引發大流行。而目前台灣對於梨形鞭毛蟲和隱胞子蟲在家畜動物的感染率研究並不多。因此本研究將使用較敏感且專一之nested PCR進行檢測及種間區分。本實驗將以梨形鞭毛蟲之b-giardin基因、隱胞子蟲之18S rDNA為藍本,來進行Nested PCR增幅,以確定梨形鞭毛蟲的基因分型及隱胞子蟲的種類。研究範疇為台灣花蓮地區,在研究期間主要採集當地家畜動物糞便檢體共373個(犬隻76個,牛隻156個,豬隻141個)進行檢查及基因型鑑定。檢測結果梨形鞭毛蟲於犬隻中感染率為9.21% (7/76)、乳牛感染率為19.87% (31/156)及豬隻感染率為4.26% (6/141)。陽性檢體經基因分析比對犬隻有6個檢體之去氧核醣核酸與已公佈之Assemblage C相近,另1個檢體之去氧核醣核酸與已公佈之Assemblage D一致;24個乳牛梨形鞭毛蟲陽性檢體之去氧核醣核酸與已公佈之Assemblage E一致或相近,1個乳牛梨形鞭毛蟲陽性檢體之去氧核醣核酸與已公佈之Assemblage D相近,另外6個梨形鞭毛蟲陽性檢體之去氧核醣核酸序列無法比對。豬隻檢體經基因分析比對有4個檢體之去氧核醣核酸與已公佈之Assemblage E相近,1個檢體之去氧核醣核酸與已公佈之Assemblage D一致,1個檢體之去氧核醣核酸與已公佈之Assemblage A一致。另外,在隱胞子蟲部分犬隻中感染率為5.26% (4/76)、乳牛感染率為26.28% (41/156)及豬隻感染率為16.31% (23/141)。陽性檢體經物種分析比對後在犬隻中4個都與已公佈之C. canis相近;乳牛41個隱胞子蟲陽性檢體中有8個屬於C. bovis、1個屬於C. canis、4個屬於C. ryanae、25個屬於C. scrofarum,3個序列無法比對。豬隻的隱胞子蟲經比對後與C. pig genotype II相近的有22個,1個與C. suis相近。結果顯示只有一個豬隻檢體為梨形鞭毛蟲人畜共通基因型(Assemblage A),其餘家畜動物找到的梨形鞭毛蟲及隱胞子蟲並無人畜共通的疑慮。本篇研究為臺灣花蓮地區第一篇針對動物感染梨形鞭毛蟲及隱胞子蟲的流行病學調查,此結果將可提供疾病防治和公共衛生等相關單位之流行病學參考依據。
Giardia lamblia and Cryptosporidium sp. are zoonotic protozoan parasites that will cause diarrhea by waterborne or fecal-oral infection. Their cysts or oocysts are resistant to the chlorination with the water, and they can pollute the drinking water and cause a pandemic disease. In Taiwan, little information is available about the epidemiology of G. lamblia and Cryptosporidium sp. in domestic animals. Therefore, this study amplified DNA sequencing of gene (G. lamblia : β-giardin and Cryptosporidium sp. : 18S rDNA) by nested PCR for both detecting and genotyping of G. lamblia and Cryptosporidium sp. from the domestic animals in Hualien of eastern Taiwan. Fecal samples were collected from 76 dogs, 156 cattle and 141 pigs in Hualien of eastern Taiwan. The infection rate of G. lamblia was 9.21% for dogs (7/76), 19.87% for cattle (31/156) and 4.26% for pigs (6/141). The genetic analysis of 7 positive samples from dogs showed that 6 belonged to assemblage C and 1 was assemblage D. The genotype of 24 infected samples of cattle belonged to assemblage E, 1 sample belonged to assemblage D and other 6 samples can’t confirm genotype. For pigs, 4 samples belonged to assemblage E, 1 was assemblage D and 1 was assemblage A. In addition, 5.26% for dogs (4/76), 26.28% for cattle (41/156) and 16.31% for pigs (23/141) were infected by Cryptosporidium sp. The species analysis was that 4 samples from dogs belonged to C. canis; 41 samples from cattle belonged to C. bovis (8), C. canis (1), C. ryanae (4), C. scrofarum (25) , other 3 samples of cattle can’t confirm infected species. The species analysis of 22 samples from pigs belonged to C. pig genotype II and 1 was C. suis. These results showed that G. lamblia assemblage A was detected in pigs, and may cause zoonotic transmission. This is the first epidemiological investigations of G. lamblia and Cryptosporidium sp .infected in animals in Hualien of Taiwan. These results could provide the epidemiological information for disease control and public health protection.
中文摘要…………………………………………………………………...I
英文摘要…………………………………………………………………III
目錄…………………………………………………………….…………IV
第一章 研究背景及目的………………………………………………….1
1.1 前言…………………………………………………..………………1
1.2 背景資料………………………………………………..……………1
1.3 形態...………………………………………………………...………3
1.4 寄生生活史及傳染途徑………………………………………..……3
1.5 疾病症狀、診斷及治療…………………………………………..…..5
1.6 研究目的……………………………………………………………..7
第二章 研究材料與方法………………………………………………….9
2.1 糞便檢體的採集…………………………………..…………………9
2.2 糞便檢體蟲株基因型鑑定……………………………..……………9
2.2.1 糞便檢體去氧核醣核酸抽取……………………...………..……9
2.2.2 糞便檢體以聚合酶連鎖反應檢測梨形鞭毛蟲..……….………10
2.2.3 糞便檢體以聚合酶連鎖反應檢測隱胞子蟲…..…….…………12
2.2.4 巢氏聚合酶連鎖反應產物檢視及定序分析…..…….…………13
第三章 實驗結果………………………………………………………...14
3.1 梨形鞭毛蟲……………………………………………..…………..14
3.1.1 犬隻糞便陽性結果檢出率…...…………………………..……..14
3.1.1.1 犬隻糞便檢體蟲株基因型鑑定結果..…………………..…..14
3.1.2 牛隻糞便陽性結果檢出率…...…………………………..……..15
3.1.2.1 牛隻糞便檢體蟲株基因型鑑定結果..………………..……..15
3.1.3 豬隻糞便陽性結果檢出率…..……………………………...…..15
3.1.3.1 豬隻糞便檢體蟲株基因型鑑定結果..………………..……..16
3.2 隱胞子蟲……………………………………………………..……..17
3.2.1 犬隻糞便陽性結果檢出率…...……………………………..…..17
3.2.1.1 犬隻糞便檢體蟲株基因型鑑定結果..………………..……..17
3.2.2 牛隻糞便陽性結果檢出率…...…………………………..……..17
3.2.2.1 牛隻糞便檢體蟲株基因型鑑定結果..………………..……..18
3.2.3 豬隻糞便陽性結果檢出率…...…………………………..……..18
3.2.3.1 豬隻糞便檢體蟲株基因型鑑定結果..………………..……..18
3.3 雙重感染,糞便檢出梨形鞭毛蟲及隱胞子蟲……………..…..…..20
3.3.1 糞便陽性結果………………...…………………………..……..21
第四章 討論……………………………………………………………...20
第五章 附圖表…………………………………………………………...28
圖一、花蓮縣市犬隻、牛隻、豬隻糞便檢體採集地點………………29
圖二、第二次巢式PCR檢測梨形鞭毛蟲之糞便檢體檢測結果……....30
圖三、第二次巢式PCR檢測隱胞子蟲之糞便檢體結果…………....…31
圖四、6個犬隻糞便陽性檢體經基因序列比對與梨形鞭毛蟲
Assemblage C (AY545646)達99.0%以上相似……………….32
圖五、各1個犬隻、牛隻、豬隻糞便陽性檢體經基因序列比對
和梨形鞭毛蟲Assemblage D (AY545648)達99.6%以上相似.31
圖六、24個乳牛糞便陽性檢體經基因序列比對和梨形鞭毛蟲
Assemblage E (DQ116608)達99.0%以上相似…….….………33
圖七、4個豬隻糞便陽性檢體經基因序列比對和梨形鞭毛蟲
Assemblage E (DQ116608)100%一致………..…………...…..34
圖八、1個豬隻糞便陽性檢體經基因序列比對和梨形鞭毛蟲
Assemblage A (AY 072723)達99.3%以上相似……..…...…....35
圖九、4個犬隻檢體與1個牛隻檢體經基因序列比對後
和隱胞子蟲C. canis (AF112576)序列相似
(犬隻99.0%以上相似,牛隻100%一致)……….……………….36
圖十、8個牛隻糞便陽性檢體經基因序列比對後和隱胞子蟲
C. bovis (AB746197)序列達98.0%以上相似……….…………..37
圖十一、4個牛隻糞便陽性檢體經基因序列比對後和隱胞子蟲
C. ryanae (AB746196)序列達99.0%以上相似……….……..…..38
圖十二、25個牛隻糞便陽性檢體經基因序列比對和隱胞子蟲
C. scrofarum (KC481229)序列達98.0%以上相似……….……...39
圖十三、22個豬隻糞便陽性檢體經基因序列比對與隱胞子蟲
C. pig genotype II (AB693944)序列100%一致…….….…………40
圖十四、1個豬隻糞便陽性檢體經基因序列比對後與隱胞子蟲
C. suis (AB449870)序列100%一致……….…………………......41
表一、隱胞子蟲物種分類與宿主之對應關係………………………….42
表二、梨形鞭毛蟲基因型分類與宿主之對應關係…………………….43
表三、花蓮地區犬糞便梨形鞭毛蟲感染率調查…………………..….44
表四、花蓮地區犬糞便隱胞子蟲感染率調查……………………..….45
表五、花蓮地區動物糞便檢出梨形鞭毛蟲陽性數目
與基因分型結果一覽……………..…………………………….46
表六、花蓮地區動物糞便檢出隱胞子蟲陽性數目與基因分型結果一
覽………………………………………………………………...47
表七、花蓮地區動物糞便檢出梨形鞭毛蟲及隱胞子蟲
雙重感染陽性檢體數目………………………………………...48
第六章 參考文獻…………………………………………………………...49

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