臺灣博碩士論文加值系統

犬艾利希體(Ehrlichia canis)在犬隻間是具有高度傳染性的疾病。是由一種棕色犬壁蝨(Rhipicephalus sanguineous)所媒介感染，被感染犬隻會造成泛血球缺乏症、貧血、出血、全身淋巴腺病變、發燒及食慾不振等，嚴重會導致死亡。為了解目前艾利希體在犬隻間的感染情況，與相關的流行病學。本調查從2011年10月至2013年9月止，共採集臺南地區家犬和流浪犬571隻的血液樣本。其中由動物醫院所採集到的病例有293例，而由民間收容所及流浪動物中心採集的有278例。應用酵素結合免疫吸附法(enzyme-linked immunosorbent assay；ELISA)檢驗套組，並且以具有特異性引子對的聚合酶連鎖反應 (polymerase chain reaction；PCR) 檢測犬隻艾利希體症的盛行率。應用酵素結合免疫吸附法檢測結果顯示家犬和流浪犬感染艾利希體症陽性率分別為15.4％(45/293)和24.5％(68/278)。總合感染率為19.8％ (113/571)。應用聚合酶連鎖反應檢測結果陽性率分別為10.9％(32/293)和17.3％(48/278)，總合感染率為14.0％(80/571)。在品種、性別與年齡的分析上並無明顯的差異與相關性。在季節變化上則以夏季和秋季兩季的感染比率為最高，春季次之、冬季最低。流浪犬感染的比率明顯高於家犬。研究結果雖然應用ELISA的檢出率較高，但容易有交叉反應的機會，而PCR檢測陽性率較低，但其敏感性與特異性較為專一，為求更精確的檢測還是以PCR的方式較準確，此結果可提供作為疾病防疫之參考依據。

關鍵字:艾利希體症、棕色犬壁蝨、酵素結合免疫吸附法、聚合酶連
鎖反應

Ehrlichia canis among dogs is a highly contagious disease. The infection is caused by the Rhipicephalus sanguineous. Ehrlichia canis causes many dog diseases including pancytopenia, anemia, bleeding, systemic lymphadenopathy, fever and loss of appetite, death in the infected dogs.The aim of this study is to understand the epidemiology of the Ehrlichia infection in the dogs. Our survey completed from October 2011 to September 2013, we collected 571 blood samples from stray dogs in Tainan. The samples were collected from the animal hospital is 293 cases and from private shelters and animal center is 278 cases. We used enzyme-linked immunosorbent assay Test Kits and the specific primers for the polymerase chain reaction to detect the prevalence of the Ehrlichia canis among dogs. The ELISA assay results showed that the positive rate of the family dog and stray dogs infected with Ehrlichi canis is 15.4% (45/293) and 24.5% (68/278) respectively, and total is 19.8% (113/571). The results of the polymerase chain reaction assay showed that the positive rate of Ehrlichia canis in the family dogs and stray dogs is 10.9% (32/293) and 17.3% (48/278) respectively and total is 14.0％(80/571) respectively. There are no difference in the species, age and gender. The rate of the infection it highest in spring and lowest in winter and the stray dogs is higher than family dogs. The results demonstrate that the detectable rate is higher by ELISA but more easily immunologic cross reaction. The detectable rate of the PCR analysis is lower but it is more sensitive and specific. In order to more accurate detection by PCR method. The results provide more information for prevention and control of disease.

Keywords: Ehrlichiosis , brown dog ticks, enzyme-linked
immunosorbent assay, polymerase chain reaction

中文摘要.................................................Ⅰ
英文摘要.................................................Ⅲ
誌謝.....................................................Ⅴ
目錄.....................................................Ⅵ
圖表目錄.................................................XI
第 1 章前言...............................................1
第 2 章文獻回顧...........................................4
2.1 艾利希體之簡介........................................4
2.2 壁蝨簡介.............................................5
2.3 艾利希體症流行病學....................................6
2.4 艾利希體症病原.......................................6
2.5 艾利希體症之傳染方式..................................8
2.6 艾莉希體症之臨床症狀與病變.............................8
2.7 艾利希體的實驗室診斷.................................10
2.7.1 臨床病理學變化....................................10
2.7.2 血液抹片檢查......................................10
2.7.3 光學顯微鏡檢查....................................11
2.7.4 免疫螢光抗體法....................................11
2.7.5 細胞培養.........................................12
2.7.6 酵素結合免疫吸附法................................12
2.7.7 聚合酶連鎖反應....................................13
2.8 艾利希體症的治療....................................13
2.9 公共衛生與預防......................................14
第3 章材料與方法........................................15
3.1 實驗材料...........................................15
3.1.1 檢體收集.........................................15
3.2.實驗方法...........................................15
3 .2 .1 酵素結合免疫吸附法.............................. 15
3 .2 .2 血液核酸抽取與純化..............................15
3.2.3 聚合酶連鎖反應....................................16
3.2.3.1 引子對的設計....................................16
3.2.3.2 犬型艾利希體聚合酶連鎖反應........................17
3.2.3.4 血小板型艾利希體聚合酶連鎖反應.....................17
3.2.3.5 馬型艾利希體聚合酶連鎖反應........................18
3.2.4 聚合酶連鎖反應產物之電泳分析........................18
3.2.5 聚合酶連鎖反應產物之純化............................18
3.2.6 接合作用..........................................19
3.2.7 勝任細胞之製備....................................19
3.2.8 轉形作用..........................................20
3.2.9 菌落之篩選與培養...................................20
3.2.10 質體DNA 的萃取與純化..............................20
3.2.11 限制酶切割確認....................................21
3.2.12 陽性菌株的保存....................................21
3.2.13 核酸定序與分析....................................21
3.3 艾利希體引子對敏感性試驗..............................21
3.3.1 犬型艾利希體引子對敏感性試驗.........................21
3.3.2 血小板型艾利希體引子對敏感性試驗.....................22
3.3.3 馬型艾利希體引子對敏感性試驗.........................22
3.4 艾利希體引子對特異性試驗..............................22
3.4.1 犬型艾利希體引子對特異性試驗.........................22
3.4.2 血小板型艾利希體引子對特異性試驗.....................23
3.4.3 馬型艾利希體引子對特異性試驗........................23
3.5 統計分析............................................23
第 4 章結果.............................................24
4.1 家犬與流浪犬基本資料.................................24
4.1.1 品種分析..........................................24
4.1.2 性別分析..........................................24
4.1.3 年齡分析..........................................24
4 . 2 病原核酸引子對的敏感性試驗與特異性試驗................26
4.2.1 Ehrl ichia canis 引子對的敏感性試驗................26
4 .2 .2 Eh r l i ch ia ca n i s 引子對的特異性試驗.......27
4.2.3 Anaplasma platys 引子對的敏感性試驗................28
4.2.4 Anaplasma platys 引子對的特異性試驗................29
4.3 ELISA 檢測家犬艾利希體症之結果統計.....................30
4.3.1 ELISA 檢測家犬艾利希體症之結果統計...................30
4.3.2 ELISA 法檢測流浪犬艾利希體症之結果統計................30
4.3.3 ELISA 檢測家犬與流浪犬艾利希體症之結果統..............30
4.4 PCR 檢測犬艾利希體症之結果統計.........................34
4.4.1 PCR 檢測家犬艾利希體症之結果統計.....................34
4.4.2 PCR 檢測流浪犬艾利希體症之結果統計...................34
4.4.3 PCR 檢測家犬合併流浪犬艾利希體症之結果統計............34
4.5 ELISA 檢測家犬與流浪犬艾利希體症之結果與品種、
性別之相關性...................................38
4.5.1 ELISA 檢測家犬艾利希體症之結果與品種之相關性..........38
4.5.2 ELISA 檢測流浪犬艾利希體症之結果與品種之相關性........38
4.5.3 ELISA 檢測家犬與流浪犬艾利希體症之結果與品種
之相關性....................................38
4.5.4 ELISA 檢測家犬艾利希體症之結果與性別之相關性..........43
4.5.5 ELISA 檢測流浪犬艾利希體症之結果與性別之相關性........43
4.5.6 ELISA 檢測家犬與流浪犬艾利希體症之結果與性別
之相關性......................................43
4 . 6 PCR 檢測家犬與流浪犬艾利希體症之結果與品種、
性別之相關性..................................47
4.6.1 PCR 檢測家犬艾利希體症之結果與品種之相關..............47
4.6.2 PCR 檢測流浪犬艾利希體症之結果與品種之相關............47
4.6.3 PCR 檢測家犬與流浪犬艾利希體症之結果與品種
之相關性......................................47
4.6.4 PCR 檢測家犬艾利希體症之結果與性別之相關性............52
4.6.5 PCR 檢測流浪犬艾利希體症之結果與性別之相關性..........52
4.6.6 PCR 檢測家犬與流浪犬艾利希體症之結果與性別
之相關性......................................52
4.7 ELISA 檢測流浪犬及家犬感染艾利希體症與年齡的關..........56
4.7.1 ELISA 檢測家犬感染艾利希體症與年齡的關係.............56
4.7.2 ELISA 檢測流浪犬感染艾利希體症與年齡的關係...........56
4.7.3 ELISA 檢測流浪犬合併家犬感染艾利希體症
與年齡的關係..................................57
4.8 PCR 檢測流浪犬及家犬感染艾利希體症與年齡的關係..........61
4.8.1 PCR 檢測家犬感染艾利希體症與年齡的關係...............61
4.8.2 PCR 檢測流浪犬感染艾利希體症與年齡的關係.............61
4.8.3 PCR 檢測流浪犬合併家犬感染艾利希體症
與年齡的關係..................................62
4.9 ELISA 檢測流浪犬及家犬感染艾利希體症與季節的關係........66
4.9.1 ELISA 檢測家犬感染艾利希體症與季節的關係.............66
4.9.2 ELISA 檢測流浪犬感染艾利希體症與季節的關係...........66
4.9.3 ELISA 檢測流浪犬合併家犬感染艾利希體症
與季節的關係................................66
4.10 PCR 檢測流浪犬及家犬感染艾利希體症與季節的關係.........68
4.10.1 PCR 檢測家犬感染艾利希體症與季節的關係..............68
4.10.2 PCR 檢測流浪犬感染艾利希體症與季節的關係............68
4.10.3 PCR 檢測流浪犬合併家犬感染艾利希體症
與季節的關係.................................68
4.11 定序結果比對與分析..................................70
4.11.1 Ehrlichia canis 序列比對分析與親源關係樹狀圖.......70
4.11.2 Anaplasma platys 序列比對分析與親源關係樹狀圖......74
第 5 章討論............................................77
參考文獻...............................................82
作者簡介...............................................90

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