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研究生:蔡依達
研究生(外文):Tsai I-Da
論文名稱:台中地區流浪犬心絲蟲及金門地區家犬血液寄生蟲疫情調查和應用鳳梨酵素溶解心絲蟲體外試驗
論文名稱(外文):Epidemiological Survey of Dirofilariasis of Stray Dogs in Taichung Area and Blood Parasites Infection of Housed Dogs in Kimmen Area and The in Vitro Lysis of Canine Heartworm by Use of Bromelain
指導教授:鄭豐邦
指導教授(外文):Cheng Feng-Pang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:犬心絲蟲症犬血液寄生蟲鳳梨酵素金門台中感染率人畜共通傳染病
外文關鍵詞:DirofilariasisCanine blood parasitesbromelainKinmenTaichunginfection ratezoonosis
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本研究的目的在於瞭解鳳梨酵素(Bromelain)對於心絲蟲成蟲體外之溶解能力,以了解未來運用於犬心絲蟲症臨床治療之可能性。在本實驗中,分別以PBS和plasma兩種溶劑及三種不同濃度的鳳梨酵素和一組對照組在 37℃無菌環境下來觀察蟲體的溶解情形。實驗結果顯示,蟲體在plasma和PBS溶液中10mg/mL鳳梨酵素的濃度下,公母蟲蟲體完全溶解的時間各為plasma:♀ 27.0 ±1.1天,♂ 25.7 ± 2.1天;PBS:♀ 26.3 ± 1.9天,♂ 24.0 ± 1.2天(n = 6)。在5mg/mL的濃度下,公母蟲蟲體完全溶解的時間各為plasma:♀ 47.3 ± 2.3天,♂ 43.7 ± 2.3天;PBS:♀ 45.8 ± 1.9天,♂ 42.2 ± 1.9天(n = 6);而對照組的蟲體在缺乏酵素溶液的環境下,可以保存六個月以上。經由組織切片發現,酵素溶解蟲體是由體內組織臟器層開始,而表皮層相對溶解速度則較慢。鳳梨酵素的濃度與溶劑的種類會影響蟲體的溶解(p<0.05)。高濃度的鳳梨酵素對於心絲蟲蟲體具有較好之溶解效果;以PBS作為溶劑其對於蟲體亦有較Plasma好之溶解能力,公母蟲之溶解情況則無顯著差異(p>0.05)。由於鳳梨酵素主要辨認切割位置為Z-Arg-Arg+NHMec,因此推測蟲體含有類似之蛋白質結構。蟲體蛋白質電泳分析則顯示有一特別之45 kDa蛋白,其可能為影響鳳梨酵素溶解蟲體之關鍵。未來研究目標應朝向活體應用鳳梨酵素於心絲蟲症之研究,進而降低因治療成蟲後所引起之肺動脈血栓栓塞及其併發症的發生。
為了瞭解台中地區流浪犬隻心絲蟲感染之情形,以隨機獲得自2000年8月~ 2001年12月間台中市動物防疫所已進行安寧處理之流浪犬82隻(公犬47隻,母犬35隻)進行疫情調查。在建立犬隻基本資料後,隨即進行採血及心、肺臟剖檢。其中44隻剖檢其心臟及肺動脈有心絲蟲成蟲存在,感染率為53.6 %(44/82)。在所有感染犬中公犬所佔比例為63.6 %(28/44)而母犬比例為36.3 %(16/44)。感染犬隻中,微絲蟲之檢出率為53.6 %(28/44)。心絲蟲方面,公成蟲平均長度為15.8 ± 2.1公分,平均重量為0.06 ± 0.01公克;母成蟲平均長度為27.1 ± 3.2公分,平均重量為0.15 ± 0.02公克(n = 100)。犬隻性別與感染率並無相關(r = 0.14;p>0.05);犬隻的毛色以及毛髮長短與感染率亦無相關(r = 0.07;p>0.05)。就品種與體重分布而言,感染族群以體重約5∼15公斤的中型混種犬隻為主。由上述調查資料顯示目前中部地區已成為犬心絲蟲症之高流行區域,相關公共衛生學上之潛在危險值得重視。
除了台灣本島,本研究進一步調查金門地區家犬血液寄生蟲之疫情以及疫苗之施打情況,以期做為金門地區未來防疫工作之參考。於2001年12月間,在金湖鎮及金城鎮隨機採樣調查96隻有植入晶片登記家犬。利用IDEXX犬心絲蟲/愛利希體/萊姆病三合一血清ELISA檢驗套組檢出心絲蟲症有4.2 %;愛利希體症為13.5 %。萊姆病抗體檢出率為零。另以Giemsa’s 染色血液抹片於高倍油鏡下檢出12.5 %犬感染愛利希體。合併ELISA及血抹片檢驗結果顯示愛利希體總感染率為15.6 %。抹片檢出感染焦蟲及肝簇蟲各有一病例。現行疫苗之施打以五合一佔1 %、七合一佔9 %及八合一佔10 %;80 %於一年內未曾施打。一年內曾施打狂犬病疫苗者佔87 %;結紮率為10 %。犬隻曾感染壁蝨高達有73 %。目前金門地區家犬心絲蟲仍屬低度感染狀態而愛利希體感染率則偏高,至於年度疫苗施打率則明顯偏低。由於這兩種血液寄生蟲為人畜共通傳染,再加上犬傳染性疾病流行之潛在危機,因此金門地區犬隻疾病之防治工作值得重視。
The aim of this study was to investigate the effect of bromelain on the lysis of canine adult heartworm body. Three concentrations (1 mg/mL, 5 mg/mL and 10 mg/mL) of bromelain were respectively prepared in two millipore-filtered (0.45 µm) mediums (plasma, and PBS). Adult heartworms were rinsed with sterilized distilled water, killed by incubation of 0.1 µg/ml RM340® (Melarsomine dichlorhydrate)for 24 hours then incubated in prepared mediums at 37°C for up to 2 months. Mediums were refreshed every 3 days till the completion of heartworm lysis. Male and female adult heartworms were separately tested according to their body weight. The adult worm body incubated in 5 mg/mL and 10 mg/mL bromelain mediums were complete dissolved. However, the worms in controls remained intact for two months onward. Bromelain dissolved male and female heartworms in a similar pattern (p>0.05). Bromelain posses more lysis potency in PBS mediums than in plasma. It might be due to plasma proteins reduce bromelain’s dissolve ability. Conclusively, Bromelain is able to dissolve the heartworm in vitro. It suggests that heartworm contains Z-Arg-Arg+NHMec protein structure.
The incidence of heartworm disease is gradually increasing in north Taiwan since 1993. This study was carried out to survey the prevalence of Dirofilaria immitis infection in stray dogs inhibited in Taichung area in August 2000 to December 2001. A total number of 82 necropsy (47 males and 35 females) was investigated. Nearly 54 % (44/82) was found with heartworm inhabited in the pulmonary artery and/or the right ventricle. The respective percent of the heartworm infection was 59.5 % (28/47) in the male dogs and 45.7 % (16/35) in the female. Only 53.6 % (28/44) of infected dogs was microscopically examined to be microfilaria positive by using direct smear technique. The length of male and female heartworms was 15.8±2.1 cm and 27.1±3.2 cm (n=100), respectively. The weight of male and female heartworms was 0.06±0.01 g and 0.15±0.02 g (n=100), respectively. Number of heartworm inhabitant was ranged from 1 to 44. The major infected age-group was those dogs within 4 to 6 years old. Besides, neither the correlation of gender and infection rate (r=0.14, p>0.05) nor the correlation of coat length/color and infection rate (r=0.07, p>0.05) was not detected. The major infected dog breed was the Mongrel. This is well possibly because Mongrel is the most common breed seen on streets. The major infested group in terms of body weight was those dogs with 5 to 15 kilograms.
To understand the present status of canine blood parasites infection among housed dogs in Kinmen area, a combined ELISA kit IDEXX (Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi) and microscopic examinations of Giemsa’s stained blood smears were conducted in randomly chosen 96 individuals (1,148 total population) in December 2001. The percentage of D. imitis, E. canis and B. burgdorferi seropositive dogs were detected to be 4.2%, 13.5% and 0% respectively and of the infected population, dual infection rate was 2.1%. Blood smear examinations revealed 12.5% of the dogs with Ehrlichia morula hosted in either monocyte or platelets. Taken together with results of ELISA kits, Ehrlichiasis was 15.6% of whole surveyed population. Infection of Hepatozoon spp. and Babesia spp. were merely observed in two single individual, respectively. Eighty percents of surveyed dogs did not receive any annual vaccine booster, whereas 87% had been immunized with the anti-Rabies vaccines within the past 12 months. Of the surveyed dogs, 10% were sterilized. Conclusively, low incidence of Dirofilariasis and relatively high prevalence of Ehrlichiasis suggest that prompt means of disease control should be undertaken to prevent from potential increasing prevalence.
圖次 --------------------------------------------------------Ⅲ
表次 --------------------------------------------------------Ⅴ
中文摘要 ----------------------------------------------------Ⅵ
英文摘要 ----------------------------------------------------Ⅷ
第一章、緒言 -------------------------------------------------1
第二章、文獻回顧 ---------------------------------------------3
第一節、犬心絲蟲之生物特性 -------------------------------3
一、心絲蟲成蟲、幼蟲、微絲蟲形態 ---------------------3
二、心絲蟲成蟲蟲體結構 -------------------------------4
三、心絲蟲生活史 -------------------------------------5
四、犬絲狀蟲D. immitis 及D. reconditum之鑑別診斷 -----7
第二節、犬心絲蟲之致病機制 -------------------------------11
一、對心肺循環系統之影響 -----------------------------11
二、對肝臟之影響 -------------------------------------14
三、對腎臟之影響 -------------------------------------14
第三節、犬心絲蟲症的臨床症狀、診斷及治療 -----------------15
一、臨床症狀 -----------------------------------------15
二、臨床診斷 -----------------------------------------17
三、犬心絲蟲症治療及預防 -----------------------------20
第四節、犬心絲蟲症之流行病學 -----------------------------23
一、國內疫情 -----------------------------------------24
二、國外疫情 -----------------------------------------25
三、公共衛生之重要性 ---------------------------------27
第五節、蟲體溶解酵素之選擇 -------------------------------28
一、酵素考慮因子 -------------------------------------28
二、鳳梨酵素(Bromelain)-----------------------------28
第六節、國內犬血液寄生蟲之流行病概況 ---------------------32
第三章、鳳梨酵素對心絲蟲成蟲溶解之體外試驗研究 ---------------34
第一節、緒言 ---------------------------------------------34
第二節、材料與方法 ---------------------------------------36
一、實驗犬隻來源 -------------------------------------36
二、血液學檢查 ---------------------------------------36
三、試劑溶液之製備 -----------------------------------36
四、蟲體溶解試驗 -------------------------------------37
五、蟲體組織切片檢查 ---------------------------------38
六、蟲體溶解之蛋白質電泳分析 -------------------------38
七、資料分析 -----------------------------------------38
第三節、結果 ---------------------------------------------39
第四節、討論 ---------------------------------------------43
第四章、台中地區流浪犬心絲蟲症之疫情調查 ---------------------49
第一節、緒言 ---------------------------------------------49
第二節、材料與方法 ---------------------------------------50
第三節、結果 ---------------------------------------------51
第四節、討論 ---------------------------------------------54
第五章、金門地區家犬血液寄生蟲疫情及免疫計畫現況調查 ---------57
第一節、緒言 ---------------------------------------------57
第二節、材料與方法 ---------------------------------------58
第三節、結果 ---------------------------------------------59
第四節、討論 ---------------------------------------------63
參考文獻 -----------------------------------------------------66
附錄 ---------------------------------------------------------78
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