應用分子診斷技術調查台灣中部地區家犬及流浪犬之人畜共通傳染病

Detection of Zoonotic Diseases in Domestic Dogs and Stray Dogs by Molecular Diagnostic Techniques in Central Taiwan

周俊宏(Chun-hung Chou)

指導教授 ： 廖明輝

國立屏東科技大學/農學院/獸醫學系所/碩士(2012年)

https://doi.org/10.6346/NPUST.2012.00254

摘要

近年來飼養寵物的風氣盛行，尤其以寵物犬隻最受大眾青睞，台灣犬隻常見的人畜共通傳染病包括弓蟲病、心絲蟲病、萊姆病、鉤端螺旋體病、Q熱等五大疾病。本研究自2009年8月至2011年7月止以逢機採樣方式，每個月從中部地區（台中、彰化、雲林等地區）採集家犬及流浪犬隻血液檢體各30隻，共計各720個樣本，應用聚合酶鏈鎖反應（polymerase chain reaction, PCR）偵測犬隻上述五種人畜共通傳染病的病原核酸之盛行率。其結果皆可檢測病原核酸至1 pg/μL 以下，顯示其皆有相當高的敏感性。在特異性試驗中，各自皆有其專一性，而對其他病原則無反應。其檢測陽性率在流浪犬分別為弓蟲3.89 %（28/720）；心絲蟲7.22 %（52/720）、伯氏疏螺旋體5.42 %（39/720）、鉤端螺旋體7.22 %（52/720）、貝氏考克斯菌5.83 %（42/720）；在家犬分別為弓蟲1.39 %（10/720）、心絲蟲7.08 %（51/720）、伯氏疏螺旋體3.33 %（24/720）、鉤端螺旋體6.25 %（45/720）、貝氏考克斯菌8.61 %（62/720），若以流浪犬及家犬合計計算則分別為弓蟲2.64 %（38/1440）、心絲蟲7.15 %（103/1440）、伯氏疏螺旋體4.38 %（63/1440）、鉤端螺旋體6.74 %（97/1440）、貝氏考克斯菌7.22 %（104/1440）。此五種病原之核酸定序與親源關係比對的部分，檢測弓蟲之陽性株與美國、德國、巴西、中國株相似度達95 ~ 100 %、心絲蟲之陽性株與台灣、中國株相似度達96 ~ 97 %，伊朗相似度93 %、伯氏疏螺旋體之陽性株與美國、德國、瑞典相似度達98~100 %、鉤端螺旋體之陽性株與中國、巴西、美國、日本株相似度達97~100 %、貝氏考克斯菌之陽性株與日本、美國株相似度達99 ~ 100 %。於統計分析相關性結果，在流浪犬部分：弓蟲、伯氏疏螺旋體、鉤端螺旋體在年齡、季節有其相關性，與品種、性別皆無相關性；心絲蟲與品種、季節有相關性，與性別、年齡則無相關性；貝氏考克斯菌在季節有其相關性，與品種、性別、年齡皆無相關性。在家犬部分:弓蟲、伯氏疏螺旋體、貝氏考克斯菌在季節有其相關性，與品種、性別、年齡皆無相關性；心絲蟲與品種、年齡、季節有相關性，與性別則無相關性；鉤端螺旋體與性別、季節有相關性；與品種、年齡無相關性。本研究結果顯示，以高敏感性及高特異性引子對以聚合酶鏈鎖反應進行人畜共通傳染病病原的檢測，可獲得準確性的結果，並對於疾病的防治提供更有用的資訊。

關鍵字

弓蟲 ； 心絲蟲 ； 伯氏疏螺旋體 ； 鉤端螺旋體 ； 貝氏考克斯菌

並列摘要

In recent years, due to the alteration of life styles, many pet owners pay more attention to their dog's health and medicine. Since domestic dogs and stray dogs live in the same environment, zoonoses had been became a serious public health problem. There are five common zoonoses in Taiwan, including toxoplasmosis,heartworm disease,lyme disease,leptospirosis and Q fever.In this study, random sampling of blood samples from domestic and stray dogs in Central Taiwan (including Taichung, Changhua and Yunlin). Blood 720 samples from 30 domestic and 30 stray dogs were collected every month from August 1, 2009 to July 31, 2011. Polymerase chain reaction (PCR)was utilized to diagnosis the five zoonotic diseases and analyze the prevalence. The sensitivity of PCR was downed to 1 pg/μL DNA. The PCR positive rate of those diseases was as followed: in stray dogs group, Toxoplasma gondii 3.89 % (28/720), Dirofilaria immitis 7.22 % (52/720), Borrelia burgdorferi 5.42 % (39/720), Leptospira spp. 7.22 % (52/720), and Coxiella burnetii 5.83 % (42/720), by which in domestic dogs group, Toxoplasma gondii 1.39 % (10/720), Dirofilaria immitis 7.08 % (51/720), Borrelia burgdorferi 3.33 % (24/720), Leptospira spp. 6.25 % (45/720), and Coxiella burnetii 8.61 % (62/720). The total positive rate was Toxoplasma gondii 2.64% (38/1440), Dirofilaria immitis 7.15 % (103/1440), Borrelia burgdorferi 4.38 % (63/1440), Leptospira spp. 6.74 % (97/1440), and Coxiella burnetii 7.22 % (104/1440). In the sequencing and phylogenetic analysis, the isolated Toxoplasma gondii and United States, Germany, Brazil and Chinese strain shared with the similarity of 95–100 %. The isolated strains and Taiwan and China strain contained 96–97 % sequence similarity, Iran contained 93 % sequence identity with Dirofilaria immitis. The isolated strains and United States, Germany and Sweden strain shared 98–100 % sequence identity with Borrelia burgdorferi. The isolated strains and China , Brazil, United States and Japan strain possessed of 97–100 % sequence identity with Leptospira spp. The isolated strains and Japan and United States strain contained 99-100 % sequence identity with Coxiella burnetii. By the statistical data analysis, we concluded: (a)for the stray dogs: Toxoplasma gondii, Borrelia burgdorferi, and Leptospira spp. are correlated with the age and seasons, but not correlated with the breed and gender ; Dirofilaria immitis species are correlated with the breed and seasons, but not correlated with the gender and age ; Coxiella burnetii is correlated with the seasons, but not correlated with the breed, gender and age ; (b)for the domestic dog: Toxoplasma gondii, Borrelia burgdorferi, Coxiella burnetii are correlated with the seasons, but not correlated with the species, gender and age ; Dirofilaria immitis is correlated with the breed, age and seasons, but not correlated with the gender ; Leptospira spp. is correlated with the gender and season, but not correlated with the age and species. The current results revealed the high sensitivity and high specificity of primer pairs by polymerase chain reaction in zoonoses diseases detection, thus, these results could be the useful information as the reference for epidemic disease prevention.

並列關鍵字

Toxoplasma gondii ； Dirofilaria immitis ； Borrelia burgdorferi ； Leptospira spp. ； Coxiella burnetii

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