應用Lig重組蛋白質以酵素結合免疫吸附法進行豬鉤端螺旋體症之血清流行病學調查

Use of recombinant Lig antigen-based enzyme-linked immunosorbent assay for the serological survey of porcine leptospirosis

黃日芳(Jih-fang Huang)

指導教授 ： 林春福 ； 潘銘正

中臺科技大學/健康科學院/生命科學研究所/碩士(2011年)

摘要

鉤端螺旋體症為鉤端螺旋體菌所引起之重要人畜共通傳染疾病，常見於熱帶及亞熱帶地區。可感染各種哺乳類動物，其中鼠類為其主要貯菌宿主。本菌多樣性的血清型種類以及引起宿主程度不一的症狀，均須經實驗室檢測才能加以確認診斷。目前只要以顯微凝集試驗(microscopic agglutination test, MAT)做為宿主鉤端螺旋體症之抗體檢測標準方法，此方法乃以各種不同血清型之活菌做為抗原，因此如用於大量的流行病學調查較為耗費時間與人力，且操作過程需小心謹慎以防感染。與其相比，酵素結合免疫吸附法有著操作較便利且適合大量樣本操作的優勢。Lig蛋白質為近年發表具有做為檢測抗原及疫苗候選者之潛力，只有具致病性的鉤端螺旋體才能表現Lig。因此本研究利用大腸桿菌表現Lig重組蛋白質作為偵測抗原，應用酵素結合免疫吸附法(enzyme-linked immunosorbent assay, ELISA)，評估Lig重組蛋白質在偵測鉤端螺旋體症與現行的顯微凝集試驗結果之一致性。本研究於2008年1月至2009年10月間採集北部與中部兩家屠宰場之屠宰豬隻之血清檢體共1,872份。以顯微凝集試驗利用21株鉤端螺旋體菌檢測，抗體力價大於100倍為陽性標準，總陽性率為51.8% (969/1,872)。力價分布由100倍至51,200倍，主要感染血清群以Australis、 Shermani、Icterohaemorrhagiae為主。以Lig重組蛋白質保守性之區域(LigBCon)做為抗原，以酵素結合免疫吸附法檢測IgG抗體，其敏感性與特異性分別為67.2%以及78.7%，評估其與顯微凝集試驗比較之結果，Kappa值為0.406介於0.4-0.7之間，兩法結果一致性中等。但是當陽性檢體MAT力價低於800倍時，敏感性明顯下低，一致性降低，歸咎於流行病學之抗體盛行率係以IgG為主要抗體，且大部份豬隻皆以低力價感染為主，因此LigBCon-IgG ELISA並不適合取代MAT，用於豬鉤端螺旋體症流行病學調查。

關鍵字

顯微凝集試驗 ； Lig重組蛋白質 ； 酵素結合免疫吸附法 ； 鉤端螺旋體症

並列摘要

Leptospirosis is an important zoonotic disease worldwide, which is common in tropical and subtropical zones. A variety of serotypes can infect almost all mammas including rodents, they are considered as one of the most important reservoir host. The diagnosis is generally difficult due to diverse clinical manifestation, and often relies on laboratory workup, especially the antibody detection for confirmation. Microscopic agglutination test (MAT) is the gold standard of antibody detection, which requires many serotypes of live bacteria as antigen, it is both time consuming and laborious, not mention there is infection hazard to laboratory personnel. Compare to MAT, enzyme-linked immunosorbent assay (ELISA) is more convenient to operate with bulk samples for epidemiological study. Leptospiral Lig proteins have been identified as a antibody detection antigen and vaccine candidate, and only present in pathogenic species. In this study, we take MAT as standard to evaluate recombinant Lig protein as an immunoglobulin G (IgG) ELISA antigen when used in serological study for swine leptospirosis. A total of 1,872 pig sera were collected from two abattoirs in northern and central Taiwan from January 2008 to October 2009, and examined with a panel of 21 reference leptospira serovars by a MAT. It showed that 51.8% (969/1,872) of sera specimens had a titer of 1:100 or greater to at least one of the serovars employed. The MAT titer range from 1:100 to 1: 51,200. The main reacting serogroups were Australis, Shermani, and Icterohaemorrhagiae. When using recombinant Lig protein conserved region (LigBCon) as ELISA antigen, the sensitivity and specificity were 67.2% and 78.8%, respectively. To evaluate the agreement between MAT and ELISA, the Kappa coefficient were 0.406 (within 0.4-0.7), indicating a fair level of agreement. The sensitivity and Kappa value were further lower when using sera with MAT titer less than 1:800 for calculation. As most of the pigs were infected with low titer; therefore, we conclude that the recombinant Lig protein-based IgG ELISA is not an appropriate MAT alternative for epidemiological survey for swine Leptospirosis.

並列關鍵字

Leptospirosis ； Lig protein ； microscopic agglutination test (MAT) ； enzyme-linked immunosorbent assay (ELISA)

參考文獻

行政院衛生署疾病管制局 2010。最新消息；颱風過後清理家園，需做好手部、腳部防護，避免鉤端螺旋體病感染。http://www.cdc.gov.tw/content.asp?cuitem=31114&mp=1.

行政院衛生署疾病管制局、中華民國比較病理學會、臺灣感染症醫學會、臺灣醫院感染管制學會。2009。人畜共通傳染疾病臨床指引 (臺北市，行政院衛生署疾病管制局)。

吳尚翰。2006。台中地區小型哺乳類動物鉤端螺旋體感染調查暨應用恆溫式圈環型核酸增幅法進行鉤端螺旋體lipL32基因偵測。國立中興大學獸醫公共衛生學研究所碩士論文。

林卉宜。2003。利用乳膠凝集試驗快速篩檢及應用LipL32重組蛋白質以 酵素結合免疫吸附法進行鉤端螺旋體感染症之血清學診斷。國立臺灣大學醫學研究所碩士論文。

Adler, B., and A. de la Pena Moctezuma. 2010. Leptospira and leptospirosis. Vet Microbiol 140:287-96.