제주도내 고양이 혈액에서 톡소포자충의 감염률 및 유전자형 조사

Abstract

톡소포자충(Toxoplasma gondii)는 세포내에 기생하는 원충으로 사람을 포함한 대부분의 포유동물이 중간숙주로 작용하며 고양이과 동물은 유일한 종숙주로 톡소포자충증의 전파에 중요한 역할을 한다. 본 연구에서는 제주도내 고양이 혈액에서의 톡소포자충 감염률과 유전자형을 조사하고 수의임상에서 톡소포자충증 진단에 사용하고 있는 상업용 IgG ELISA kit와 ImmunoComb?? kit의 시행 결과를 비교하였으며, 실시간 PCR법을 확립하여 톡소포자충증의 진단을 보다 신속히 하였다. 실험에 사용된 시료는 제주도내 야생고양이 66마리와 집고양이 11마리의 혈액에서 추출한 gDNA와 혈청이며 gDNA를 이용하여 톡소포자충 SAG2유전자의 PCR과 PCR-RFLP를 시행하였고 혈청을 이용하여 ELISA와 ImmunoComb??kit을 실시하였다. 실험 결과, 총 77개의 시료 중 14개의 시료(20%)가 톡소포자충 양성이었으며 그 중 길고양이에서 11개체(17%), 집고양이에서 3개체(27%)가 나타났다. SAG2유전자 PCR-RFLP 결과 모든 양성 시료의 유전자형이 가장 침해도가 높은 제 1형으로 나타났다. 항체가 검출 결과, 70개의 혈청시료를 대상으로 사람의 2차 항체를 사용한 상업용 ELISA kit에서 모든 시료가 음성으로 나타났다. 반면, 고양이의 2차 항체를 사용한 ImmunoComb??kit의 경우 PCR 결과 양성시료 14개 중 12개, PCR 음성 시료 10개 중 5개에서 양성을 나타냄으로써 고양이의 톡소포자충증 진단에는 고양이의 2차 항체를 사용한 ImmunoComb??kit를 사용하는 것이 적합할 것이다. 또한 초고속 실시간 PCR의 반복 실험을 통한 결과, 일반 PCR에 비해 적은 시간에 동일한 결과를 얻어낼 수 있었으며 실제 임상에서 톡소포자충증의 진단에 유용하게 사용될 수 있을 것이라 사료된다.

Toxoplasma gondii is a widespread protozoan parasite that may cause serious diseases in various mammalian hosts including humans and small ruminants. It has been well-known that cats play an important role in the spread of toxoplasmosis, and they act as the definitive host of this parasite. This study is designed to investigate prevalence and genotypes of Toxoplasma gondii, which is detected in blood of cats of Jeju island. In this process, both antibody-detecting and antigen-detecting methods, which are widely used for diagnosis of Toxoplasmosis, were evaluated, and their detection rates were compared. The antibody-detection methods include a commercial kit involving Toxoplasma gondii IgG ELISA and ImmunoComb?? kit detecting feline specific IgG, while PCR was used as the antigen detection method in this study. Furthermore, ultra rapid real-time PCR method has been adopted to diagnose T. gondii more promptly. Blood samples were collected from 66 stray cats and 11 house-kept cats. Genomic DNA was extracted for detection of T. gondii by PCR method. Meanwhile, serum samples were also collected for ELISA kit and ImmunoComb?? kit. Based on PCR results, the positive samples were used for demonstration of PCR-RFLP patterns of SAG2 gene. On the other hand, ultra rapid real-time PCR method was used to provide a prompt diagnosis of toxoplasmosis. In total, 14 of the 77 blood samples (20%) were positive in which 11(17%) were from stray cats and 3(27%) were from house-kept cats in PCR method, respectively. Meanwhile, 70 serum samples showed negative results in ELISA kit. On the other hand, ImmunoComb??tests were performed on both 14 positive and 10 negative samples from PCR methods, and 12 out of 14 positive and 5 out of 10 negative samples in PCR were demonstrate das positive. PCR-RFLP revealed that all samples belong to Type I clonal lineage. In addition, the results obtained using ultra rapid real-time PCR method well correlated with those from conventional PCR method, and it is also much less time-consuming. In this study, it was possible to know the prevalence of T. gondii in the cat population of Jeju island. Meanwhile, all positive samples were belong to Type I clonal lineage which is the most virulent type. As toxoplasmosis is one of the major zoonotic diseases and it poses a threat on public health, the prevalence rate needs to be continuously monitored. In addition, it can be suggested that the mmunoComb?? kit is superior to the commercial ELISA kit in diagnosis of feline toxoplasmosis, as the former uses the feline secondary antibody in contrast to the latter using the human secondary antibody.