생명과학회지 (Journal of Life Science)

  • 제19권9호
  • /
  • Pages.1328-1332
  • /
  • 2009
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

고래회충유충증 감별 진단을 위한 18S ribosomal DNA (rDNA) PCR-RFLP 법 적용

Application of the 18S Ribosomal DNA (rDNA) PCR-RFLP Technique for the Differential Diagnosis of Anisakidosis

  • 김선미 (부산대학교 의학대학원 약리학교실) ;
  • 조민경 (부산대학교 의학대학원 기생충학교실) ;
  • 유학선 (부산대학교 의학대학원 기생충학교실) ;
  • 차희재 (고신대학교 의과대학 기생충학 유전학교실) ;
  • 옥미선 (고신대학교 의과대학 기생충학 유전학교실)
  • Kim, Sun-Mee (Department of pharmacology, Pusan National University College of Medicine) ;
  • Cho, Min-Kyung (Department of Parasitology, Pusan National University College of Medicine) ;
  • Yu, Hak-Sun (Department of Parasitology, Pusan National University College of Medicine) ;
  • Cha, Hee-Jae (Department of Parasitology and Genetics, Kosin University College of Medicine) ;
  • Ock, Mee-Sun (Department of Parasitology and Genetics, Kosin University College of Medicine)
  • 발행 : 2009.09.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

고래회충유충증은 해산어류에 기생하는 고래회충과(family Anisakidae)에 속하는 선충류 유충에 의한 질병으로 유충의 직접적인 위장관내 침입으로 인한 병변과 더불어 유충의 분비 배설물에 의한 알레르기 질환도 유발될 수 있다. 고래회충유충증은 A. simplex를 비롯하여 Contracaecum, Pseudoterranova, Hysterothylacium 등의 유충에 의해 야기될 수 있으나 이들에 대한 형태학적 감별 진단은 유충의 형태적 유사성으로 인하여 매우 어려운 경우가 많다. 이러한 형태학적 진단의 어려움을 극복하고 분자생물학적 감별진단 방법을 확립하기 위하여 A. simplex, Contracaecum type A. type C' 및 Goezia 유충을 숭어, 도다리, 고등어, 아나고, 참돔 등 5종의 어류에서 분리하였다. 각각의 유충으로부터 분리한 18S rDNA를 PCR로 증폭한 후 Taq 1, Hinf I, Hha I, Alu 1, Dde I, Hae III, Sau 96I, Sau 3AI 등 8종의 제한효소를 사용하여 PCR-RFLP를 시행하였다. PCR product의 크기는 약 2.0 Kb였으며 Hinf l, Alu 1, Hha I, Dde 1 및 Hae III로 A. simplex와 Contracaecum type C'을 구분할 수 있었다. 그러나 Contracaecum type A의 경우에는 Taq I, Hinf I, Alu I 및 Dde I의 경우에는 2가지 패턴으로 나타났으며 이들 가운데 일부는 A. simplex, Contracaecum type C', 및 Goezia와 동일한 분석 패턴을 보이기도 하였다. Goezia는 사용한 8개의 제한 효소 모두에서 A. simplex 및 Contracaecum type A 및 type C'과 각기 다른 양상을 보였다. 이러한 결과로 18S rDNA PCR-RFLP 방법은 A. simplex와 Contracaecum type C'의 감별 진단에 유용한 것으로 밝혀졌으며, Contracaecum type A의 분류에는 제한적으로 사용되어야 함은 물론 형태학적 분류 기준에 대한 재검토가 뒤따라야 할 것으로 사료되었다.

Anisakidosis is caused by anisakid nematodes (family Anisakidae) larvae which can cause not only direct tissue damage but also a severe allergic response related to excretory-secretion products. Lots of different species of anisakid larvae, including Anisakis simplex, Contracaecum, Goezia, Pseudoterranova, and Hysterothylacium, cause the anisakidosis. But it is difficult to diagnosis the species of larvae since the morphologies of larval anisakid nematodes are almost indistinguishable. In order to diagnosis the differential infections of larval anisakid nematodes, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 18S rDNA - was conducted. Three major species of anisakid larvae including A. simplex, C.ontracaecum spp, and Goezia spp. were collected from mackerel (Scomber japonicus), mullet (Mugil cephalus), founder (Paralichthys olivaceus), eel (Astroconger myriaster) and red sea bream (Pagrus major). PCR amplified 18S rDNA from each species of anisakid larvae was digested with eight restriction enzymes including Taq I, Hinf I, Hha I, Alu I, Dde I, Hae III, Sau96 I, and Sau3A I. The original sizes of PCR amplified 18S rDNA were 2.0Kb in both anisakid larvaes and Goezia. Restrction enzymes including Hinf 1, Alu 1, Hha I, Dde 1 and Hae III cut differently and distinguished the A. simplex and Contracaecum type C'. However, Contracaecum type A showed two different restriction enzyme cutting patterns by Taq 1, Hinf I, Alu 1, and Dde 1. One of the patterns was the same as those of A. simplex, Contracaecum type C' and Goezia and the other was unique. These results suggest that PCR-RFLP pattern by Hinf 1, Alu 1, Hae I, Dde 1 and Hae III can be applied to differential diagnosis of human infection with A. simplex and Contracaecum type C'. Contracaecum type A needs further study of classification by morphological characteristics and genetic analysis.

키워드

참고문헌

  1. Audicana, M. T., L. Fernandez de Corres, D. Munoz, E. Fernandez, J. A. Navarro, and M. D. del Pozo. 1995. Recurrent anaphylaxis due to Anisakis simplex parasitizing sea- fish. J. Allergy Clin. Immunol. 96, 558-560 https://doi.org/10.1016/S0091-6749(95)70301-2
  2. Chai, J. Y., Y. M. Chu, W. M. Shon, and S. H. Lee. 1986. Larval Anisakids collected from the yellow corvina in Korea. Korean J. Parasitol. 24, 1-11
  3. D' Amelio, S, K. D. Mathiopoulos, C. P. Santos, O. N. Pugachev, S. C. Webb, M. Picanco, and L. Paggi. 2000. Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase chain reaction-based restriction fragment length polymorphism. Int. J. Parasitol. 30, 223-226 https://doi.org/10.1016/S0020-7519(99)00178-2
  4. D' Amelio, S, K. D. Mathiopoulos, O. Brandonisio, G. Lucarelli, D. Foronzo, and L. Paggi. 1999. Diagnosis of a case of gastric anisakidosis by PCR-based restriction fragment length polymorphism analysis. Parassitologia. 41, 591-593
  5. Del Pozo, M. D., M. Audicana, J. M. Diez, D. Munoz, I. J. Ansotegui, E. Fernandez, M. Garcia, M. Etxenagusia, I. Moneo, and L. Fernandez de Corres. 1997. Anisakis simplex, a relevant etiologic factor in acute urticaria. Allergy 52, 576-579 https://doi.org/10.1111/j.1398-9995.1997.tb02603.x
  6. Dobson, S. J. and S. C Baker. 1999. Phylogeny of the hard ticks (Ixodidae) inferred from 18S rRNA indicates that the Genus Aponomma is Paraphyletic. Mol. Phylogenet. Evol. 11, 288-295 https://doi.org/10.1006/mpev.1998.0565
  7. Dorris, M, P. De Ley, and M. L. Blaxter. 1999. Molecular analysis of nematode diversity and the evolution of parasitism. Parasitol. today. 15, 188-193 https://doi.org/10.1016/S0169-4758(99)01439-8
  8. Fernandez De Corres, L, M. Audicana, M. D. Del Pozo, D. Munoz, E. Fernandez, J. A Navarro, M. Garcia, and J. Diez. 1996. Anisakis simplex induces not only anisakiasis: report on 28 cases of allergy caused by this nematode. J. Invest. Allergol. Clin. Inmunol. 6, 315-319
  9. Hasegawa, H, S. Hayashida, Y. Ikeda, and H Sato. 2009. Hyper-variable regions in 18S rDNA of Strongyloides spp. as markers for species-specific diagnosis. Parasitol. Res. 104, 869-874 https://doi.org/10.1007/s00436-008-1269-9
  10. Hwang, U. W. and W. Kim. 1999. General properties and phylogenetic utilities of nuclear ribosomal DNA and mitochondrial DNA commonly used in molecular systematics. Korean J. Parasitol. 37, 215-228 https://doi.org/10.3347/kjp.1999.37.4.215
  11. Kennedy, M. W., J. Tierney, P. Ye., F. A McMonagle, A McIntosh, D. McLaughlin, and J. W. Smith. 1988. The secreted and somatic antigens of the third stage larva of Anisakis simplex, and antigenic relationship with Ascaris suum, Ascaris lumbricoides, and Toxocara canis. Mol. Biochem. Parasitol. 31, 35-46 https://doi.org/10.1016/0166-6851(88)90143-0
  12. Kieht E, V. Walldorf, S. Klimpet S. AI-Quraishy, and H Mehlhorn. 2009. The European vectors of Bluetongue virus: are there species complexes, single species or races in Culicoides obsoletus and C. pulicaris detectable by sequencing ITS-I, ITS-2 and 18S-rDNA? Parasitol. Res. 105, 331-336 https://doi.org/10.1007/s00436-009-1414-0
  13. Kijewska, A, M. Stominska, G. Wegrzyn, and J. Rokicki. 2000. A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions. Mol. Cell Probes 14, 349-354 https://doi.org/10.1006/mcpr.2000.0322
  14. Li, A X, S. D' Amelio, L. Paggi, F. He, R. B. Gasser, Z. R. Lun, E. Abollo, M. Turchetto, and X. Q. Zhu. 2005. Genetic evidence for the existence of sibling species within Contracaecum rudolphi (Hartwich, 1964) and the validity of Contracaecum septentrionale (Kreis, 1955) (Nematoda: Anisakidae). Parasitol. Res. 96, 361-366 https://doi.org/10.1007/s00436-005-1366-y
  15. Lozano, M. J., H. L. Martin, S. V. Diaz, A I. Manas, L. A Valero, and B. M. Campos. 2004. Cross-reactivity between antigens of Anisakis simplex s1 and other ascarid nematodes. Parasite. 11, 219-223 https://doi.org/10.1051/parasite/2004112219
  16. Mattiucci, S., M. Paoletti, S. C. Webb, N. Sardella, J. T. Timi, B. Berland, and G. Nascetti. 2008. Genetic relationships among species of Contracaecum Railliet & Henry, 1912 and Phocascaris Host, 1932 (Nematoda: Anisakidae) from pinnipeds inferred from mitochondrial cox2 sequences, and congruence with allozyme data. Parasite. 15, 408-419 https://doi.org/10.1051/parasite/2008153408
  17. Nadler, S. A, S. D' Amelio, M. D. Dailey, L. Paggi, S. Siu, and J. A Sakanari. 2007. Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals. J. Parasitol. 91, 1413-1429 https://doi.org/10.1016/j.parint.2007.08.003
  18. Paggi, L., S. Mattiucci, and S. D' Amelio. 2001. Allozyme and PCR-RFLP markers in anisakid nematodes, aethiological agents of human anisakidosis. Parassitologia. 43(Suppl 1), 21-27
  19. Perteguer, M. J., C. Cuellar, J. L. Guillen, C. Aguila, S. Fenoy, T. Chivato, and R. Laguna. 2003. Cross-reactivity between Anisakis simplex sensitization and visceral larva migrans by Toxocara canis. Acta. Trap. 89, 85-89 https://doi.org/10.1016/S0001-706X(03)00201-8
  20. Sakanari, J. A, and J. H McKerrow. 1989. Anisakiasis. Clin. Microbiol. Rev. 2, 278-284
  21. Shamsi, S, R. Norman, R. Gasser, and I. Beveridge. 2009. Redescription and genetic characterization of selected Contracaecum spp. (Nematoda: Anisakidae) from various hosts in Australia. Parasitol. Res. 104, 1507-1525 https://doi.org/10.1007/s00436-009-1357-5
  22. Shamsi, S., R. Norman, R. Gasser, and I. Beveridge. 2009. Genetic and morphological evidences for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) (Nematoda: Anisakidae) in Australia. Parasitol. Res. 105, 529-538 https://doi.org/10.1007/s00436-009-1424-y
  23. Sugimachi, K K. Inokuchi, T. Ooiwa, T. Fujino, and Y. Ishii. 1985. Acute gastric anisakiasis. Analysis of 178 cases. JAMA 253, 1012-1013 https://doi.org/10.1001/jama.253.7.1012
  24. Szostakowska, B., P. Myjak, and J. Kur. 2002. dentification of anisakid nematodes from the Southern Baltic Sea using PCR-based methods. Mol. Cell Probes. 16, 111-118 https://doi.org/10.1006/mcpr.2001.0391
  25. Zhu, X., R. B. Gasser, M. Podolska, and N. B. Chilton. 1998. Characterization of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences. Int. J. Parasitol. 28, 1911-1921 https://doi.org/10.1016/S0020-7519(98)00150-7