Çukurova ve Şanlıurfa bölgelerinde deri lezyonlarından izole edilen leishmania sp. DNA’larının restriksiyon endonükleazlarla karşılaştırılması

Öz

Amaç: Leishmaniasis, dünyanın tropikal ve subtropikal bölgelerinde yaygın olarak görülmektedir. Leishmania türlerinin tiplendirilmesinde, günümüzde klasik mikroskobik teknikler yanında DNA’ya dayalı teknikler kullanılmaktadır. Bu çalışmada, Çukurova ve Şanlıurfa bölgeleri deri lezyonlarından izole edilen Leishmania suşları arasında genotipik farklılıkların olup olmadığını saptamak amaçlanmıştır. Gereç-Yöntem: Hastaların deri lezyonu kenarlarından iğne aspirasyonu ile alınan örnekler NNN besiyerine ekilerek promastigotlar elde edilmiştir. Büyük hacimde promastigot kültürü yapıldıktan sonra promastigotlardan DNA izole edilmiş ve DNA; Eco RI, Hind III, Hae III, Hinf I, Alu I ve Rsa I restriksiyon endonükleazları ile kesilmiş ve agaroz jelinde elde edilen DNA profilleri karşılaştırılmıştır. Bulgular: 18’i Çukurova, 7’si Şanlıurfa bölgelerinden elde edilen suşlardan ve 14 referans suştan izole edilen toplam 39 DNA örneği RFLP Restriction fragment lengt polymorphisms analizi ile karşılaştırılmış ve Çukurova bölgesinden izole edilen deri Leishmania örneklerinin, Şanlıurfa bölgesi Leishmania örneklerinden daha heterojen oldukları belirlenmiştir. Sonuç: Her iki bölgeden izole edilen cutaneous leishmaniasis etkenleri arasında genotipik farklılıklar tespit edilmiştir. Tiplendirme için, PCR Polymerase Chain Reaction tekniğinin uygulanması şarttır

Anahtar Kelimeler

• Jel elektroforezi,Restriksiyon endonükleaz,RFLP analizi,DNA,total DNA İzolasyonu

Comparision of leishmania sp. DNA’s isolated in the regions of Çukurova and Şanlıurfa with the restriction endonucleases

Öz

Objective: Leishmaniasis is widely seen in tropical and subtropical regions of the world. Today, DNA-based techniques are used in the identification of Leishmania sp. beside microscopic techniques. In this study, it has been aimed whether there are genotypic differences among Leishmania strains isolated from cutaneous lesions in Çukurova and Şanlıurfa regions. Materials-Methods: The samples taken by needle aspiration from the edges of skin lesion have been inoculated to change to promastigotes in NNN medium. After the promastigote culture was achieved for the large volume, DNAs from promastigotes have been isolated and they have been cut with the restriction endonucleases, such as Eco RI, Hind III, Hae III, Hinf I, Alu I, and Rsa I, and their profiles obtained on agarose gel have been compared. Results: The samples of 39 DNA isolated from 18 of Çukurova, 7 of Şanlıurfa regions, and 14 of reference strain have been compared with RFLP Restriction Fragment Length Polymorphism analysis and the cutaneous Leishmania samples isolated from Çukurova have been designated more heterogenicity than Leishmania samples taken from Şanlıurfa region. Conclusions: The genotypic differencies have been determined among the cutaneous Leishmaniasis agents isolated from either regions. PCR Polymerase Chain Reaction technique should be applied for the identification

Anahtar Kelimeler

• Gel electrophoresis,Restriction endonucleases,RFLP analyses,DNA,Total DNA isolation

Kaynakça

1. Memişoğlu HR, Kotoğyan A, Acar MA ve ark: Leishmaniasis. Tüzün Y, Kotoğyan A, Aydemir EH ve ark (eds): Dermatoloji, 2.baskı, Nobel Tıp Kitabevi, Istanbul, 1994, 221-231.
2. van Eys GJJM, Schoone GJ, Lighthart GS, et al: Identification of world leishmania by DNA recombinant probes. Molecular and Biochemical Parasitology, 1989; 34: 53-62.
3. Kreutzer RD, Grogl M, Neva FA, et al: Identification and genetic comparition of leishmanial parasites causing viscerotropic and cutaneous disease in soldiers returning from operation desert storm. Am J Trop Med Hyg, 1993; 49(3): 357-363.
4. Guessous N, Berrag B, Riyad M, et al: Short report: L.tropica etiologic agent of a case of canine visceral leishmaniasis in Northern Morocco. Am J Trop Med Hyg, 1997; 57(2): 172-173.
5. Hashiguchi Y, Gomez EA, De Coronel VV, et al: Andean leishmaniasis in Ecuador caused by infection with Leishmania mexicana and Leishmania major-like parasites. Am J Trop Med Hyg, 1991; 44(2): 205-217.
6. Momen H, Grimaldi JrG, Pacheco RS, et al: Brazilian leishmania stocks phenotypically similar to L.major. Am J Trop Med Hyg, 1985; 34(6): 1076-1084.
7. Beverley SM, Ismach RB, McMahon-Pratt D: Evoluation of genus leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns. Proc Natl Sci USA, 1987; 84: 484-488.
8. Guizani I, van Eys GJJM, Ben Ismail R, et al: Use of recombinant DNA probes for species identification of world leishmania isolates. Am J Trop Med Hyg, 1994; 50(5): 632-640.

9. Zvala-Castro J, Velasco-Castrejon O, and Hernandez R: Molecular characterization of mexican stocks of Trypanosoma cruzi using total DNA. Am J Trop Med Hyg, 1992; 47(2): 201- 209.
10. Berzunza-Cruz M, Bricaire G, Zuluoaga Romero SZ, Perez-Becker R, Saavedra-Lira E, Perez-Montfort R, Crippa-Rossi M, Velasco- Castrejon O, and Becker I. Leishmania mexicana mexicana: Genetic Heterogeneity of Mexican. Isolates Revealed by Restriction Length Polymorphism Analysis of Kinetoplast DNA. Experimental Parasitology, 2000; 95: 277–284.
11. Jackson PR, Wohlhieter JA, Jackson JE, et al: Restriction endonuclease analyses of leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease. Am J Trop Med Hyg, 1984; 33(5): 808-819.
12. Lu HG, Zhong L, Guan L, et al: Separation of Chinese leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity. Am J Trop Med Hyg, 1994; 50(6): 763-770.
13. Reiner NE, Lo R, Llanos-Cuentas A, et al: Genetic heterogeneity in Peruvian leishmania isolates. Am. J Trop Med Hyg, 1989; 41(4): 416- 421.
14. Dilmeç F, Matur A, Uzun S, Karakaş M. Leishmania promastigotlarının üretilmesinde kullanılan besiyerlerinin standardizasyonu ve üreme eğrisinin tespiti. Çukurova Üniversitesi Tıp Fakültesi Dergisi, 1999; 24(4): 165-171.
15. Dilmeç F, Matur A, Alhan E ve ark: Çukurova bölgesi visseral Leyişmaniya etkenlerinde total DNA restriksiyon endonükleaz profilleri. ÇÜ Tıp Fakültesi Dergisi, 1999; 24 (4): 158-164.
16. Wincker P, Ravel C, Blaineau C, et al: The leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species. Nucleic Acids Research, 1996; 24(9): 1688-1694.
17. Spithill TW, and Samaras N: Genomic organization, chromosomal location and transcription of dispersed and repeated tubulin genes in Leishmania major. Molecular and Biochemical Parasitology, 1987; 24: 23-37.
18. Pacheco RS, Martinez JE, Valderrama L, et al: Genotypic polymorphisms in experimental metastatic dermal leishmaniasis. Molecular and Biochemical Parasitology, 1995; 69: 197-209.
19. Strelkova MV, Pacheco RS, Bulat SA, et al: Comparison of some molecular-genetic techniques for identification of leishmania circulating in the central Asia region. Mem Inst Oswaldo Cruz, Rio de Janeiro, 1997; 92(1): 104- 114.
20. Dobner P, Löscher T, Rinder H: Intra and interspesific polymorphisms of Leishmania donovani and Leishmania tropica minicircle DNA. Parasitol Res, 1994; 8: 474-477.
21. Özbel Y, Turgay N, Özensoy S ve ark: Klinik materyallerde leishmania parazitlerinin PCR (Polymerase Chain Reaction) ve Southern Blot hibridizasyon teknikleri yardımıyla saptanması. Türkiye Parazitoloji Dergisi, 1997; 21(4): 350- 356.
22. Noyes HA, Belli AA, and Maingon R: Appraisal of various amplified polymorphic DNA, polymerase chain reaction for leishmania identification. Am J Trop Med Hyg, 1996; 55(1): 98-105.
23. Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, and Felger I. Identification and Differentiation of Leishmania Species in Clinical Samples by PCR Amplification of the Miniexon Sequence and Subsequent Restriction Fragment Length Polymorphism Analysis. Journal of clinical microbiology, 2003; 41(7): 3147–3153.
24. Schonian G, Nasereddin A, Dinse N, Schweynocha C, Schallig HDFH, Presbera W, Jaffeb CL. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagnostic Microbiology and Infectious Disease, 2003; 47: 349–358.
25. Gomez-Eichelmann MC, Holz JrG, Beach D, et al: Comparison of several lizard leishmania species
26. and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids. Molecular and Biochemical Parasitology, 1988; 27: 143- 158.
27. Miller SA, Dykes DD, and Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 1988; 16(3):1215.