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Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis

  • Nemati, Sara (Department of Medical Parasitology and Mycology, School of Medicine, Zanjan University of Medical Sciences) ;
  • Fazaeli, Asghar (Department of Medical Parasitology and Mycology, School of Medicine, Zanjan University of Medical Sciences) ;
  • Hajjaran, Homa (Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences) ;
  • Khamesipour, Ali (Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences) ;
  • Anbaran, Mohsen Falahati (School of Biology, University of Tehran) ;
  • Bozorgomid, Arezoo (Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences) ;
  • Zarei, Fatah (Department of Animal Sciences, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University)
  • Received : 2017.03.19
  • Accepted : 2017.05.23
  • Published : 2017.08.31

Abstract

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Keywords

References

  1. Yaghoobi-Ershadi MR, Hanafi-Bojd AA, Javadian E, Jafari R, Zahraei-Ramazani AR, Mohebali M. A new focus of cutaneous leishmaniasis caused by Leishmania tropica. Saudi Med J 2002; 23: 291-294.
  2. Edrisian GhH, Nadim A, Alborzi AV, Aredehhali S. Visceral leishmaniasis: the Iranian experience. Arch Iran Med 1998; 1: 22-26.
  3. Mohebali M, Edrissian GhH, Nadim A, Hajjaran H, Akhoundi B, Hooshmand B, Zarei Z, Arshi Sh, Mirsamadi N, Naeini KM, Mamishi S, Sanati AA, Moshfe AA, Charehadar S, Fakhar M. Application of direct agglutination test (DAT) for the diagnosis and seroepide-miological studies of visceral leishmaniasis in Iran. Iran J Parasitol 2006; 1: 15-25.
  4. Nadim A, Navid-Hamidi A, Javadian E, Tahvildari Bidruni GH, Amini H. Present status of kala-azar in Iran. Am J Trop Med Hyg 1978; 27: 25-28. https://doi.org/10.4269/ajtmh.1978.27.25
  5. Schonian G, Cupolillo E, Mauricio I. Molecular evolution and phylogeny of Leishmania. In Ponte-Sucre A, Diaz E, Padron-Nieves M eds, Drug Resistance in Leishmania Parasites. London, UK. Springer. 2013, pp. 15-44.
  6. Schonian G, Mauricio I, Cupolillo E. Is it time to revise the nomenclature of Leishmania? Trends Parasitol 2010; 26: 466-469. https://doi.org/10.1016/j.pt.2010.06.013
  7. Cupolillo E, Grimaldi G, Momen H, Beverley SM. Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol 1995; 73: 145-155. https://doi.org/10.1016/0166-6851(95)00108-D
  8. Cupolillo E, Grimaldi Jr G, Momen H. A general classification of new world Leishmania using numerical zymotaxonomy. Am J Trop Med Hyg 1994; 50: 296-311. https://doi.org/10.4269/ajtmh.1994.50.296
  9. Montalvo AM, Fraga J, Maes I, Dujardin JC, Van der Auwera G. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol Infect Dis 2012; 31: 1453-1461. https://doi.org/10.1007/s10096-011-1463-z
  10. Degrave W, Fernandes O, Campbell D, Bozza M, Lopes U. Use of molecular probes and PCR for detection and typing of Leishmania: a mini-review. Mem Inst Oswaldo Cruz 1994; 89: 463-469. https://doi.org/10.1590/S0074-02761994000300032
  11. Schonian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HDFH, Presber W, Jaffe CL. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 2003; 47: 349-358. https://doi.org/10.1016/S0732-8893(03)00093-2
  12. Wilson SM. DNA-based methods in the detection of Leishmania parasites: field applications and practicalities. Ann Trop Med Parasitol 1995; 89: 95-100.
  13. Croan DG, Morrison DA, Ellis JT. Evolution of the genus Leishmania revealed by comparison of DNA and RNA polymerase gene sequences. Mol Biochem Parasitol 1997; 89: 149-159. https://doi.org/10.1016/S0166-6851(97)00111-4
  14. Zelazny AM, Fedorko DP, Li L, Neva FA, Fischer SH. Evaluation of 7SL RNA gene sequences for the identification of Leishmania spp. Am J Trop Med Hyg 2005; 72: 415-420.
  15. Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Sosa Cabrera T, Perez-Montfort R, Becker I. Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana. Parasitol Res 2002; 88: 918-925. https://doi.org/10.1007/s00436-002-0672-x
  16. Davila AM, Momen H. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol 2000; 94: 651-654. https://doi.org/10.1080/00034983.2000.11813588
  17. Spanakos G, Piperaki ET, Menounos PG, Tegos N, Flemetakis A, Vakalis NC. Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method. Trans R Soc Trop Med Hyg 2008; 102: 46-53. https://doi.org/10.1016/j.trstmh.2007.05.019
  18. Waki K, Dutta S, Ray D, Kolli BK, Akman L, Kawazu SI, Lin CP, Chang KP. Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans-endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase. Eukaryot Cell 2007; 6: 198-210. https://doi.org/10.1128/EC.00282-06
  19. Asato Y, Oshiro M, Myint CK, Yamamoto Y, Kato H, Marco JD, Mimori T, Gomez EA, Hashiguchi Y, Uezato H. Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing. Exp Parasitol 2009; 121: 352-361. https://doi.org/10.1016/j.exppara.2008.12.013
  20. Fraga J, Montalvo AM, De Doncker S, Dujardin JC, Van der Auwera G. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect Genet Evol 2010; 10: 238-245. https://doi.org/10.1016/j.meegid.2009.11.007
  21. Garcia L, Kindt A, Bermudez H, Llanos-Cuentas A, De Doncker S, Arevalo J, Wilber Quispe Tintaya K, Dujardin JC. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol 2004; 42: 2294-2297. https://doi.org/10.1128/JCM.42.5.2294-2297.2004
  22. Fotouhi-Ardakani R, Dabiri S, Ajdari S, Alimohammadian MH, AlaeeNovin E, Taleshi N, Parvizi P. Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites. Infect Genet Evol 2016; 46: 33-41. https://doi.org/10.1016/j.meegid.2016.10.011
  23. Hajjaran H, Mohebali M, Mamishi S, Vasigheh F, Oshaghi MA, Naddaf SR, Teimouri A, Edrissian GH, Zarei Z. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed Res Int 2013; 789326.
  24. Mirahmadi H, Salimi Khorashad A, Sohrabnahad A, Heydarian P, Bizhani N. Species identification and molecular typing of Leishmania spp. using targeting HSP70 gene in suspected patients of cutaneous leishmaniasis from Sistan and Baluchestan Province, Southeast Iran. Iran J Parasitol 2016; 11: 489-498.
  25. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 1999; 41: 95-98.
  26. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30: 2725-2729. https://doi.org/10.1093/molbev/mst197
  27. Hurvich CM, Tsai CL. Regression and time series model selection in small samples. Biometrika 1989; 76: 297-307. https://doi.org/10.1093/biomet/76.2.297
  28. Posada D. jModelTest: phylogenetic model averaging. Mol Biol Evol 2008; 25: 1253-1256. https://doi.org/10.1093/molbev/msn083
  29. Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 2010; 10: 564-567. https://doi.org/10.1111/j.1755-0998.2010.02847.x
  30. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 1989; 123: 585-595.
  31. Fu YX. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 1997; 147: 915-925.
  32. Cupolillo E, Medina-Acosta E, Noyes H, Momen H, Grimaldi G Jr. A revised classification for Leishmania and Endotrypanum. Parasitol Today 2000; 16: 142-143. https://doi.org/10.1016/S0169-4758(99)01609-9
  33. Lainson R. Evolution, Classification and Geographical Distribution. Cambridge, Massachusetts, USA. Academic Press. 1987.
  34. Rioux JA, Lanotte G, Serres E, Pratlong F, Bastien P, Perieres J. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann Parasitol Hum Comp 1990; 65: 111-125. https://doi.org/10.1051/parasite/1990653111
  35. Van der Auwera G, Fraga J, Montalvo AM, Dujardin JC. Leishmania taxonomy up for promotion? Trends Parasitol 2011; 27: 49-50. https://doi.org/10.1016/j.pt.2010.11.007
  36. da Silva LA, de Sousa CdoS, da Graca GC, Porrozzi R, Cupolillo E. Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil. Infect Genet Evol 2010; 10: 77-83. https://doi.org/10.1016/j.meegid.2009.11.001

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