Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing
-
Sara Nemati
,
Homa Hajjaran
,
Ali Khamesipour
,
Mohsen Falahati Anbaran
,
Hamed Mirjalali
,
Asghar Fazaeli
Abstract
Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST).
Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes.
Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite.
Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.
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9. Degrave W. Use of molecular probes and PCR for detection and typing of Leishmania-a mini-review. Mem Inst Oswaldo Cruz. 1994; 89(3):463-9.
10. 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(4):415-420.
11. Wilson S. DNA-based methods in the detection of Leishmania parasites: field applications and practicalities. Ann Trop Med Parasitol. 1995;89 Suppl 1:95-100.
12. 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(2):149-159.
13. Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Cabrera TS, Pérez-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(10):918-925.
14. Dávila A, Momen H. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol. 2000;94(6):651-654.
15. Nemati S, Fazaeli A, Hajjaran H, et al. Genetic diversity and phylogenetic analysis of the Iranian Leishmania parasites based on HSP70 gene PCR-RFLP and sequence analysis. Korean J Parasitol. 2017;55(4):367-374.
16. Mauricio IL, Yeo M, Baghaei M, et al. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol. 2006;36(7):757-769.
17. Bañuls A-L, Brisse S, Sidibé I, Noël S, Tibayrenc M. A phylogenetic analysis by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting of the Leishmania genome project Friedlin reference strain. Folia Parasitol (Praha). 1999;46(1):10-14.
18. Urwin R, Maiden MC. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 2003;11(10):479-487.
19. Sullivan CB, Diggle MA, Clarke SC. Multilocus sequence typing. Mol Biotechnol. 2005;29(3):245-54.
20. Bougnoux M-E, Morand S, d'Enfert C. Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans. J Clin Microbiol. 2002;40(4):1290-1297.
21. Guerbouj S, Victoir K, Guizani I, et al. Gp63 gene polymorphism and population structure of Leishmania donovani complex: influence of the host selection pressure? Parasitology. 2001; 122 Pt 1:25-35.
22. Hajjaran H, Kazemi Rad E, Mohebali M, et al. Expression analysis of activated protein kinase C gene (LACK 1) in antimony sensitive and resistant Leishmania tropica clinical isolates using real‐time RT‐PCR. Int J Dermatol. 2016;55(9):1020-1026.
23. Hajjaran H, Mohebali M, Teimouri A, et al. Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene. Infect Genet Evol. 2014; 26:203-212.
24. Castilho TM, Shaw JJ, Floeter-Winter LM. New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol. 2003;41(2):540-6.
25. Fotouhi-Ardakani R, Dabiri S, Ajdari S, et al. 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.
26. Zhang C-Y, Lu X-J, Du X-Q, Jian J, Shu L, Ma Y. Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing. PLoS One. 2013;8(4): e63124.
27. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. in Nucleic acids symposium series. 1999. (London): Information Retrieval Ltd. c1979-c2000.
28. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725-2729.
29. Huson DH. Splits Tree: analyzing and visualizing evolutionary data. Bioinformatics. 1998;14(1):68-73.
30. Leigh JW, Bryant D. Popart: full‐feature software for haplotype network construction. Methods Ecol Evol. 2015;6(9):1110-1116.
31. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585-595.
32. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451-1452.
33. Grundmann H, Hori S, Tanner G. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol. 2001;39(11):4190-4192.
34. Seridi N, Belkaid M, Quispe-Tintaya W, Zidane C, Dujardin JC. Application of PCR-RFLP for the exploration of the molecular diversity of Leishmania infantum in Algeria. Trans R Soc Trop Med Hyg. 2008;102(6):556-563.
35. Hide M, Banuls AL. Species-specific PCR assay for L. infantum/L. donovani discrimination. Acta Trop. 2006;100(3):241-245.
36. Oshaghi MA, Ravasan NM, Hide M, et al. Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L. infantum/L. donovani discrimination. Exp Parasitol. 2009;122(1):61-65.
37. Hajjaran H, Mohebali M, Mamishi S, et al. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed Res Int. 2013;2013:789326.
38. Mirzaei A, Rouhani S, Taherkhani H, et al. Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol. 2011;129(4):375-380.
39. Hammoudeh N, Kweider M, Abbady A-Q, Soukkarieh C. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene. Iran J Parasitol. 2014;9(4):574-83.
40. Fraga J, Montalvo AM, De Doncker S, Dujardin J-C, Van der Auwera G. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect Genet Evol. 2010;10(2):238-245.
41. Hosseini M, Rostami MN, Hosseini Doust R. Khamesipour A. Multilocus sequence typing analysis of Leishmania clinical isolates from cutaneous leishmaniasis patients of Iran. Infect Genet Evol. 2020; 85:104533.
2. Organization WH. Control of the leishmaniases: report of a meeting of the WHO Expert commitee on the control of leishmaniases, geneva, 22-26 March 2010. 2010: World Health Organization. https://apps.who.int/iris/handle/10665/44412
3. Postigo JAR. Leishmaniasis in the world health organization eastern mediterranean region. Int J Antimicrob Agents, 2010; 36 Suppl 1:S62-5.
4. Mohebali M, Edrisian GH, Nadim A, et al. Application of direct agglutination test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iran J Parasitol. 2006;1(1):15-25.
5. Badirzadeh A, Mohebali M, Asadgol Z, et al. The burden of leishmaniasis in Iran, acquired from the global burden of disease during 1990–2010. Asian Pac J Trop Dis. 2017;7(9):513-518
6. Mohebali M, Edrissian G, Akhoundi B, et al. Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022. Iran J Parasitol. 2023;18(3):279-293.
7. Mohebali M. Visceral leishmaniasis in Iran: review of the epidemiological and clinical features. Iran J Parasitol. 2013; 8(3):348-58.
8. Razmjou S, Hejazy H, Motazedian MH, Baghaei M, Emamy M, Kalantary M. A new focus of zoonotic cutaneous leishmaniasis in Shiraz, Iran. Trans R Soc Trop Med Hyg. 2009;103(7):727-730.
9. Degrave W. Use of molecular probes and PCR for detection and typing of Leishmania-a mini-review. Mem Inst Oswaldo Cruz. 1994; 89(3):463-9.
10. 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(4):415-420.
11. Wilson S. DNA-based methods in the detection of Leishmania parasites: field applications and practicalities. Ann Trop Med Parasitol. 1995;89 Suppl 1:95-100.
12. 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(2):149-159.
13. Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Cabrera TS, Pérez-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(10):918-925.
14. Dávila A, Momen H. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol. 2000;94(6):651-654.
15. Nemati S, Fazaeli A, Hajjaran H, et al. Genetic diversity and phylogenetic analysis of the Iranian Leishmania parasites based on HSP70 gene PCR-RFLP and sequence analysis. Korean J Parasitol. 2017;55(4):367-374.
16. Mauricio IL, Yeo M, Baghaei M, et al. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol. 2006;36(7):757-769.
17. Bañuls A-L, Brisse S, Sidibé I, Noël S, Tibayrenc M. A phylogenetic analysis by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting of the Leishmania genome project Friedlin reference strain. Folia Parasitol (Praha). 1999;46(1):10-14.
18. Urwin R, Maiden MC. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 2003;11(10):479-487.
19. Sullivan CB, Diggle MA, Clarke SC. Multilocus sequence typing. Mol Biotechnol. 2005;29(3):245-54.
20. Bougnoux M-E, Morand S, d'Enfert C. Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans. J Clin Microbiol. 2002;40(4):1290-1297.
21. Guerbouj S, Victoir K, Guizani I, et al. Gp63 gene polymorphism and population structure of Leishmania donovani complex: influence of the host selection pressure? Parasitology. 2001; 122 Pt 1:25-35.
22. Hajjaran H, Kazemi Rad E, Mohebali M, et al. Expression analysis of activated protein kinase C gene (LACK 1) in antimony sensitive and resistant Leishmania tropica clinical isolates using real‐time RT‐PCR. Int J Dermatol. 2016;55(9):1020-1026.
23. Hajjaran H, Mohebali M, Teimouri A, et al. Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene. Infect Genet Evol. 2014; 26:203-212.
24. Castilho TM, Shaw JJ, Floeter-Winter LM. New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol. 2003;41(2):540-6.
25. Fotouhi-Ardakani R, Dabiri S, Ajdari S, et al. 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.
26. Zhang C-Y, Lu X-J, Du X-Q, Jian J, Shu L, Ma Y. Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing. PLoS One. 2013;8(4): e63124.
27. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. in Nucleic acids symposium series. 1999. (London): Information Retrieval Ltd. c1979-c2000.
28. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725-2729.
29. Huson DH. Splits Tree: analyzing and visualizing evolutionary data. Bioinformatics. 1998;14(1):68-73.
30. Leigh JW, Bryant D. Popart: full‐feature software for haplotype network construction. Methods Ecol Evol. 2015;6(9):1110-1116.
31. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585-595.
32. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451-1452.
33. Grundmann H, Hori S, Tanner G. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol. 2001;39(11):4190-4192.
34. Seridi N, Belkaid M, Quispe-Tintaya W, Zidane C, Dujardin JC. Application of PCR-RFLP for the exploration of the molecular diversity of Leishmania infantum in Algeria. Trans R Soc Trop Med Hyg. 2008;102(6):556-563.
35. Hide M, Banuls AL. Species-specific PCR assay for L. infantum/L. donovani discrimination. Acta Trop. 2006;100(3):241-245.
36. Oshaghi MA, Ravasan NM, Hide M, et al. Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L. infantum/L. donovani discrimination. Exp Parasitol. 2009;122(1):61-65.
37. Hajjaran H, Mohebali M, Mamishi S, et al. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed Res Int. 2013;2013:789326.
38. Mirzaei A, Rouhani S, Taherkhani H, et al. Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol. 2011;129(4):375-380.
39. Hammoudeh N, Kweider M, Abbady A-Q, Soukkarieh C. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene. Iran J Parasitol. 2014;9(4):574-83.
40. Fraga J, Montalvo AM, De Doncker S, Dujardin J-C, Van der Auwera G. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect Genet Evol. 2010;10(2):238-245.
41. Hosseini M, Rostami MN, Hosseini Doust R. Khamesipour A. Multilocus sequence typing analysis of Leishmania clinical isolates from cutaneous leishmaniasis patients of Iran. Infect Genet Evol. 2020; 85:104533.
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| Issue | Vol 19 No 2 (2024) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v19i2.15853 | |
| Keywords | ||
| Leishmania spp. Phylogeny Multilocus sequence typing Iran | ||
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How to Cite
1.
Nemati S, Hajjaran H, Khamesipour A, Falahati Anbaran M, Mirjalali H, Fazaeli A. Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing. Iran J Parasitol. 2024;19(2):171-182.
