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Volume 8/ Issue 2/ July-December 2018 T ropical Parasitology • V olume 8 • Issue 2 • July-December 2018 • Pages 61-** Original Article

Determination of genetic diversity of Leishmania species using mini‑circle kDNA, in ‑Iraq countries border

Tahereh Mousavi, Sahar Shokohi, Jahangir Abdi, Razi Naserifar, Mahmoud Ahmadi1, Asad Mirzaei Department of Medical Parasitology, Paramedical School, Ilam University of Medical Sciences, Ilam, 1Department of Medical Parasitology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract Background and Objective: Cutaneous leishmaniasis (CL) is one of the most important diseases worldwide, with a different range of prevalence in endemic areas. Anthroponotic and zoonotic CL are two epidemiological forms of CL, in Iran. Although in the west of Iran is one of the main endemic areas of the disease, there is no inclusive study to determine the genetic variations of parasite in these areas. The objective of this study was to determine the genetic diversity of Leishmania species in Ilam Province, using mini‑circle kDNA gene. Materials and Methods: Direct smears were taken from skin lesions of 200 suspected cases of CL. Smears were stained, screened under light microscope. Polymerase chain reaction (PCR) was performed, using specific kinetoplast DNA primers. Data were analyzed, using the molecular bio‑software. Results: All the samples were positive by direct examination. PCR results showed all cases were positive for Leishmania major. Although all isolated cases belong to a different county of Ilam province, all were positive for L. major with intra‑species genetic diversity, divided into four clades in the dendrogram. Interpretation and Conclusion: This variation can affect drug resistance and controlling strategies of parasite. It is possible that different species of sand flies and rodents are the vector and reservoir of parasite, respectively; however, further studies are needed to validate this.

Keywords: Heterogeneity, Iran, Kinetoplast DNA, Leishmania major

Address for correspondence: Prof. Asad Mirzaei, Department of Parasitology, Paramedical School, Ilam University of Medical Sciences, Ilam, Iran. E‑mail: [email protected] DOA: 19-11-18, DOP: 27-12-2018

INTRODUCTION areas.[5] Among all neglected tropical diseases, leishmaniasis is the most important protozoan infection, in the Middle Leishmaniasis is a vector‑borne disease, caused by obligate East and North Africa region.[6] intracellular parasite belongs to the order of Kinetoplastida, transmitted by phlebotomine sand flies (Diptera: This disease is endemic in tropical and subtropical regions Psychodidae).[1‑4] Cutaneous leishmaniasis (CL) is an of 98 countries in Africa, Asia, Europe, and the Americas.[7] important health problem in many countries of the It is a major global health problem in five continents, and it southern and eastern Mediterranean.[2,5] In fact, the World is estimated that 350 million people are at risk and currently Health Organization has announced, leishmaniasis as the 12 million individuals are infected, worldwide.[2,8‑10] Annually sixth most significant disease in tropical and subtropical This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to Access this article online remix, tweak, and build upon the work non-commercially, as long as appropriate credit Quick Response Code: is given and the new creations are licensed under the identical terms. Website: For reprints contact: [email protected] www.tropicalparasitology.org

How to cite this article: Mousavi T, Shokohi S, Abdi J, Naserifar R, DOI: Ahmadi M, Mirzaei A. Determination of genetic diversity of Leishmania 10.4103/tp.TP_3_18 species using mini-circle kDNA, in Iran-Iraq countries border. Trop Parasitol 2018;8:77-82.

© 2018 Tropical Parasitology | Published by Wolters Kluwer - Medknow 77 Mousavi, et al.: Determination of genetic diversity of Leishmania species two million new clinical cases occur globally, and incidence Sampling of CL is 0.7 to1.2 million.[4,11] The samples [Table 1] were collected from cutaneous lesions [Figure 2], suspected with CL, and then transferred Unorganized urbanization and human migration in these to a clinical laboratory. From all cases, medical records regions are the two main factors affecting leishmaniasis, were collected. Direct smear was taken from each lesion [6] regarding epidemiology and morbidity. Despite the of the suspected cases. Slides were fixed with methanol, existence of a universal program for vectors control stained with Giemsa and examined with light microscopy. and leishmaniasis treatment, the disease continues to Serous sample from lesion prepared in microtube, with a [6] spread. In recent years, factors, such as new settlements, final volume of 250µ l phosphate‑buffered saline. Samples urbanization, agricultural development, migration, regional were stored at −20°C, for DNA extraction. wars, improvement of reporting systems, and ecological changes, resulted in a significant increase in CL.[2] Genomic DNA extraction Total DNA was extracted from all samples. 100 µl of each Annually, more than 20 thousand cases of CL are reported sample was used for DNA extraction process, using the from different parts of Iran, while the actual number of DNA extraction kit (DNG‑plus™, Cinna Gene, Iran). [5] cases is several times more than the official reports. In Then the DNA extraction was performed, according to Iran, three species of Leishmania, such as Leishmania major, the manufacturer’s instructions. DNA was stored at −20°C, Leishmania tropica, and somewhat Leishmania infantum, have for further analysis. caused CL lesion.[12,13] Polymerase chain reaction amplification Thus, identification of Leishmania species is important for Extracted DNA was amplified by PCR, using specific planning control program and treatment protocols of the primers for kDNA mini‑circles. The primers were [14] disease. Many polymerase chain reaction (PCR)‑based designed, as previously reported by Noyes et al.: forward techniques have been used for Leishmania diagnosis and been primer: 13Z (ACTGGGGGTTGGTGTAAAATAG), validated. PCR technique, using kDNA gene is the most homologous to conserved sequence block 3, and reverse sensitive method for the detection of Leishmania parasite, and primer: LiR (TCGCAGAACGCCCCT), complementary it is superior to parasitological methods for identifying the cases, missed by either microscopic examination or culture.[15] Kinetoplast is a unique DNA containing structure in the mitochondrion of the cell, consisting of two components, maxi‑circle (present in a number of 30–50 copies/parasite, with 20–40 kb in length) and mini‑circle kDNA (present in a number of 10,000–20,000 copies/parasite with 1 kb in length).[16‑19] Kinetoplast DNA minicircles are often the first choice for leishmaniasis diagnosis, containing three highly conserved blocks conserved sequence blocks (CSB) (CSB‑1, conserved blocks of synteny‑2 and CSB‑3).[20] The main advantage of minicircles is high copy number and variation of kDNA mini‑circles, used for the analysis of genetic Figure 1: Geographical location of study area, Ilam province where [4,16,17,21] polymorphism. Therefore, it is an ideal target for samples collected to be screened for Leishmania the identification of Leishmania species.[22] Hence, we used kDNA‑PCR to determine the genetic diversity of this Table 1: Samples information received from different protozoon, in Ilam Province. counties in the Ilam province that studied Province County Number of Direct Microtube with MATERIALS AND METHODS samples smear lesion serous Ilam 2 * * Malekshahi 1 * * Study area Chardavol 5 * * Ilam province is located in the west of Iran, in border Dehloran 137 * * of Iraq‑Iran, covering 19,086 km2, including eight 3 * * Ilam 1 * * counties, in which Mehran, Dehloran, and Abdanan Mehran 50 * * with a high‑temperature climate, has a joint border with Sirvan 1 * * Iraq [Figure 1]. Total 200

78 Tropical Parasitology | Volume 8 | Issue 2 | July-December 2018 Mousavi, et al.: Determination of genetic diversity of Leishmania species to conserved sequence block 1, amplifying 560, 750, presented in GenBank. Only 20 out of 80 (25%) PCR and 680 bp fragments for L. major, L. tropica, and products had sufficient amount of amplified DNA, L. infantum, respectively.[23] Each 25 μl reaction mixture, which have been used for direct sequencing [Table 2]. contained 1x PCR,1.5 mM MgCl2, 0.2 mM dNTP, Dendrogram of kDNA gene sequences of isolates, 2.5 U Taq (Qbiogene, Irvine, CA, USA) and 1 μM of constructed by the neighbor‑joining method, using Mega each primer. A total of, 35 cycles were performed, using a 6 software [Figure 4]. Sequences designated as L. major IR/ PCR‑Thermal‑Cycler (SensoQuest, Göttingen, Germany), IL. L. tropica (accession number AB901375), was used as using the following conditions: an Initial denaturation of the out‑group. Bootstrap values (%) are indicated at the 5 min at 94°C, followed by 30 cycles of 94°C for 30 s, internal nodes (1000 replicates). 55°C for 60 s, 72°C for 90 s, and subsequent extension at 72°C for 10 min. Positive and negative control were used DISCUSSION for each PCR reaction. Due to the heterogeneity among Leishmania species, and The PCR products were separated on 1.2% agarose various clinical manifestation, as well as different types gel electrophoresis and visualized by Molecular Imager of lesions in humans, the diagnosis is difficult or this has Chemi Doc™ XRST (imaging system, Bio‑Rad, USA), led to a misdiagnosis. Different molecular techniques, after staining with DNA safe. Single fragments of 560 bp, such as various types of PCR, have been used, as accurate 750 bp, and 680 bp are indicative of the species L. major, tools for definitive diagnosis of the disease. Therefore, L. tropica, and L. infantum, respectively. Table 2: Details of patients from whom Leishmania ware RESULTS isolated Sample code Host Age/sex Number of All lesions were ulcerative with different size. Isolates lesions that caused great scars, located in the same clade in the Leishmania major IR/IL11fv Human 4/male 2 Leishmania major IR/IL10fv Human 25/male 1 dendrogram. According to the questionnaires, all the Leishmania major IR/IL158fv Human 2/male 2 patients, suffering from L. major infections were from Leishmania major IR/IL23fv Human 2/female 2 endemic areas or had a history of travel to endemic areas. Leishmania major IR/IL22fv Human 2/male 6 Leishmania major IR/IL16d Human 45/male 4 Leishmania amastigote was identified, using Giemsa‑stained Leishmania major IR/IL144d Human 2/male 1 smears, obtained from all isolated samples. Some PCR Leishmania major IR/IL140d Human 1/male 2 products represent a mixed infection, from two to three Leishmania major IR/IL319d Human 21/female 3 species of Leishmania. All the samples were examined, Leishmania major IR/IL411sa Human 28/female 1 Leishmania major IR/IL393m Human 30/male 4 using PCR. Species‑specific band observed in all the Leishmania major IR/IL392m Human 27/female 3 samples [Figure 3]. Leishmania major IR/IL377sa Human 27/male 2 Leishmania major IR/IL7d Human 11/male 5 Leishmania major IR/IL155db Human 24/male 2 At least, 20 L. major kDNA haplotypes have been identified, Leishmania major IR/IL17d Human 32/male 3 by sequence alignment, in comparison with sequences, Leishmania major IR/IL20d Human 53/male 2 Leishmania major IR/IL259ca Human 42/male 1 Leishmania major IR/IL258da Human 7/female 2 Leishmania major IR/IL252pf Human 19/male 7 Leishmania major IR/IL331cd Human 32/male 1 Leishmania major IR/IL253pf Human 19/male 7

Figure 2: Cutaneous lesions of patient with suspected cutaneous Figure 3: Detection of Leishmania parasite using kDNA gene, leishmaniasis lanes1‑7 patients samples, neg: Negative control, pos: Positive control

Tropical Parasitology | Volume 8 | Issue 2 | July-December 2018 79 Mousavi, et al.: Determination of genetic diversity of Leishmania species high copy number (10000–26000) of the Leishmania minicircles, makes it as a reliable target for molecular analysis of Leishmania spp.[4,24,29,31,32] The conserved region of these minicircles has been used as a diagnostic PCR target, since 1990s.[33] The kDNA‑PCR seems to be the most sensitive approach for the diagnosis of leishmaniasis, suggested being implemented as the standard method for routine diagnosis, if the identification of species does not require.[34]

In this study, despite the fact that all isolates belonged to a different county in Ilam province, but all were positive for L. major. Our analysis showed an intra‑species genetic diversity, and 20 isolates were divided into four clades in the dendrogram. So that, Clade 1 contains (10fv, 11 fv, 22 fv, 23 fv, and 158 fv taken from the Farrukhabad village in Dehloran county and 16d belongs to the Dehloran city), geographically closed to each other. Clade 2 contains 319 d, 140 d, and 144d taken from the city of Dehloran, 411sa from Salehabad in Mehran county, 392 m and 393 m in Mehran county, which they joined together in the dendrogram. Dehloran and Mehran are geographically close, located at Figure 4: Phylogenetic tree of the kDNA gene sequences of Leishmania the joint border of Iraq‑Iran. Clade 3 contains (7 d, 17 d, isolates constructed by the neighbor‑joining method using Mega6 and 20 d taken from Dehloran county, 155, db that is in software (version 6). Isolates or strains names are as provided in the proximity of Dhiraj dham near Dehloran, and 377, sa Genbank as available. Sequences generated in this study named as Leishmania major IR/Il. Leishmania tropica (accession number in Mehran county). Clade 4 contains 5 strains, belonging AB901375.1) used as the out‑group to different areas of Dashtabas and Dehloran county. Rodent and sand flies as the reservoirs and a vector of characterization and identification of Leishmania species the disease, respectively, can easily be transferred between are necessary, because of different forms of CL, caused Iran and Iraq, by pilgrimage travelers and border control by different species, which may require specific control officers, changing the disease epidemiology and genetic measures and treatment programs.[22] diversity between different isolates and spread of imported strains between Iran and Iraq. Interestingly, some isolates In this study, 200 samples of suspected ulcer were collected from officers in the joint border between Iran and used, 130 (65%) were from males, and 70 (35%) Iraq, created a big and serious ulcer, and joined together in were from females. The age of the patients was in the the same clades (Clad 1) in the dendrogram, showing that range of 6 months to 86 years old. In addition, the diameter pathogenicity of the parasite related to the genetic diversity. of the lesions was from 2 mm to 10 cm, and the number Furthermore, some mix infections have been observed that of lesions was from 1 to 13. PCR amplification of kDNA could not be diagnosed, decisively. gene was used for the diagnosis and characterization of Leishmania species. All the samples were positive, using Many studies reported intra‑species genetic diversity among direct examination and PCR method. L. major, using different target genes, based on factors, such as geographic distribution of parasite, vectors, and host In this study, we have used kDNA gene as a tool to reservoir.[12,35‑37] Ayari et al. showed two different clusters evaluate the genetic diversity of Leishmania spp. The of L. major, based on mitochondrial cytochrome b gene.[38] kDNA contains thousands of minicircles and dozens Oryan et al. showed six clusters of L. major in southern Iran, of maxicircles, concatenated in a giant network.[24] The based on mini‑circle kDNA.[39] Nasereddin et al. showed structure of kinetoplast DNA is unique for a mitochondrial two main clusters in central Israel and Palestine, based on genome.[23,25,26] The 10,000 kinetoplast minicircles are kDNA.[21] Ali et al. showed two clusters (LmA and LmB) distributed among 10 different sequence classes.[4,23,27‑29] for L. major and two clusters (LtA and LtB) for L. tropica The number of mini‑circles in each class is very variable, in Iraq, based on ITS gene.[40] Studies of the Leishmania dividing into conserved and variable region.[4,23,30] The genome, using different genes, showed some degrees of

80 Tropical Parasitology | Volume 8 | Issue 2 | July-December 2018 Mousavi, et al.: Determination of genetic diversity of Leishmania species diversity, affecting drug resistance and the controlling the patients for their cooperation and all the individuals strategies for the parasite. Our data indicate the dominance participating in this study. of L. major and presence of intra‑species genetic variation in Ilam province, requiring the codification of a precise, Financial support and sponsorship specific, and coherent program for disease control and Nil. further investigations, in this field. Conflicts of interest Treatment of patients with CL depends not merely There are no conflicts of interest. on direct examination and clinical manifestations, but REFERENCES molecular differentiation has a more important role in this proceeding.[15,22] The result of this study provides a 1. Karimi A, Nabipour A. 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