J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 162 http://jad.tums.ac.ir Published Online: June 12, 2018 Original Article Molecular Identification of Agents of Human Cutaneous Leishmaniasis and Canine Visceral Leishmaniasis in Different Areas of Iran Using Internal Transcribed Spacer 1 PCR-RFLP Aref Teimouri 1, 2, *Mehdi Mohebali 1, 3, Elham Kazemirad 1, *Homa Hajjaran 1 1Department of Medical Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran 2Students Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran 3Center for Research of Endemic Parasites of Iran, Tehran University of Medical Sciences,Tehran, Iran (Received 6 June 2017; accepted 16 May 2018) Abstract Background: Leishmaniasis is a major medical health problem and distributes in nearly half of 31 provinces of Iran. We aimed to identify cutaneous and visceral Leishmania spp. isolated from infected humans and domestic dogs in various regions of Iran, 2010‒2013. Methods: DNA was extracted from 108 lesion exudate samples of suspected patients to cutaneous leishmaniasis and nine liver and spleen aspirates of infected dogs cultured in RPMI-1640 and amplified using partial sequence of ITS1 gene. The PCR amplicons were digested using HaeIII endonuclease enzyme and used in restriction fragment length polymorphism (RFLP) assay. Then, 48 amplicons representing various hosts were sequenced and compared to se- quences from GenBank databases using BLAST. Results: PCR-RFLP analysis showed that 60 and 48 CL patients were infected by Leishmania tropica and L. major, respectively. From nine canine visceral leishmaniasis (CVL) isolates, eight isolates were identified as L. infantum and one as L. tropica. The greatest similarity of 95.7% in ITS1 region was seen between L. infantum and L. major. Furthermore, the lowest similarity with 65.7% was seen between L. tropica and L. major. Intra-species comparison of ITS1 region in L. infantum, L. major and L. tropica isolates were showed 100%, 98.2% and 72.4 % similarities, respectively. Conclusion: PCR-RFLP based on ITS1 region is an appropriate method to distinguish three Leishmania spp. of L. major, L. tropica, and L. infantum. In intra-species comparison of ITS1 region, genotypic variations showed that L. tropica isolates were more heterogeneous than L. major and L. infantum isolates. Keywords: Leishmania, ITS1 gene, PCR- RFLP, Iran Introduction Cutaneous (CL) and visceral leishmaniasis (VL) include some of the world most neglect- ed diseases in tropical and subtropical areas with an estimated incidence of 0.6‒1.0 and 0.05‒0.09 million new cases of CL and VL occur worldwide each year, respectively (1). In Iran, CL is considered as a medical health problem in nearly half of 31 provinces (2). Both epidemiological forms of CL are report- ed in Iran, anthroponotic cutaneous leishman- iasis (ACL) and zoonotic cutaneous leishman- iasis (ZCL) caused by Leishmania tropica and L. major, respectively (3). Nearly 20000 CL cases are annually reported and the prevalence of leishmaniasis in provinces of Iran sugges- tively ranges 1.8‒37.9% (2). Furthermore, VL is caused by L. donovani complex and L. in- fantum is the main agent of VL in endemic areas of Iran with about 100–300 new cases annually. Infections due to L. infantum have been reported in humans, domestic dogs and phlebotomine vectors (4, 5). Human, rodents (mainly great Gerbils (Rhombomys opimus)) and domestic canines (Canis familiaris) are the main reservoir hosts of ACL, ZCL and VL, respectively (6-8). *Corresponding authors: Dr Mehdi Mohebali, E- mail: mohebali@tums.ac.ir, Dr Homa Hajjaran, Email: hajaranh@tums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 163 http://jad.tums.ac.ir Published Online: June 12, 2018 Recently, a few cases of viscerotropic leish- maniasis caused by L. tropica and L. major have been reported in immunocompromised patients in Iran (9, 10). Furthermore, L. in- fantum is involved in sporadic CL in the en- demic areas of VL in Northwestern Iran (11). Because of wide clinical diversities of leish- maniasis, various responses of patients to treat- ment, various reservoir hosts of the parasite and to develop effective control strategies in endemic areas, distinguish between Leishma- nia spp. and find dominant spp. in provinces are critical. Since Leishmania spp. are mor- phologically identical; therefore, species iden- tification using microscopy or culture methods is not easily possible. For species character- ization, additional methods must be used. One of these methods, MLEE (multilocus enzyme electrophoresis) analysis, remains the current gold standard but requires mass culture of the parasites which is not simply carried out (12). Relatively, molecular methods are sen- sitive for the detection of low amounts of the parasite. Nowadays, molecular based analyses have extensively been used for the identification of Leishmania spp. as well as other parasites. Identification and phylogenetic implication with various targets such as kDNA genes and introns are routine (13). Furthermore, the ITS region of rDNA has been used in several stud- ies to resolve taxonomic questions and to de- termine phylogenetic affinities among close- ly related Leishmania species (14, 15). The ITS1 gene is considered as the most appro- priate region and gives the best results in Leishmania differentiation in the old world (16). The ITS1 locates between 18S and 5.8S rRNA genes and possesses conservative loci targets for the PCR (17). Additionally, it in- cludes satisfactory polymorphisms to facili- tate species identification. The aim of this study was to achieve a better understanding of the current status of various isolates of Leishmania spp. in Iranian provinces using ITS1-PCR-RFLP. Furthermore, molecular findings of this study were used to assess phylogenetic relationships between the isolates. Materials and Methods Sampling Samples were collected from 148 lesion exudates of suspected patients to CL referred to Leishmaniasis Laboratory of the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, and District Health Cen- ters of Iran, 2010‒2013. Most of the patients were referred from endemic areas in north- east, southeast, center, west, and southwest of Iran (Fig. 1). Serosity materials of the le- sions were smeared on a microscope slide, air-dried, fixed with absolute methanol and stained with Giemsa in 10% phosphate buff- er (pH 7.4) for 25min and then examined for amastigotes using light microscope with 1000× magnification. Serosity materials from lesions of CL were cultured in RPMI-1640. Twelve dogs suspected to CVL living in endemic areas were tested serologically using direct agglu- tination test (DAT). Liver and spleen aspi- rates of the infected dogs positive for DAT were cultured in RPMI-1640. The parasito- logical positive dogs showed clinical manifes- tations of VL including dermatological wounds, ocular variations, weight loss, laziness, lym- phadenopathy and splenomegaly (18, 19). Ethical approval The study was approved by the Ethics Committee of the Tehran University of Med- ical Sciences (Approval No. 13462). No in- formation of the patients was revealed in the study. Verbal informed consents were re- ceived from the patients and the dog owners. Direct agglutination test (DAT) Anti Leishmania antibodies in dog sera were detected using DAT. Briefly, pro- mastigotes of L. infantum, Iranian strain (MCAN/IR/07/Moheb-ghو GenBank Acces- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 164 http://jad.tums.ac.ir Published Online: June 12, 2018 sion No. FJ555210), were cultured in RPMI- 1640 with 10% heat-inactivated fetal bovine serum (FBS), trypsinized, fixed with 1.2% formaldehyde (Merck, Germany) and stained with Coomassie brilliant blue (Sigma, USA). Negative and positive controls were used in each experiment. Antibody titers ≥ 1:320 were considered as positive in canines (18). Parasite culture and cryopreservation All samples including lesion exudates of the suspected patients to CL and liver and spleen aspirates of the infected dogs were cultured in RPMI-1640 media supplemented with 10–15% heat-inactivated FBS, 100U/ml of penicillin and 100µg/ml of streptomycin (Gibco, Life technologies, Germany) and in- cubated at 24–25 °C. Five days after the last sub-culture, parasites were harvested, washed in sterile phosphate buffered saline (PBS, pH 7.2–7.4), stored at -20 °C until use and were preserved in liquid nitrogen for further studies. DNA extraction, PCR, and RFLP Genomic DNA was extracted from pellets of the Leishmania cultures using High Pure PCR Template Preparation Kit (Roche Diag- nostics, Germany) according to the manufac- turer′s instructions. Samples were stored at - 20 °C until use. DNA samples from Iranian reference strains of L. tropica (MHOM/IR/ 02/Mash10/Accession No. EF653267), L. major (MRHO/IR/11/GOL-2/ Accession No. JN860745) and L. infantum (MCAN/IR/ 07/Mash-ir1/ Accession No. EU810776) were used as positive controls. The DNA samples were assessed for the Leishmania-specific ri- bosomal internal transcribed spacer 1 region (ITS1) by PCR amplification using primer pairs of LITSR (F: 5′-CTGGATCATTTTCCGATG- 3′) and L5.8S (R: 5′-TGATACCACTTAT CGCACTT-3′). Amplification was carried out using PCR-Ready Premix (Roche, Germany) in a 25μl reaction. The amplification condi- tions included those described previously (20). PCR products (8μl) were digested with the restriction endonuclease enzyme HaeIII (BsuRI) (Fermentas, Germany) for the species identification according to the manufactur- er′s instructions. Amplicons of nearly 300– 350bp and restriction fragments were ana- lyzed using 1.5‒3% agarose gels containing safe stain, visualized under UV and com- pared with those from reference strains of L. tropica, L. major and L. infantum. Nucleotide sequence and phylogenetic anal- ysis PCR products from 44 CL and four CVL samples were sequenced using LITSR as for- ward primers. DNA sequences obtained from the current study were compared to sequenc- es from GenBank database using Basic Local Alignment Search Tool (BLAST). These se- quences have been annotated to GenBank and aligned using Clustal X Software and MEGA Software v.6.0 (21). Various statisti- cal methods (maximum likelihood, UPGMA and Neighbor-Joining (NJ) trees) were digital- ized for phylogenetic analysis of the aligned sequences. The Tamura 3-parameter option of the neighbor-joining method was shown as the best phylogenetic tree (22). Inter and intra- species similarities of ITS1 region were cal- culated for L. major, L. infantum and L. trop- ica isolates using MEGA Software v.6.0. Bootstrap analysis was carried out with 1000 replicates. Accession numbers of 48 Leish- mania isolates used in this study are record- ed in GenBank as follows: JX289844-JX 289881, JN860713-JN860714 and JN860718- JN860725. Results Microscopic, culture and DAT analysis Microscopic study demonstrated Leish- mania amastigotes in 112 out of 148 smears (75.6%) collected from patients suspected to CL. Furthermore, 108 samples (72.9%) were positive for Leishmania parasites in culture method. DAT analysis of domestic dog sera http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 165 http://jad.tums.ac.ir Published Online: June 12, 2018 showed that 10 sera (83.3%) were positive with titers ≥ 1:320 out of 12 samples. Nine out of 12 dog samples (75.00%) were positive in microscopy and aspirate culture methods. Leishmania spp identification using RFLP analysis In general, ITS1 PCR was carried out for the diagnosis of Leishmania spp, in which positive samples produced amplicons of 300– 350bp (Fig. 2A). Digestion of the PCR prod- ucts with HaeIII endonuclease produced two bands of 220 and 140bp for L. major refer- ence strain. Furthermore, bands of 200, 60 and 5bp and 200, 80 and 40bp were pro- duced for L. tropica and L. infantum ref- erence strains, respectively (Fig. 2B). Com- pared to patterns produced in reference strains and CL patients, two profiles were clearly distinguishable as follows: 1) 48 amplicons (44.5%) produced two bands (220 and 140bp) indicating L. major, and 2) 60 amplicons (55.5%) produced three bands (200, 60 and 50bp) indicating L. tropica. RFLP analysis on dog samples revealed L. infantum with three bands of 200, 80 and 40bp in eight cases (88.88%) and L. tropica in one case (11.12%). Sequencing and phylogenetic tree analysis Forty eight amplicons were successfully sequenced. The phylogenetic trees from the sequences of ITS1 fragments clearly showed divergence between L. major, L. infantum, and L. tropica. Furthermore, phylogenetic analy- sis demonstrated variations by sequencing as 21 haplotypes were shown in 48 Iranian iso- lates including L. major (H1–H7), L. infan- tum formed independent and equal clusters with 94% bootstrap (H1) and L. tropica (H1‒H13) (Fig. 3). Intra-species similarities of ITS1 region in isolates of L. infantum, L. major, and L. tropica included 100%, 98.2% and 72.4%, respectively (Fig. 4). The highest inter-species similarity of ITS1 region with 95.7% was reported in isolates of L. infan- tum and L. major followed by a 68.5% of similarity between L. infantum and L. tropica. The lowest similarity with 65.7% was reported between L. tropica and L. major (Fig. 5). Fig. 1. Geographical origins of 148 Leishmania spp isolated from cutaneous and visceral leishmaniasis cases identified by ITS1-PCR RFLP (2010-2013) Fig. 2. A) Electrophoresis of Leishmania DNA am- plified a 350bp fragment using primers of LITSR and L5.8S. DNA was collected from reference strains and lesion exudates of suspected patients to cutaneous leishmaniasis and liver and spleen aspirates of infect- ed dogs cultured in RPMI-1640. B) Digestion patterns of ITS1 amplicons of Leishmania spp. from refer- ences and patients using HaeIII enzyme. Line 1, negative control, Line 2, L. tropica (Accession Num- ber: EF653267); Line 3, L. major (Accession Num- ber: EU810776), Line 8, L. infantum (Accession Number: JN860745) as references strain; Line 4‒7, patient samples; Line 9, dog sample, M, 50bp ladder http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 166 http://jad.tums.ac.ir Published Online: June 12, 2018 Fig. 3. The neighbor-joining tree constructed from ITS1 regions of 48 isolates using Tamura 3-parameter. Numbers above branches correspond to bootstrap valued based on 1000 replicates. Branches without numbers include values of less than 70%. Genbank accession numbers are shown in parentheses Fig. 4. Intra-species mean similarities for Leishmania spp. isolated from CL and VL cases based on ITS1 region sequence, 2010‒2013 Fig. 5. Inter-species mean similarities among Leish- mania spp. isolated from CL and VL cases based on ITS1 region sequence, 2010–2013 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 167 http://jad.tums.ac.ir Published Online: June 12, 2018 Discussion Species identification is critical in diag- nosis, treatment and epidemiological studies of Leishmania spp. Several studies have am- plified ITS-1 region of the ribosomal DNA repeat unit (rDNA-ITS1) for the recognition of Iranian Leishmania spp. (8, 23-25). In the present study, a number of unknown Leish- mania spp. isolated from CL and CVL cases in various endemic regions of Iran was iden- tified by PCR-RFLP and sequencing based on ITS1 region of rRNA gene. In the current study, the major foci of 60 L. tropica CL iso- lates belonged to Khorasan Razavi Province, Bam City (Kerman Province), Kashan City (Isfahan Province). Moreover, one isolate be- longed to a CL lupoid patient residing in Shahr- e-Rey in southern Tehran (ACC, JX289854). Forty-eight L. major isolates belonged to Go- lestan, Isfahan, Kermanshah, Khuzestan, Ilam and Semnan Provinces of Iran (Fig. 1). Two isolates recovered from diffuse cutaneous leish- maniasis (DCL) patients in Damghan City of Semnan Province in central (ACC, JN860713) and Dehloran City of Ilam Province in South- western Iran (ACC, JN860714) were identi- fied as L. major. Of nine CVL isolates, eight isolates were identified as L. infantum (six isolates from Ardabil, one isolate from Go- lestan and one isolate from Tehran provinces) and one isolate as L. tropica (from Golestan Province). The phylogenetic analysis revealed 21 haplotypes within the isolates. No correla- tions were seen between the haplotypes and geographic distribution of the species com- plexes. In similar studies in Iran, no correla- tions were demonstrated between the intra- species divergence and geographical distri- bution based on RAPD-PCR and ITS1 and N-acetylglucosamine-1-phosphate transferase (NAGT) genes with PCR-RFLP methods (3, 26). In the phylogenetic analysis, L. tropica isolates showed 13 haplotypes (H1–H13) in- dicating a significant divergence between L. tropica isolates from the other two species (Fig. 3). Although the ITS region is one of the best candidates for the differentiation of Leishmania at species and strain levels, limited studies have used ITS sequence analysis for L. tropica isolates to compare (27-29). Leishmania tropica (a diploid microorgan- ism) is known as a heterogenous species, quite observed in alignment of the PCR product se- quences and in low bootstrap frequencies found in the phylogenetic tree (30-32). The existence of at least two alleles for ITS in rDNA of Leishmania spp. may be a good explanation for this phenomenon (33). In the current study, sequences from three L. tropica isolates were technically unreadable excluded from the phy- logenetic analysis. However, unreadable se- quences of ITS-rDNA fragments belonged to positive leishmaniasis cases (24). This can prove high heterogeneity or sometimes mixed infections with two or three Leishmania spp. (23, 24, 34, 35). Similar to pilot studies, a high degree of heterogeneity was seen in L. tropica in our study (30, 33, 36, 37). In intra-species analy- sis, a varied heterogeneity was found at vari- ous levels in Leishmania spp. using ITS-PCR- RFLP analysis. This included a highest to lowest order of L. tropica > L. major > L. in- fantum with similarities of 72.4%, 98.2% and 100%, respectively. Relatively, L. donovani, L. infantum and L. major were reported as the less and L. tropica, L. turanica and L. gerbilli as the most divergent complexes (38). In 2005, a heterogeneity variation at various levels in Old World Leishmania spp. Was reported with a highest to lowest order of L. tropica > L. ae- thiopica > L. major > L. donovani (39). These findings are similar to studies reported that L. tropica isolates included the highest diver- gence in ITS1 genes (26). Similar to a study on L. major with 98.2% similarity, results of the current study showed a limited genetic variation with seven haplotypes seen in L. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 162–171 A Teimouri et al.: Molecular Identification … 168 http://jad.tums.ac.ir Published Online: June 12, 2018 major (H1‒H7) (25). Four haplotypes of L. major were found in a distinct clade in nine rodent isolates in Central Iran (23) and six haplotypes of L. major were identified through PCR-RFLP of rodent samples using ITS1 markers (8). Results of the present study on L. infantum isolates showed no diversities while formed independent and equal clusters with the high- est bootstrap values (94%) in the phylogenetic analysis. Moreover, heterogeneity of L. trop- ica with various groups was detected in the current phylogenetic analysis while L. infan- tum isolates consisted of only one haplotype. In a similar study in Turkey using an ITS1 based real-time PCR, genotypic variations of Leishmania spp. at species and intra-species levels were observed and heterogeneities were found in L. tropica isolates while L. infan- tum isolates formed single group (40). In the present study, the lowest and highest inter- species varieties were seen between L. major and L. infantum and between L. tropica and L. major with 4.3% and 34.3% variations, re- spectively. In the tree, L. major was more as- sociated to L. infantum than L. tropica was. These findings were in contrast to those from studies based on NAGT genesو in which, the lowest inter-species similarity of 95.7% were reported between L. major and L. infantum (3). Conclusion Sequencing results of Leishmania spp. iso- lated from CL and CVL cases showed 93–98% similarities with other annotated sequences in GenBank database. Therefore, PCR-RFLP based on ITS1 region can be suggested as an adequate method to distinguish L. major, L. tropica and L. infantum which are the most prevalent Leishmania spp. in Middle East. Fur- thermore, genotypic variations based on ITS1 region within inter and intra-species of Leish- mania spp. have shown that L. tropica in- cludes more heterogeneity than L. major and L. infantum do. Acknowledgements The authors would like to thank all staff within the leishmaniose laboratory for their collaboration in isolation of Leishmania spp., School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 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