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Tatera indica (Rodentia: Muridae) as the Prior Concern and the Main Reservoir Host of Zoonotic Cutaneous Leishmaniasis on the Border of Iran and Iraq


Somayeh Mohammadi 1 , Babak Vazirianzadeh 2 , Reza Fotouhi-Ardakani 1 , Elnaz Alaee Novin 1 , Aref Amirkhani 3 , Javad Samii 1 , Ahmad Reza Esmaeili Rastaghi 1 , Parviz Parvizi 1 , *


1 Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, 69 Pasteur Ave., Tehran, Iran

2 Social Determinants of Health Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Department of Epidemiology, Medical Sciences of Tehran branch, Islamic Azad University, Tehran, Iran


Jundishapur Journal of Microbiology: 10 (1); e42452
Published Online: December 5, 2016
Article Type: Research Article
Received: September 25, 2016
Revised: November 18, 2016
Accepted: November 26, 2016




Background: Zoonotic cutaneous leishmaniasis (ZCL) is increasing in many parts of the world including Iran. Rodents are the most important reservoirs of Leishmania parasites in many remote areas of ZCL. Identification and molecular characterization of Leishmania parasites in reservoir hosts (rodents and dogs), potential vectors (sandflies), and suspected patients in leishmaniasis foci should be clarified for different controlling measurements and treatments.

Objectives: This study aimed to determine the main reservoir hosts of ZCL in Khuzestan province bordering Iran and Iraq.

Methods: Rodents were captured and identified using morphological and molecular techniques. Leishmania species were sampled from both ears of rodents and DNA was extracted. Leishmania detection was based on PCR and sequencing of ITS-rDNA of infected rodents. Phylogenetic analyses were conducted to understand the relationship, homology, and haplotype variations among Leishmania major parasites and identify the causative agents of leishmaniasis in the area. The maximum likelihood and neighbor-Joining with alternative Kimura 2-Parameter models were employed for phylogenetic analyses.

Results: Leishmania major was firmly recognized by conducting molecular analysis on 121 captured rodents, from which 45 samples unequivocally were identified as Tatera indica. Leishmania parasites obtained from T. indica were sequenced to analyze genetic polymorphism and/or similarity using ITS-rDNA genotype. Phylogenies revealed that one common haplotype of L. major (GenBank accession no. EF413075) was the most haplotype variant dominated among seven infected T. indica.

Conclusions: The widespread distribution of L. major parasites in human suggests not only T. indica was the main reservoir but also other rodents and mammalian hosts might be the reservoir hosts of ZCL in the region. Molecular and phylogenetic analyses confirmed the strength of haplotype variation maintaining the circulation of Leishmania species in their reservoir hosts.


Leishmania major Tatera indica Zoonotic Cutaneous Leishmaniasis Phylogenetic Analyses ITS-rDNA Iran

Copyright © 2016, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
1. Background

Leishmaniasis as one of the emerging and neglected infectious diseases has been largely distributed in the world. Khuzestan province is situated on the border of Iran and Iraq, having a tropical climate with high prevalence rate of leishmaniasis in the five past years (1). Although, it is believed that three species of Leishmania parasites have been incriminated as the causative agents of human leishmaniasis in Iran, other mammals’ Leishmania species have been isolated and identified from sandflies, rodents, and humans (2).

Zoonotic cutaneous leishmaniasis (ZCL) is an endemic disease in more than 80 countries in the world including Iran (3, 4). Zoonotic cutaneous leishmaniasis is distributed in more than half of Iranian provinces and Khuzestan province in the southwest has been affected by the disease with new and unknown foci. The life cycle of Leishmania parasites in ZCL depends on some criteria such as sandfly species as vectors, wild rodent species as reservoir hosts, and the geographical locations as natural important foci of the disease (5, 6). Among different species of wild rodents, a few are identified as the main species of ZCL reservoir hosts in Iran. A number of rodent species are widespread; some of them in Asia and some others in smaller areas (7).

There are many important species of rodents from different ZCL regions in Iran such as Rhombomys opimus, Meriones libycus, M. persicus, Tatera indica, Nesokia indica, M. hurrianae, and Rattus norvegicus which have been reported as the main hosts of ZCL parasites. But there is no sufficiently precise and consistent evidence to confirm that all of the species are main reservoirs of ZCL in Iran and other regions (4, 8-20).

Currently, T. indica has drawn more attention not only as a principal ZCL reservoir but also due to our recent finding revealing that four subspecies of T. indica exist in Iran based on morphological and molecular characteristics. In spite of the expectation that two of which exist in Khuzestan province, only one subspecies was found (21).

2. Objectives

The objective of this investigation was to find the potential and/or prominent reservoir hosts of ZCL and define the role of T. indica in the transmission of Leishmania major in southwestern Iran. Another objective of this study was to explore (i) genetic variation, (ii) polymorphism, and (iii) genetic similarity as effective factors in maintaining equilibrium of mutation due to natural selection or genetic drift in the population of L. major isolated from sandfly species as vectors, wild rodent species as reservoirs and human at the border of Iran and Iraq (22).

3. Methods
3.1. Origin and Sampling of Leishmania From Reservoir Hosts

Rodents were collected from the active colonies of rodent burrows around the villages in the study area using 40 wooden and wire live traps in 2012 - 2014. Cucumbers and dates were used as baits for each location of ten villages in four districts of Khuzestan province. The areas’ altitude was 18 meters above the sea level (a.s.l) with geographical coordinates as: 31.3273°N; 48.6940°E; (Figure 1). The rodent traps were set up in active colonies in 6 cities (12 villages) of Khuzestan Province early in the morning. The traps were left for several days in each location. However, T. indica was captured only from 6 villages. The samples was collected from the colonies of rodents’ burrows located around the villages where ZCL was endemic using 50 wooden and wire live traps for each location.

The traps were checked and the baits were changed consecutively. Collected rodents were transferred to Pasteur Institute of Iran and maintained to be identified morphologically as well as molecularly.

3.2. DNA Extraction and Amplification of Leishmania Infection

The ears of each rodent were scratched and two impression smears were taken. Routine laboratory procedures and molecular methods of impression smears prepared from rodents’ ears were followed according to the Parvizi et al. (2008) protocol to detect Leishmania infection. Leishmania sampling from rodents, isolation of parasites, impression smears from each ear of rodents, light microscope observation, culturing in Novy-MacNeal-Nicolle (NNN), and inoculation in Balb/C from the captured rodents were followed by the methods of Mirzaei et al. (2011) (17).

Whole genomic DNA of T. indica was extracted using Genet Bio kit (Takapoo Zist), Phenol-Chloroform (CinnaGen Co. Tehran. Iran) and ISH Horovize methods based on Parvizi et al. (13). The DNA of all T. indica samples was extracted and the ITS-rDNA gene fragment of Leishmania parasites was amplified using PCR to find the precise species of Leishmania parasite causing leishmaniasis. The forward primer IR1 and the reverse primer IR2 were exerted for the first-stage of PCR; while for the second-stage of the nested PCR, the forward primer ITS1F and the reverse primer ITS2R4 were used (23, 24).

3.3. Sequences and Phylogenetic Analyses

The positive PCR products obtained from Leishmania parasites were directly sequenced, aligned, and edited to determine Leishmania species and haplotype variations in individual rodents using Sequencher 4.1.4TM for PC. Phylogenetic analysis was performed in MEGA 6 software. Maximum likelihood (ML) and neighbor-joining (NJ) were the two statistical methods to draw trees and determine the genetic relationship of Leishmania species with different original hosts using alternative Kimura 2-Parameter (K2P) models (25). The DnaSP5 software was used to indicate polymorphisms and haplotype diversities.

4. Results
4.1. Morphological and Molecular Identification

Among 121 rodents sampled from six districts around 12 villages and sites in Khuzestan province, Iran, in 2012 - 2014, 45 were identified as T. indica (Table 1 and Figure 1). All T. indica samples (20 female, 25 male) were identified first morphologically and then molecularly using Cyt b gene in the method of Parvizi et al. 2008 (13). Tatera indica was captured more in Behbahan than other locations; but no significant difference was found in Leishmania infections based on different locations. 41 live-captured T. indica samples were examined for Leishmania infections using both conventional and molecular methods; but only 4 dead captured T. indica were used to isolate and detect Leishmania parasites in molecular methods.

Table 1. Distributions and Leishmania Infections in T. indica, Khozestan Province, Iran
LocationHabitatT. indicaAge GroupsSeasonTotal (+ve)
TownLeishmania (+ve)0 - 2 (+ve)2 - 4 (+ve)> 4 (+ve)SpringSummerEarly FallLast Winter
AhvazF (+ve)M (+ve)F (+ve)M (+ve)F (+ve)M (+ve)F (+ve)M (+ve)F (+ve)M (+ve)10 (2)
Jadeh Hamideyeha25 (1) LR124 (1)12 (1)130000
Jadeh Abadana12 (1) LR01 (1)21001 (1)0001
ShushtarGhalehnoub01 (1) L01 (1)00001 (1)00001 (10)
BehbahanKharestana7 (1) LR10 (1) LR3 (2)593534 (1)1 (1)10021 (3)
Ab Amirb0110000010000
Heyat Abadb1 (1) R01 (1)00001 (1)00000
DezfulGavmish Abadb521241030120011 (1)
Bonyeh Abadb21 (1) R1 (1)202001 (1)0000
Seyed Nurb0101001000000
Total No (+ve)20 (2)25 (5)9 (4)17 (2)19 (1)10 (0)8 (1)8 (1)13 (4)2 (1)3 (0)01 (0)45 (7)
45 (7)45 (7)45 (7)45 (7)45 (7)18 (1)18 (1)21 (5)21 (5)5 (1)5 (1)1 (0)1 (0)

Abbreviations: F, female; M, male; L, left ear; R, right ear; LR, right and left ear.

aRodent burrow, desert.

bRodent burrow, around villages.

Location of Villages and Districts in Khuzestan Province, Iran, Where T. indica Was Sampled and Screened for Leishmania Infections
Figure 1. Location of Villages and Districts in Khuzestan Province, Iran, Where T. indica Was Sampled and Screened for Leishmania Infections
4.2. Detection of Leishmania Infection Using Molecular Analyses

Seven out of 45 T. indica were found with Leishmania infection (Table 1). Live-captured T. indica were more infected with Leishmania (7 out of 41) than those captured dead (zero out of 4). No significant difference was found in Leishmania infection between left and right ears of T. indica. But concurrent Leishmania infection in both ears was more prevalent.

This is the first time to detect Leishmania parasites focusing only on T. indica in large scales of Khuzestan province. Leishmania major firmly was identified first time in 7 T. indica by amplifying 460 bp fragment of ITS1-5.8S rRNA -ITS2 gene, RFLP, sequences, aligning, and comparing our sequences with homologous ones from the GenBank database. In addition, this finding was important because Khuzestan province has up to 1609 kilometres shared border with Iraq. Twenty one sequences of ITS-rDNA of Leishmania parasites were analyzed for genetic polymorphism and genetic similarity. Leishmania were isolated from reservoir hosts of rodents, sandflies, and humans. 16 sequences of L. major, 4 sequences of L. tropica, and one sequence of L. infantum were employed for phylogenetic analysis trees reconstruction, and determination of the evolution of Leishmania parasites.

In terms of evolutionary relationships, molecular phylogenetic analysis by maximum likelihood and neighbor-joining methods revealed that taxa and two old world Leishmania species (L. major and L. tropica) had share common ancestors (Figures 2 and 3). In topology of trees, more variations were observed in L. major isolated from sandflies, followed by those from rodents and humans. L. tropica had more diversity than L. major and placed as out group (Table 2).

Maximum Likelihood Bootstrap Tree Showing the Relationships of the Haplotypes of the ITS1-rDNA Gene Fragment for the Isolates of Three Leishmania species
Figure 2. Maximum Likelihood Bootstrap Tree Showing the Relationships of the Haplotypes of the ITS1-rDNA Gene Fragment for the Isolates of Three Leishmania species
Neighbor-Joining Tree Showing the Relationships of the Haplotypes of the ITS1-rDNA Gene Fragment for the Isolates of Leishmania Species
Figure 3. Neighbor-Joining Tree Showing the Relationships of the Haplotypes of the ITS1-rDNA Gene Fragment for the Isolates of Leishmania Species
Table 2. Comparison of Leishmania Infection in T. indica Based on Epidemiological Factors Using Cross Tab, Chi-Square and Adjusted Logistic Regression Statistical Testsa
VariablesUninfected, (n= 38)Infected, (n = 7)OR (CI 95 %)P Value
Age groups
0 - 25 (55.6)4 (44.4)10.105
2 - 415 (88.2)2 (11.8)0.27 (0.026 - 2.916)0.284
> 418 (94.7)1 (5.3)0.052 (0.003 - 0.81)0.035
Female17 (85.0)3 (15.0)11
Male21 (84.0)4 (16.0)1.05 (0.090 - 12.228)0.969
Spring17 (94.4)1 (5.6)10.480
Summer18 (78.3)5 (21.7)7.1 (0.43 - 111.15)0.170
Late fall2 (66.7)1 (33.3)20.35 (0.22 - 1861.4)0.191
Early winter1 (100)011
RBAVb15 (88.2)2 (11.8)11
RBDc23 (82.1)5 (17.9)2.96 (0.33 - 26.1)0.328

aValues are expressed as No. (%).

bRodent burrow around village.

cRodent burrow desert.

5. Discussion

Research on rodents as leishmaniasis reservoirs has revealed a diverse array of transmission cycles where epidemic cutaneous disease caused by L. major occurs near colonies of reservoir gerbil rodents in Asia including Khuzestan province on the border of Iran and Iraq (19). Rodents are not always well distinguished in literature; although distinguishing closely-related members of T. indica species is complex, it is important for transmission cycle and epidemiological aspects (13, 26). This is crucial to separate reservoirs that are biomedically important from the rodent species that are competent reservoirs but without reservoir capacity to cause much ZCL disease (7, 24). The ecological associations with infected reservoir hosts or humans, and the descriptive eco-epidemiology could suggest a potential reservoir role. Modeling of transmission cycling associations is required to identify the reservoirs that are a real public health priority (6, 27).

Most ZCL infections are diagnosed clinically and microscopically in patients; but in reservoir hosts, a combination of molecular, biochemical, and serological tests can demonstrate significant numbers of Leishmania infections in endemic areas of ZCL foci (28, 29). The incidence of ZCL associated with the transmission of L. major by rodents has declined in many foci where living standards have been improved (4). Only one report on Leishmania infection in one T. indica has been presented in a very small area of Khuzestan province named Roffaye, although it is not clear how authors confirmed L. major without sequencing and aliment and molecular analyses (19). Rhombomys opimus as the main reservoir host of ZCL has been trapped frequently in many areas of Iran while they were more with Leishmania infections; despite expecting the same situation in the case of T. indica as the second main reservoir host of ZCL in south of Iran, the minority of T. indica were found with Leishmania infection in the conducted investigations (17, 30, 31).

Despite low Leishmania infection in T. indica, the prevalence of human disease and infections is relatively high in some districts of Khuzestan province, so that these districts appear to have a transmission cycle typical of the ZCL, while T. indica were incriminated as the reservoir hosts of L. major (1, 10, 17, 20). Using cross tab, chi-square, and adjusted logistic regression statistical tests, some epidemiological factors such as age, gender, season, and habitat were analyzed to compare any epidemiological factor affecting Leishmania infection in T. indica. No significant factor was found to change the situation of disease; however, small changes in some factors were shown (Table 2). Statistical analyses showed the first age group of T. indica had significantly high Leishmania infection. This may be due to that younger T. indica search for food and show more activities around the rodent borrows and this can increase the chance for biting by sandflies.

Using ITS-rDNA gene and two statistical methods (NJ and ML), a few old world Leishmania species were identified in Iran and elsewhere under similar conditions (22, 32-34).

The number of pairwise differences among sequences was compared with the expected number of segregating sites in Leishmania species and using Tajima’s D index analysis, a negative evolution process was found; also, the number of observed mutations was lower than the number of expecting mutations. Majority of mutations were not informative but unique. The gape in alignments of ITS-rDNA gene increased with the number of different haplotypes while by ignoring or removing the gap, the number of haplotypes decreased (Table 3).

Table 3. The Role of ITS-rDNA Gene Mutations in the Specified Host of Leishmania Species, and Comparison of Nucleotide Diversity and Genetic Variationa
HostParasite Sp.No. SeqNo Nucleotide2S (%)KPer SeqbπcPer SitebTajima’s DSingleton VariableParsimony VariableHHd
With GapWithout GapWith GapWithout Gap
RodentL. major6373 (336)5 (1.488)1.662.180.00490.006-1.3350420.80.33
SandflyL. major6373 (333)15 (4.504)5.26.560.0150.019-1.221506510.93
L. tropica1-------------
HumanL. major4373 (337)0--0----310.830
L. tropica3373 (332)17 (5.120)11.3311.330.0340.03411703311
TotalAll21373 (306)33 (10.784)

Abbreviations: S, segregation of variable nucleotide sites; K, average number of pairwise nucleotide difference between pairs of sequences; H, No. of haplotype; Hd, haplotype diversity.

aNumber of sequence used in this study.

bThe amount of genetic variation.

cNucleotide diversity, Tajima’s D: the D test statistic proposed by Tajima, (35).

Using the maximum likelihood method based on the Kimura 2-parameter model, a tree with the highest log likelihood (-838.0810) was constructed. Initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2030)). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 21 nucleotide sequences. There were a total of 373 positions in the final dataset (Figure 2). Using the neighbor-joining method, the optimal tree with the sum of branch length of 0.13416000 was drawn. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). The analysis involved 21 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 306 positions in the final dataset (Table 3, Figures 2 and 3). Phylogenetic trees for L. major are supported on their specific clades and L. tropica on own clades.

The current report established that T. indica is not the only reservoir host of ZCL circulating in Khuzestan province. Our investigation raises the possibility that the role of some rodents or other mammals in the incidence of ZCL might be due to some changes in the transmission rate of L. major. Phylogenetic analysis of ITS-rDNA gene is recommended for firm identification and separation of Leishmania species.

1 Spotin A, Rouhani S, Ghaemmaghami P, Haghighi A. , Zolfaghari MR, Amirkhani A. Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran. Iran Biomed J. 2015; 19(3): 149-59[ PubMed ]
2 Bordbar A, Parvizi P. High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran. Acta Trop. 2014; 133: 69-72[DOI][ PubMed ]
3 Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases 2010;
4 Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012; 7(5): 35671[DOI][ PubMed ]
5 Najafzadeh N, Sedaghat MM, Sultan SS, Spotin A, Zamani A, Taslimian R, et al. The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran. Rev Soc Bras Med Trop. 2014; 47(5): 599-606[ PubMed ]
6 Gholamrezaei M, Mohebali M, Hanafi-Bojd AA, Sedaghat MM, Shirzadi M. Ecological Niche Modeling of main reservoir hosts of zoonotic cutaneous leishmaniasis in Iran. Acta Trop. 2016; 160: 44-52[DOI]
7 Bates PA, Depaquit J, Galati EA, Kamhawi S, Maroli M, McDowell MA, et al. Recent advances in phlebotomine sand fly research related to leishmaniasis control. Parasit Vectors. 2015; 8: 131[DOI][ PubMed ]
8 Seyedi-Rashti MA, Nadim A. Epidemiology of cutaneous leishmaniasis in Iran. B. Khorassan area. I. The reservoirs. Bull Soc Pathol Exot Filiales. 1967; 60(6): 510-4[ PubMed ]
9 Yaghoobi-Ershadi MR, Akhavan AA, Mohebali M. Meriones libycus and Rhombomys opimus (Rodentia: Gerbillidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in Iran. Trans R Soc Trop Med Hyg. 1996; 90(5): 503-4
10 Mohebali M, Javadian E, Yaghoobi-Ershadi MR, Akhavan AA, Hajjaran H, Abaei MR. Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran. East Mediterr Health J. 2004; 10(4-5): 591-9[ PubMed ]
11 Mehrabani D, Motazedian MH, Oryan A, Asgari Q, Hatam GR, Karamian M. A search for the rodent hosts of Leishmania major in the Larestan region of southern Iran: demonstration of the parasite in Tatera indica and Gerbillus sp., by microscopy, culture and PCR. Ann Trop Med Parasitol. 2007; 101(4): 315-22[DOI][ PubMed ]
12 Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS-rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 2008; 13(9): 1159-71[DOI]
13 Parvizi P, Moradi G, Akbari G, Farahmand M, Ready PD, Piazak N, et al. PCR detection and sequencing of parasite ITS-rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central Iran. Parasitol Res. 2008; 103(6): 1273-8[DOI][ PubMed ]
14 Pourmohammadi B, Motazedian MH, Kalantari M. Rodent infection with Leishmania in a new focus of human cutaneous leishmaniasis, in northern Iran. Ann Trop Med Parasitol. 2008; 102(2): 127-33[DOI][ PubMed ]
15 Hajjaran H, Mohebali M, Alimoradi S, Abaei MR, Edrissian GH. Isolation and characterization of pathogenic Leishmania turanica from Nesokia indica (Rodentia, Muridae) by PCR-RFLP and ITS1 sequencing in Iran. Trans R Soc Trop Med Hyg. 2009; 103(11): 1177-9[DOI][ PubMed ]
16 Motazedian MH, Parhizkari M, Mehrabani D, Hatam G, Asgari Q. First detection of Leishmania major in Rattus norvegicus from Fars Province, Southern Iran. Vector Borne Zoonotic Dis. 2010; 10(10): 969-75[DOI]
17 Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M. 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): 375-80[DOI]
18 Azizi K, Moemenbellah-Fard MD, Kalantari M, Fakoorziba MR. Molecular detection of Leishmania major kDNA from wild rodents in a new focus of zoonotic cutaneous leishmaniasis in an Oriental region of Iran. Vector Borne Zoonotic Dis. 2012; 12(10): 844-50[DOI]
19 Vazirianzadeh B, Saki J, Jahanifard E, Zarean M, Amraee K, Pour SN. Isolation and Identification of Leishmania Species From Sandflies and Rodents Collected From Roffaye District, Khuzestan Province, Southwest of Iran. Jundishapur J Microb. 2013; 6(6): 10025[DOI]
20 Rouhani S, Mirzaei A, Spotin A, Parvizi P. Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran. J Infect Public Health. 2014; 7(3): 210-7[DOI]
21 Mohammadi S, Parvizi P. Simultaneous Morphological and Molecular Characterization of Tatera indica in Southwestern Iran. J Arthropod Borne Dis. 2016; 10(1): 55-64[ PubMed ]
22 Spotin A, Rouhani S, Parvizi P. The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan Province, Iran. Biomed Res Int. 2014; : 913510-3
23 Cupolillo E, Grimaldi Junior 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(1-2): 145-55[DOI][ PubMed ]
24 Ready PD. Leishmaniasis emergence and climate change. Rev Sci Tech. 2008; 27(2): 399-412[DOI][ PubMed ]
25 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12): 2725-9[DOI][ PubMed ]
26 Mirshamsi O, Darvish J, Kayvanfar N. A preliminary study on Indian Gerbils, Tatera indica Hardwicke, 1807 at population level in eastern and southern parts of Iran (Rodentia: Muridae). Iran J Animal Biosystematics. 2007; 3(1): 49-61
27 Ebrahimi S, Bordbar A, Rastaghi AR, Parvizi P. Spatial distribution of sand fly species (Psychodidae: Phlebtominae), ecological niche, and climatic regionalization in zoonotic foci of cutaneous leishmaniasis, southwest of Iran. J Vector Ecol. 2016; 41(1): 103-9[DOI]
28 Mehrabani D, Motazedian MH, Hatam GR, Asgari Q,, Owji SM, Oryan A. Leishmania Major in Tatera indica in Fasa, Southern Iran, microscopy, culture, isoenzyme, PCR and morphologic study. Asian J Anim Vet Adv. 2011; 6(3): 255-64[DOI]
29 Srivastava P, Gidwani K, Picado A. , Van der Auwera G, Tiwary P, Ostyn B. Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India. Trop Med Int Health. 2013; 18(5): 548-54[DOI]
30 Akhavan AA, Yaghoobi-Ershadi MR, Hasibi F, Jafari R, Abdoli H, Arandian MH. Emergence of cutaneous leishmaniasis due to Leishmania major in a new focus of southern Iran. Iran J Arthropod Borne Dis. 2007; 1(1): 1-8
31 Asgari Q, Motazedian MH, Mehrabani D, Oryan A, Hatam GR, Owji SM. Zoonotic cutaneous leishmaniasis in Shiraz, Southern Iran: A molecular, isoenzyme and morphologic approach. J Res Med Sci. 2007; 12(1): 7-15
32 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(2): 238-45[DOI][ PubMed ]
33 Maraghi S, Mardanshah O, Rafiei A, Samarbafzadeh A, Vazirianzadeh B. Identification of cutaneous leishmaniasis agents in four geographical regions of Khuzestan province using Nested PCR. Jundishapur J Microb. 2013; 6(4)[DOI]
34 Sharbatkhori M, Spotin A, Taherkhani H, Roshanghalb M, Parvizi P. Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis. 2014; 51(1): 16-21[ PubMed ]
35 Tajima F. The amount of DNA polymorphism maintained in a finite population when the neutral mutation rate varies among sites. Genetics. 1996; 143(3): 1457-65