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

Introduction: Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species. Methods: All rodents captured in 2011 and 2012 from Abarkouh district were identifi ed based on morphological characteristics and by amplifi cation of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplifi ed by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confi rm the Leishmania identifi cation. Results: Of 68 captured rodents in the region, 55 Rhombomys opimus were identifi ed and nine Leishmania infections (9/55) were found. In addition, eight Meriones libycus and two Tatera indica were sampled, and one of each was confi rmed to be infected. Two Meriones persicus and one Mus musculus were sampled with no infection. Conclusions: The results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major (GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Leishmaniasis is one of the nine emerging individual infectious diseases that have been largely neglected around the world and in the Middle East 1 .
In Iran, leishmaniasis is observed in three clinical forms: zoonotic cutaneous leishmaniasis (ZCL), anthroponotic cutaneous leishmaniasis (ACL) and zoonotic visceral leishmaniasis (ZVL).ZCL caused by Leishmania major has a health as well as socioeconomic impacts in Iran.ZCL has been reported in rural regions of Iran in 15 of 31 Provinces,including: Bushehr, Hormozgan and Fars in the south 2,3 ; Ilam and Khuzestan in the southwest and west 4,5 ; Golestan, Khorasan and Semnan in the northeast and north [4][5][6] ; and Isfahan in the central region 7 .
During the life cycle of ZCL, which depends on the geographical location of the disease, sandfl ies act as vectors of Leishmania, Bartonella bacilliformis and some arboviruses, and wild rodents are considered to be the reservoir hosts 8 .Many investigations have been conducted on different aspects of ZCL in naturally important foci in Iran [9][10][11] , although some areas of neighboring Provinces have been neglected for unknown reasons .Predisposing factors, such as increasing migration of patients from endemic foci to potential areas, irregular construction and urbanization and changing sandfl y fauna in the region affect the distribution and survival of ZCL 9,12 .
Yazd Province in central Iran is one of these regions, and the number of cases of ZCL has been increasing since 1981 10 .The offi cial reports from the health center in Yazd Province has demonstrated that the number of cases of CL in the Ardakan area (which is an important potential focus in southwestern Yazd) increased from 1996 to 1997 (total: 372 cases), which

Major Article
Najafzadeh N et al. -L.major in reservoirs of a ZCL focus, Iran. in the neighboring Provinces of Isfahan and Sh iraz 14,15 .The geographical distribution of reservoir hosts of ZCL in different regions of Iran is shown in Figure 1 11 .

Study area, sampling and laboratory methods
This cross-sectional/descriptive study was performed in 2011 and 2012, and rodent samples were obtained from 5 villages across Abarkouh district, Yazd Province, central Iran, including: Abarghasr, Haroni, Khorram abad, Gonabad and Chahgir.
Abarkouh district is situated between Fars (southern Iran) and Isfahan (central Iran) Provinces (Figure 1).These Provinces are considered hyper-endemic regions that are important sites for ZCL 7 .Abarkouh district, with an altitude of 1,510 meters above sea level (a.s.l.) (4,954 feet), geographic coordinates of "31°07′44″N 53°16′57″E31.13°N53.28°E" (Figure 1) and a population of approximately 21,818 people, is located in Yazd Province in central Iran.Due to its hot and dry climate and its proximity to Isfahan and Fars Provinces, Abarkouh is considered a new and emerging focus of ZCL as well.
The rodents were sampled in Abarkouh area 120km southwest of Yazd Province, using wooden and wire traps.To identify active colonies of rodents within a diameter of 1-1.5km around villages in Abarkouh, approximately 30-40 live traps were used, and the rodents were captured monthly by baiting with dates and cucumbers.The genus and species of each rodent were determined based on external features, including: ears, color, tail, body measurements, teeth, feet and cranium 15,24,25 .
Each protocol and method applied in this survey was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Human and Animal Research Ethics Committee of the Pasteur Institute of Iran.contains nearly 10,000 small circular DNAs (kDNA minicircle).This minicircle comprises a variable region (600bp) and a conserved region (120bp).Microsatellite markers in Leishmania parasites are co-dominant and allelic and combine 1-7 nucleotide units into short, tandemly repeated DNA sequences 19,20 .Currently, multilocus microsatellite typing (MLMT) is being used widely in population genetic studies in different species of Leishmania parasites 21,22 .Minicircle kDNA and microsatellite ITS-rDNA are also well known as molecular markers for the detection of Leishmania infections 7,21 .
The Cyt b gene encodes the central catalytic subunit of an enzyme present in the respiratory chain of mitochondria and exists in almost all organisms.This gene has been broadly used for phylogenetic studies and identifi cation of animals and plants 23 .The Cyt b gene of the genus Leishmania consists of two regions: the edited region (the most 5′ region of 23bp), which undergoes ribonucleic acid (RNA) editing, and the non-edited region (the 3′ region of 1,056bp) 23 .
Since the rodent fauna, the Leishmania species and their infection rate in Abarkouh district of Yazd Province, Iran, haves not been elucidated completely; we have designed this study in order to investigate these aspects of ZCL.
Two impression touch slides were obtained from both ears of each rodent by scratching.For brief microscopic observation, rodent samples were collected from the ears after removing the hair and making small scratches from which to extract serous fl uid, which was then fi xed on a microscopic slide with methanol and stained for 30min with Giemsa diluted 1:10.The slides were then observed under a light microscope to detect the presence of Leishman bodies.
Furthermore, serous fl uid from the rodents' ears was injected into Balb/C mice to monitor for the appearance of Leishmania infection lesions.Prepared serous fl uid from infected Balb/C mice accompanied by serous fl uid from scratches from each ear of rodents was cultured in Novy-MacNeal-Nicolle (NNN) medium.Subsequently, the cultures were incubated at 22°C for 6 weeks.The cultures were checked at two-day intervals until they reached the growth phase (log phase) based on observation using an inverted microscope.Positive cultures were confi rmed by the presence of promastigotes, which were sub-cultured into restriction fragment length polymorphism (RFLP) medium weekly.
The harvested promastigotes from the early stationary phase (approximately 2×10 6 promastigotes/ml) and serous fl uid from each ear of the rodent were injected subcutaneously into the base tail of a Balb/C mouse.Inoculated Balb/C mice were examined weekly for the appearance of lesions at the injection site for 6 months.Samples from infected Balb/C mice with cutaneous lesions were used for DNA extraction.

Molecular methods
Smears prepared from infected Balb/C mice, with serous fl uid and/or cuts from each rodent ear, were kept in separate 1.5ml microtubes containing 100μl phosphate-buffered saline (PBS) and then centrifuged briefl y three times at 13,000rpm.The PBS was discarded.Each rodent ear was placed in a 1.5ml microtube and placed in liquid nitrogen 3 times for 3min each.The genomic DNA of each rodent and any parasite within was extracted using the ISH-Horovize method and a GeNet BIO kit (Global Gene Network South Korea); these procedures were carried out in the systematic molecular laboratory of the Pasteur Institute in a room where amplifi ed and cloned DNAs were never processed 11,25 .
The DNA samples extracted from rodent tissues were used in polymerase chain reaction (PCR) to amplify a 624bp fragment of the cyto chrome b gene (Cyt b) from the mitochondrial DNA to accurately identify the rodent species.We followed the protocol of Kent and Norris (Table 1) 26 .
The internal transcribed spacer-ribosomal deoxyribonucleic acid gene was amplified for the detection of Leishmania infection using ITS1-5.8SrRNA-ITS2fragments, with ITS1F as the forward primer and ITS2R4 as the reverse primer (Table 1) 7 .
To perform RFLP analysis, the PCR products were blunt digested using endonuclease BsuR1 (HaeIII) (Fermentas, Life Sciences, Germany) in the recognition site pattern GG↓CC, as recommended by the manufacturer.Enzyme selection was performed by analyzing sequences of Leishmania reference species with CLC DNA Workbench 5.2 software (CLC bio A/s, Aarhus, Denmark) 27 .The primer sets LINR4 (forward), LIN17 (fi rst-step reverse) and LIN19 (second-step reverse) were used in the semi-nested PCR for the minicircle kDNA gene 16 .The primers anneal within the conserved area of the minicircle and are based on the conserved sequence blocks recognized by Brewster and Baker (Table 1) 28 .
The third method used for Leishmania infection identifi cation was microsatellite ITS-rDNA analysis; the protocol used in this assay was designed by Parvizi et al. 25 .The primers were ITSMF1 (forward) and ITSMR2 (reverse) (Table 1).
To detect Leishmania infection, we also used a fragment of the cytochrome b gene from mitochondrial DNA, and the primers used in this amplifi cation were LCBF1 (forward) and LCBR2 (reverse) (Table 1) 17 .
After amplification, the DNA samples were excised, purifi ed and sequenced using an ABI PRISM TM310 automated sequencer (Applied Biosystems, USA).The sequences obtained were edited and aligned with database sequences using Sequencher TM v. 4.4 software to identify unique sequences (= haplotypes), which were analyzed phylogenetically using MEGA5.05software 29,30 .

Ethical considerations
This study was approved by the D epartment of Parasitology, Pasteur Institute of Iran, Tehran, Iran.

RESULTS
In total, 68 rodents were captured in five villages in Abarkouh district (Figure 1; Table 2).Thirty-three of 68 rodents were alive and transported to the Pasteur Institute of Iran, Tehran, for further studies using conventional and molecular methods.Thirty-fi ve rodents were dead after being caught by wire and wooden live traps at sampling sites.The ears of these dead rodents were used only for molecular methods.Based on morphological characteristics and rodent molecular markers (Cyt b sequences), fi ve species were identifi ed.The most abundant rodent was R. opimus (55/68: 81%).The frequencies and abundances of the other rodent species were as follows: M. libycus (8/68: 12%), Meriones persicus (2/68: 3%), Tatera indica (2/68: 3%) and Mus musculus (1/68: 1%), respectively (Table 2).
The most interesting result was that despite the low number of captured rodents, fi ve different rodent species were collected and identifi ed.Leishmania infection was detected from three of these species, and for the fi rst time, T. indica was captured in Abarkouh district and identifi ed both morphologically through diagnostic keys and molecularly by sequencing of the Cyt b gene.In addition, one of the two T. indica specimens was infected with L. major.
To fi nd identify additional Leishmania parasite infections and molecular variation rates among collected samples, different genes were employed.Standard and semi-nested PCR were used to amplify ITS-rDNA, microsatellites, kDNA and Cyt b genes from Leishmania parasites (Figure 2).All 11 Leishmania-positive samples were analyzed using RFLP and sequencing to defi nitively identify Leishmania species (Figure 2).With RFLP, which allows for the differentiation of each species unambiguously, two fragments of 120 and 310bp belonging to L. major were obtained (Figure 2).
All sequences from positive samples by ITS-rDNA gene were blasted and confi rmed to be most similar to L. major, and only L. major with one common haplotype (GenBank accession No. EF413075) was found after direct sequencing, editing, aligning and comparing with the sequences submitted to GenBank using Sequencher TM v. 4.4 and phylogenetic analysis by MEGA5.05 software.

DISCUSSION
In our study, only the L. major parasite with one common haplotype (GenBank access No. EF413075) was fi rmly identifi ed in three rodent species.Leishmania parasites have been isolated from all three species in other ZCL foci in Iran 11,15,25,31 .This is the fi rst report of L. major in only a small area of ZCL focus in Abarkouh.In our current publication, we also found L. major www.scielo.br/rsbmtI www.rsbmt.org.br in P. papatasi, a proven vector of ZCL in Iran, in the same area in Abarkouh where L. major was isolated in rodents 7,10,32 .
Finding additional Leishmania infections in different rodent species compared with only one sandfl y species can be explained by the fact that among sandfl ies, we mainly examined P. papatasi, and only a small number of other sandfl y species were tested and found to be Leishmania negative, which did not provide suffi cient for a precise result 10 .In addition, only P. papatasi is able to develop L. major in its midgut and transfer the parasite to salivary glands to cause ZCL 33 .However, we analyzed all the captured rodent samples from different species, and therefore, we were able to identify Leishmania infections in at least three rodent species.The Leishmania infection rate in rodents as the reservoir host of ZCL is much higher than in sandfl ies as vectors, and in some cases, more than 50% of the samples were found to be infected with Leishmania parasites 14 .
Based on our experience in different ZCL foci in Iran, we expected to identify more Leishmania infections in reservoir hosts in Abarkouh district and to observe at least a small amount of variation in the ITS-rDNA gene of L. major in rodents.However, only one haplotype was found, and approximately 16% (11/68) of the tested rodents were infected with Leishmania parasites 11,15,25 .After sequencing, only one haplotype of L. major, which is also the common haplotype present in Iran, including Fars and Isfahan Provinces, was detected from Abarkouh rodent samples (GenBank accession No. EF413075).
The low density of sampled rodents as well as Leishmania parasites may be due to a control program of the health care authorities of Abarkouh district that uses zinc phosphate poison and the destruction of rodents' barrowers to control ZCL.
According to reports of different ZCL foci in Iran, many haplotypes of L. major have been identifi ed in sandfl ies, rodents and humans 7,11,15,27 .The objective of the present study was to use molecular methods and different genes to identify additional Leishmania infections and various haplotypes; to this end, four different genes were employed to detect Leishmania infections in rodents and/or the numbers of haplotypes circulating in the area, but this method relies on a few sequences from all of the genes from our samples, and no variations were identifi ed.We also used routine laboratory (conventional) methods, such as NNN cultures, microscopic observation and Balb/C mouse injection.Because most captured rodents died before being transferred to our lab, only a few live rodents were used for the conventional methods, and the infection rate was low.Only 2 infected samples were found by microscopic observation of the presence of amastigotes on slides and the appearance of a lesion after Balb/C mouse injection.Because the NNN cultures were prepared in the fi eld and due to fungal infection in some cultures, no growth was shown in any of the cultures.
We employed fi ve different genes during this investigation; the rodent Cyt b gene was amplifi ed for accurate determination of the rodent's genus and species.For Leishmania infection, two mitochondrial genes (kDNA and Leishmania Cyt b) and two nuclear genes (ITS-rDNA and microsatellite ITS-rDNA) were used.In this investigation, the highest infection rate among rodents (8/68) was detected using the microsatellite ITS-rDNA gene because of its short tandemly repeated DNA sequence fragments and because it is highly specifi c.A comparison of the rest of the genes demonstrated that Cyt b as a mitochondrial gene is more sensitive for Leishmania detection (4/68) because of its high copy numbers per cell; however, nuclear genes are more specifi c, and of those, the ITS-rDNA gene (3/68), because it is homogenous and highly conserved with few intracellular polymorphisms, a linear genome and has readable sequences, is considered a suitable gene for sequencing, genus, species, strain and/or even haplotype detection.
Our Leishmania infection data in rodents are similar to the results of a parallel study among sandfl ies and suspected patients that was carried out near the time of our investigation 10 (Parvizi P et al: unpublished data). .In previous investigations, reservoir hosts of ZCL have been distributed in different regions.R. opimus and M. libycus are dominant in the northeastern and central regions; M. libycus and T. indica in the central and southwestern regions and T. indica in southwestern and southern Iran 11,34 .R. opimus and M. libycus have previously been found to be infected with L. major parasites from Golestan and Isfahan Provinces 11,14,15,25 .In addition, T. indica was found to be infected with L. major in Fars Province, Iran 31 .Interestingly, we were able to identify L. major infections in all three of these rodents within Abarkouh district of Yazd Province in central Iran.
In this survey, T. indica was captured for the fi rst time in Abarkouh district; the existence of this rodent in the area may be explained by the fact that Abarkouh neighbors Fars Province, which is a known habitat for T. indica 31 , and the rodents can be transported and/or migrate to Abarkouh from Fars and vice versa.The simultaneous existence of T. indica along with R. opimus and M. libycus as main and potential reservoir hosts of ZCL in Abarkouh district and the fact that Abarkouh has been largely neglected as an important ZCL focus gives this district an important role in the ZCL life cycle, epidemiology, prognosis and disease-control programs.
Leishmania major was firmly identified in R. opimus, M. libycus and T. indica, which indicates that at least these three rodent species can be incriminated as reservoir hosts of ZCL in this location.R. opimus was abundant and had a greater rate of L. major infection and should be incriminated as the main reservoir host of ZCL 35 .

FIGURE 1 -
FIGURE 1 -Map of Abarkouh showing sampling regions and the geographical distribution of reservoir hosts of ZCL in different regions of Iran.L: Leishmania; ZCL: zoonotic cutaneous leishmaniasis.

FIGURE 2 -
FIGURE 2 -A: Electrophoresis image of Cyt b gene amplifi cation in Leishmania infection among different rodent species of Abarkouh district, Yazd Province, Iran.B: RFLP of ITS-rDNA gene electrophoresis image after digestion with BsuR1 (HaeIII) enzyme of PCR products using In-Silico software (CLC bio A/s, Aarhus, Denmark) of Leishmania infection among different rodent species of Abarkouh district, Yazd Province, Iran (Bordbar and Parvizi 2013) (+Ve contains Leishmania major parasite PCR product without the enzyme effect, and +Ve (enz) is a Leishmania major parasite PCR product with the enzyme effect).Cyt b: cytochrome b; RFLP: Restriction fragment length polymorphism; ITS-rDNA: internal transcribed spacer-ribosomal deoxyribonucleic acid; PCR: polymerase chain reaction.+ve: positive sample, enz: with enzyme.

TABLE 2 -Leishmania infections among different rodents captured in Abarkouh district in Yazd Province, Iran, using conventional and molecular methods.
ITS-rDNA: internal transcribed spacer-ribosomal deoxyribonucleic acid; kDNA: kinetoplast deoxyribonucleic acid.+ve: Positive sample; PCR: polymerase chain reaction; Cyt b: Cytochorome b. *3 of 13 positive samples were also tested via one additional gene and were confi rmed to have Leishmania major infection; **The positive microscopic and BALB/C injected samples also tested positive in molecular tests (ITS-rDNA, microsatellite and kDNA gene amplifi cation).