Molecular Identification and Polymorphism Determination of Cutaneous and Visceral Leishmaniasis Agents Isolated from Human and Animal Hosts in Iran

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.


Introduction
Leishmaniases are a group of diseases caused by the parasites belonging to the genus Leishmania. The disease affects 98 tropical and Mediterranean countries, and 1.5 to 2 million cases are estimated to occur annually worldwide, with up to 350 million people at risk of infection [1]. Leishmaniases present a wide spectrum of clinical manifestations including cutaneous (CL), diffuse cutaneous (DCL), mucocutaneous (MCL), and potentially fatal visceral leishmaniasis (VL), also known as "Kala-Azar, " that are largely caused by different Leishmania species. In old world, three species caused CL: L. tropica, L. major (both usually cause localized skin lesions), and L. aethiopica that cause nonulcerative disseminated cutaneous lesions. VL is caused by parasites of the L. donovani-L. infantum complex. Recently, L. tropica has been implicated in several human cases with VL manifestations [2]. In Iran, the etiological agents of anthroponotic CL (ACL) and zoonotic CL (ZCL) are known to be L. tropica and L. major, respectively. L. infantum, the principal agent of VL, causes splenomegaly and hepatomegaly [3]. Most of ACL are reported from northeastern and central parts of Iran [4][5][6], while ZCL extends from northeast to center and west of Iran and covers all southern provinces as well [7][8][9]. More than 90% of the visceral cases in Iran are reported from the northwest and some southern provinces including Fars and Bushehr [10]. All the three species of Leishmania could be found in some areas like Fars province in the south [11]. In Iran, the main reservoir hosts for ACL are human infected patients, for ZCL are some of the desert rodents particularly the great gerbils (Rhombomys opimus), and for VL are domestic and wild canines (mainly domestic dogs) [4,[12][13][14][15]. In addition to the classic clinical leishmaniasis manifestations, there is a variety of atypical signs in CL including lupoid, mucosal lesions, and disseminated forms and in VL including symptomatic and asymptomatic forms [16][17][18]. The diagnosis of clinical forms of leishmaniasis is commonly based on visualization of the parasite in Giemsastained smears by microscopy and culture. Despite sensitivity and specificity, such methods fail to identify the infecting Leishmania species [19,20]. Over the last two decades, DNAbased approaches including PCR followed by sequencing or RFLP analysis and species-specific PCR have been widely used for the identification of Leishmania species [20][21][22][23].
Our study aimed to identify different Leishmania species in some foci of Iran and obtain polymorphisms among the species using RAPD-PCR.

Sampling
2.1.1. Cutaneous Leishmaniasis. Lesion aspirates were obtained from 173 suspected CL patients referred either to leishmaniasis laboratory at the School of Public Health, Tehran University of Medical Sciences (TUMS), or district health centers (DHCs) and hospitals in various regions of Iran from 2004 to 2012. Most patients were from northern, northeastern, western, central, and southern areas of the country. Samples were collected by injecting 0.2 mL of sterile saline into the dermis of the internal border of skin lesions with sterile insulin syringes. After suction, the sample was transferred to RPMI-1640 medium culture and some portion was checked for Leishmania infection by microscopy following the preparation of Giemsa-stained smears [22].

Rodents. Sixty rodents including 36
Rhombomys opimus, 5 Meriones libycus, 13 Nesokia indica, 5 Tatera indica, and one Mus musculus were trapped by live baits traps from CL endemic areas in the northeast and west of Iran. None of the animals had any acute cutaneous lesion. For preparation of samples, the external edges of the ear lobes were cut with scissors after washing and disinfection, and then low amounts of the exudates were transferred to culture media [24]. Giemsa-stained smears were prepared from the same samples.

Visceral Leishmaniasis.
Blood samples were taken from 49 suspected VL cases, presented with fever, weakness, and hepatosplenomegaly, at the medical health centers or leishmaniasis laboratory at the School of Public Health (SPH), TUMS. Most patients were from northwestern, western, central, and southern areas of the country. Also, the bone marrow aspirate was taken from iliac of the same patients under local anesthesia by physicians.
Also, blood samples were taken from eighty dogs living in VL endemic area in the northwest and localities in the northeast and center of the country. Of the 80 dogs, 59 presented one or several of the typical clinical signs (such as lymphadenopathy, hair loss, dermal lesions, onychogryposis, and cachexia) and 56 had showed anti-L. infantum antibodies. All of the 59 dogs with typical clinical signs were sacrificed after obtaining the consent of their owners, and Giemsa stained-smears were prepared from liver and spleen tissues.
Peripheral blood samples were collected into tubes with sodium citrate anticoagulant 4% (Merck, Germany) for PCR testing and into tubes without anticoagulant for DAT. All samples used for serology and PCR were stored at −20 ∘ C until use.
All experiments on the humans and animals were performed according to the guidelines of the Ethical Board of Tehran University of Medical Sciences, Iran.

Parasite Culture and Cryopreservation.
All the specimens including exudates from human skin lesions and rodents ears, aspirates from bone marrow of human patients, and liver and spleen of dogs were cultured in RPMI-1640 medium (Gibco, Life technologies GmbH, Frankfurt, Germany) supplemented with 10-15% fetal bovine serum (Gibco, Germany), 100 U/mL penicillin, and 100 ug/mL streptomycin (Gibco, Germany) and incubated at 24-25 ∘ C. Five days later the last subcultured parasites were harvested, washed in sterile phosphate buffered saline (PBS, pH: 7.2-7.4), and kept in −20 ∘ until used. All the cultures specimens were preserved in liquid nitrogen for further possible use.

Preparation of DAT Antigen and Performance of the Test.
L. infantum (MCAN/IR/07/Moheb-gh; GenBank accession number FJ555210) was cultured in RPMI1640. The parasites were harvested at early stage of stationary phase (about 120 h later), washed with PBS (pH = 7.2), and subjected to trypsin digestion. The parasites were stained with Coomassie brilliant blue R-250 dye (Sigma, USA), fixed with formaldehyde (Merck, Germany) 1.2%, and used for DAT on humans, and dogs' sera. DAT was performed to determine the presence of the anti-Leishmania antibodies [14,25,26]. The titers ≥ 1 : 3200 and ≥320 were considered positive for humans and dogs, respectively [10].
2.1.6. DNA Extraction, PCR, and RFLP. DNA was extracted from cultured parasites from human skin lesions, rodents' ears exudates, and whole blood samples of suspected VL patients and canine visceral leishmanissis dogs by using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Partial ITS1 of ribosomal RNA (ITSI-rRNA) gene of the parasites was amplified using the primers LITSR (5 -CTGGATCATTTTCCGATG-3 ) and L5.8S (5 -TGATACCACTTATCGCACTT-3 ) and PCR conditions outlined by others [19]. Amounts of 10 L of amplicons were digested with Fast digestion HAEIII (BsuRI) enzyme (Fermentas GmbH, Thermo Scientific, Germany) according to the manufacturer's instructions, and the fragments were visualized on 3% agarose gels [22]. Leishmania species were identified based on obtained patterns alongside reference species including L. tropica (GenBank accession number EF653267), L. major (GenBank accession number JN860745), L. turanica (GenBank accession number EU395712), and L. infantum (GenBank accession number EU810776).

Variation
Analyses by RAPD-PCR. RAPD-PCR was performed for 46 DNA extracted from cutaneous and visceral cultured parasites samples with the primer AB1-O7:5 -GGTGACGCAG-3 and PCR conditions outlined by others [4,27]. This primer was chosen due to its reproducibility and its ability to reveal inter-and intraspecies variation.
Variations among isolates of different species were calculated based on profile of bands produced by AB1-O7 primer ( Figure 1). A matrix for each of the isolates was constructed with the numbers "1" and "0" corresponding to the presence and absence of each possible band. For determining the polymorphism rate among different Leishmania species, we identified the bands that were common in less than 50% of the isolates (polymorphic bands) using the following equations, where was calculated by (1) for each polymorphic band.
-test was used for estimating the relative risk between two independent groups [28,29]. If the obtained value for in a band is less than <1.64 with a confidence interval of more than 95% ( < 0.05), then it will be included as a polymorphic band in the matrix. After determining the polymorphic bands, polymorphism rate was calculated via (2), where is the number of isolates which contain a polymorphic band, is the total number of isolates in each group, and is the number of total bands in each isolate Polymorphism rate = × × 100.
2.1.8. Nucleotide Sequence and Phylogenic Analyses. Partial sequences of ITS1 gene from 20 cutaneous and visceral Leishmania isolates (Table 1) were sequenced using LITSR as the forward primer. The sequences were aligned by ClustalX software, the similarities among them were calculated, and a phylogenic tree was constructed by using Tamura 3parameter option of the maximum likelihood method by MEGA 5 software.  Figure 3). Almost all of the L. tropica isolates originated from northeastern areas, while L. major isolates belonged to the center, west, southern, and southwest of the country (Figure 4).   L. major and 3 L. turanica. The infecting species in N. indica was L. turanica ( Figure 2).  Figure 3).

RAPD-PCR and Polymorphism
Rate. The polymorphism rate among L. tropica isolates was 3.6%. The rate among L. major isolates was 7.3% and among human VL isolates was 2.8%. The highest rate (9.8%) was found among canine VL isolates.  the other three species. The numbers above the branches indicate the percentage of bootstrap samplings. There was no clear grouping among the 20 isolates according to their geographical origin ( Figure 5). Details of the specimens sequenced and submitted to GenBank are shown in Table 1.

Discussion
In this study, we could identify four species of Leishmania including L. major, L. tropica, L. infantum, and L. turanica by ITS1 gene followed by RFLP and sequencing. The molecular results on the Leishmania isolated from human cutaneous lesions are consistent with the epidemiologic studies committed in recent decades [3]. The results showed the major causes of CL in Iran were L. major (67%) and L. tropica (33%). The major foci of ZCL transmission were in the northern (Golestan), northeastern near Turkmenistan's border, central (Esfahan), western (Kermanshah) near the Iraqi border, and southern (Khuzestan) provinces of Iran. The major foci of ACL transmission were in the northeast (Razavi Khorasan province) and center of Iran in the city of Bam (Kerman province) especially after the earthquake in 2003.
L. major was found to be the most prevalent species in R. opimus (the main reservoirs of ZCL) followed by L. turanica as the second agent [13]. L. turanica is incapable of infecting humans, but it causes lesions in laboratory animals [30].
In suspected VL patients, DAT showed presence of anti-L. infantum antibodies in 29 (59.2%) of the human cases. PCR detected Leishmania DNA in blood samples of 28 (57.14%) individuals. RFLP analyses revealed L. infantum as the main causative agent of VL (92.86%) and L. tropica as the secondary agent (7.14%). This finding is in agreement with the results of other studies performed in Iran [10,16]. The main endemic areas of VL were Ardebil province (Meshkin-Shahr district) in the northwest and Fars provinces in the south. HIV-Leishmania coinfection has been reported recently from northeastern Iran [31].
Of the 49 bone marrow aspirates from human VL, 25 (51.02%) showed amastigote forms of Leishmania sp. by microscopy. DAT and PCR were both positive in all the 25 human patients.
In a similar study, PCR and microscopy of bone marrow aspirates were shown to be equally sensitive in patients with microscopically confirmed VL. Moreover, PCR could detect Leishmania DNA in bone marrow aspirates in 66.7% of suspected VL patients, while microscopy of the same material was negative [32].
Serology was more sensitive than PCR in diagnosis of suspected VL dogs. As in humans, L. infantum was the main causing agent of VL in dogs (91.5%) and L. tropica was found in 8.5% of the cases. One of the important findings of this study was molecular detection of L. tropica DNA in a considerable number of humans and dogs with VL.
In a similar study, microscopy detected parasite in liver and spleen of 93.2% of symptomatic dogs, while serology was positive in 94.9% and PCR in 76.6% of cases [17]. PCR seems to be less sensitive than microscopy or serology for the detection of Leishmania in blood of suspected VL dogs [33]. Phylogenic trees derived from the ITS1 sequences support a clear divergence between L. tropica from the other three species, but there was no clear grouping among the isolates according to their geographical origin. In RAPD-PCR assay, we used the primer AB1-07, which had the most consistency rate between different populations of Leishmania strains in different regions.

Conclusions
Characterization of Leishmania isolates collected from different parts of Iran showed that L. tropica with 3.6% polymorphisms was the primary agent in the ACL foci and L. major with 7.3% genetic variations was the predominant agent in the ZCL areas of Iran. Moreover, L. infantum with 2.8% genetic variations in human VL and 9.8% polymorphisms in canine VL was found in VL endemic areas of Iran. The results of this study showed that PCR-RFLP and RAPD-PCR, despite some limitations, are simple and powerful tools for the characterization and determination of Leishmania species polymorphisms.