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Document heading doi: 10.12980/APJTB.4.2014APJTB-2014-0199 襃 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

Laboratory based diagnosis of leishmaniasis in rodents as the reservoir hosts in southern Iran, 2012

Amin Masoumeh1,2, Azizi Kourosh1,2, Kalantari Mohsen3*, Motazedian Mohammad Hossein4, Asgari Qasem4, Moemenbellah-Fard Mohammad Djaefar1,2, Najafi Mohammad Esmaeil5, Dabaghmanesh Tahereh2

1Research Centre for Health Sciences, Shiraz University of Medical Sciences, Shiraz, Iran2Department of Medical Entomology and Vector Control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran3Department of Public Health, Mamasani Paramedical School, Shiraz University of Medical Sciences, Shiraz, Iran4Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran5Shiraz University of Medical Sciences, Shiraz, Iran

Asian Pac J Trop Biomed 2014; 4(Suppl 2): S575-S580

Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.apjtb.com

*Corresponding author: Kalantari Mohsen, Mamasani Paramedical School, Shiraz University of Medical Sciences, Shiraz, Iran Tel: +98 (0) 9177041090 E-mail: kalantari22@yahoo.com Foundation Project: Supported by the Research Council of Shiraz University of Medical Sciences (No. 90-01-42-3034, 02/06/2012).

1. Introduction

Leishmaniasis is a term allocated to a range of infections caused by human pathogens like Leishmania spp. (Kinetoplastida: Trypanosomatidae). It is one of eight important neglected tropical diseases of World Health Organization. About two million people are annually exposed to this disease.

Cutaneous leishmaniasis (CL) is a sand fly-borne skin infection caused by the blood parasite Leishmania[1]. It is still a major public health issue in Iran, where most CL cases are zoonotic with rodents acting as reservoir hosts in many rural areas. Zoonotic cutaneous leishmaniasis (ZCL) is essentially an infection of rodents with natural foci. In Iran, the causative agent of ZCL, Leishmania major (L. major), is often transmitted to humans by the bites of the female sand fly, Phlebotomus papatasi (P. papatasi), mostly from rodent hosts that act as reservoirs for the human infection[2]. ZCL occurs in most rural areas of the arid and semi-arid regions but the northwest provinces of Iran. Definitely, P. papatasi is the main vector and

ARTICLE INFO ABSTRACT

Keywords:LeishmaniaGerbilPCRIran

Objective: To examine the fauna of rodents as zoonotic cutaneous leishmaniasis reservoir hosts in Zarqan County, Fars Province, south of Iran, during 2012.Methods: During 2012, wild rodents from different parts of this region were caught by Sherman traps and checked by the examination of liver and spleen smears, for Leishmania infection, to see which species were acting as reservoir hosts; the slides were then processed to extract DNA for molecular test using PCR assay. Results: From 108 rodent species caught, 63% were male and 37% identified as female. Meriones libycus was the most abundant species caught (80.5%) and 5.7% of them were found to be smear-positive for Leishmania amastigotes. The other species were Rattus rattus (14.8%) and Mus musculus (4.7%), but none of them were found positive. Leishmania infection was observed in male and female samples microscopically. Moreover, molecular results revealed Leishmania major in three male and two female specimens.Conclusions: Based on our knowledge, Meriones libycus is incriminated as the main reservoir hosts of Leishmania major in the rural area of Zarqan.

Article history:Received 21 Apr 2014Received in revised form 22 May 2014Accepted 24 Jun 2014 Available online 2 Jul 2014

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various feral rodents, particularly but not exclusively those in the subfamily Gerbillinae (Rodentia: Muridae), are reservoir hosts. Rodents are the largest order of mammals with a population of more than any other mammal and are the reason of many health and economic losses. One of the major health problems among these animals is their role as reservoir hosts of zoonotic disease and one of the most important diseases is CL[3]. So far, two species of parasites have been identified as human pathogens. Among the four major forms of the diseases caused by different flagellated protozoan species of Leishmania type, two forms of skin CL and visceral leishmaniasis in Iran are endemic. There are two forms of CL caused by Leishmania tropica (L. tropica) (dry or urban) and L. major (wet or rural). In general, CL in recent decades has been expanding its geographic region and is endemic in 15 provinces of the country and there are sporadic cases in other regions as well. The annual registered number of cases in Iran is 20 000-30 000 and its annual incidence is 28 per 100 000 individuals under risk[4]. ZCL has high incidence in arid and semi-arid regions of North Africa, the Middle East and Central Asia, where P. papatasi is the main vector and various species of tailed rodents have been introduced as the main reservoirs. Rhombomys opimus (R. opimus) is the main reservoir in Central Asia and Psammomys obesus is the main reservoir in North Africa. Despite all efforts by health authorities in controlling CL, unfortunately, the spread of the disease in different areas of country is evident in recent years. In the wet or ZCL form, tailed rodents in subfamily of Gerbillinae have been introduced as the main reservoirs in Iran and the world[5]. In the central and north eastern foci of the country and the south of Tehran, the species R. opimus has been reported as proven and primary reservoir and Meriones libycus (M. libycus) species has been reported as the secondary reservoir of the disease. Recently, a group of researchers reported 37.5% infection in the R. opimus species in Kalale, Golestan Province, Iran[6]. In West and South west regions of the country, Tatera indica (T. indica) is the main reservoir and Nesokia indica and M. libycus are introduced as the secondary reservoir of the disease[7]. In the southeast of the country, Meriones hurrianae has been reported as proven and primary reservoir and T. indica is the secondary reservoir. In rural areas of the cities Arsanjan and Neiriz, M. libycus species plays the role of primary and proven reservoir of the disease[8,9]. The same species with 8.4% infection has been reported in Marvdasht, Fars Province, south of Iran[10]. T. indica species infected with L. major has been

reported in Kharameh, Fars Province, south of Iran[11]. A recent report on Leishmania infection in Jahrom region of Fars Province and closest focus to the studied area, found four Marinobacter persicus and two T. indica infected and one infection in each of the two rodents of Mus musculus (M. musculus) and Rattus rattus (R. rattus)[12]. Another report on Leishmania infection in Larestan region of Fars closest focused on the studied area and found four cases of T. indica and two cases of infection in two rodent samples of Gerbillus spp[13]. In addition, recently, Leishmania turanica has been detected from Nesokia indica in Kermanshah Province. Also for the first time, L. major was separated from Gerbillus nanus in Jask county in Hormozgan Province, south of Iran[14,15]. A gerbil is a small mammal of the order Rodentia. Once known simply as “desert rats”, the gerbil subfamily includes about 110 species of African, Indian, and Asian rodents, including sand rats and jirds, all of which are adapted to arid habitats. M. libycus is a species of rodent in the family Muridae. It is found in Afghanistan, Algeria, China, Egypt, Iran, Iraq, Jordan, Kazakhstan, Libya, Mauritania, Morocco, Saudi Arabia, Syria, Tunisia, Turkmenistan, and Uzbekistan. M. libycus occupies desert habitats, generally in areas with stabilized dunes (desert and semi-desert habitats). It is sometimes found in arable land. It is a highly mobile species, frequently changing burrows or even migrating should forage conditions deteriorate. In recent years, in Zarqan County, the number of cases of ZCL has been increased. Considering that in the control of ZCL wild animals are involved, the control of this disease is complicated. So the only justified control method is the identification of rodents and determination of ZCL reservoirs around public settlements within a radius of 500 meters based on the flight range of vectors. Considering that some 1 300 cases of ZCL were recorded in 2012 in Zarqan district, Fars Province, Iran, this research was conducted. The present study was designed to examine the fauna of rodents as ZCL reservoir hosts in Zarqan county, Fars Province, south of Iran, during 2012.

2. Materials and methods

2.1. Study area

Fars Province placed in south of Iran covers an area of about 122 400 km2. Zarqan county is located 25 km to the northeast of Shiraz, the capital city of Fars Province (29°46′

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N, 52°43′ E) at an altitude of 1 600 m above sea level (Figure 1).

IRAN

FARS

FARS

Figure 1. A map of Iran, showing the location of the Zarqan County, Fars Province, South of Iran, 2012.

2.2. Sample collection and rodent identification

In a descriptive cross-sectional study, colonies of gerbil rodents were located on the periphery (0.5-1.5 km away) of the selected villages. Sample collections were performed from March to January 2012. All colonies of wild gerbils were checked to find the most active burrows characterized by the external activity marks (fresh food remains, stool, footprints and loose soil on burrow pores). Rodents were captured once every month using 20 standard Sherman live traps (30 cm伊15 cm伊15 cm wire mesh cage traps) placed next to burrow holes, and baited with a mixture of dates, cucumbers or millet seeds. The traps were set after sunset and checked for gerbils early every morning. Each trap set on one night was considered a ‘trap-night’ and there were 20 ‘trap-nights’ per month. The studied areas were Lapooee, Band-Amir, Rahmat-Abad, Shahriyar, Dormanezar and Shool. All samples of rodent were reviewed microscopically to detect Leishmania infection as well as to determine DNA of Leishmania parasite in their liver and spleen. After anaesthesia of the caught rodents with chloroform, several slides were prepared from the ear pinnae, tail base, liver, spleen and any patent skin lesions of every gerbil, air-dried, Giemsa-stained and examined under a compound light microscope for detection of Leishmania amastigotes[16]. The slides were then processed to extract DNA and tested molecularly by PCR method. Rodents were subsequently sent to the Natural History and Technology Museum of Shiraz University to determine their identity using diagnostic keys (Figure 2).

Figure 2. Rodent net, Zarqan County, Fars Province, South of Iran, 2012.

2.3. DNA extraction

DNA was extracted from the slides containing parasites and liver and spleen samples of rodents following the method described elsewhere[14,15]. The surface of slides were scatched by a scalpel and the materials resulting from slides were added into an sterile Eppendorf tube containing 200 µL of lysis buffer [50 mmol/L Tris-HCl (pH=7.6), 1 mol/L ethylene diamine tetraacetic acid, 1% (v/v) Tween-20] containing 8.5 µL proteinase solution K (19 µL/mL) in tubes of 1.5 mL. Each tube was incubated for two hours at 56 °C and then 200 µL of mixture of phenol: chloroform: isoamyl alcohol (25:24:1) was added to it. After intense shaking, the tube was centrifuged for 10 min at 6 000 r/min. Then, two to three times the volume of the solution containing DNA, cold ethanol 100% (absolute) was added, slowly mixed several times and put in freezer at -20 °C for 2 h. Then, it was centrifuged at 10 000 r/min for 6 minutes. Then, proportional to the amount DNA (in this study 50 λ) double-distilled sterile water was added to the tubes and they were kept in the refrigerator at 4 °C until PCR performance. Extraction of DNA from liver and spleen samples of reservoirs was carried out by cutting a small piece of these organs which was put in sterile 1.5 mL Eppendorf tube and was well crushed with a sterile pastor pipette to be homogenized. Then, adding lysis buffer and proteinase K, other steps were performed as the described method.

2.4. Nested PCR assay

The specific and sensitive nested-PCR was used to

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amplify the variable area of the DNA of Leishmania spp. in the rodent liver, spleen, ear and footpad as previously described[17]. The primer sequences were as follow; CSB1XR (reverse for 1st round): (5’-ATT TTT CGC GAT TTT CGC AGA ACG-3), CSB2XF (forward for 1st round): (5’-CGAGTAGCAGAAACTCCCGTTCA-3’). 13Z (forward for 2nd round): (5’-ACT GGG GGT TGG TGT AAA ATA G-3’), LIR (reverse for 2nd round): (5’-TCGCAGAACGCCCCT-3’). First stage product (2 µL) with two primers CSB1XR and CSB2XF, with ratio 9:1 was diluted with deuterium depleted water and was used as template for the second stage. The required amount of materials and thermal profiles in the second stage was like the first one.

2.5. Electrophoresis

For detection of PCR products, 5 µL of the product of the second stage on agarose gel 1.5%, mixed with ethidium bromide, was put in the electrophoresis and the gel was transferred to the device UV transilluminator after 45 min and the resulting bands were examined and photographed. It was interpreted comparing with bands obtained from reference strains and the molecular weight marker. The bands resulting from these two methods for standard species of Leishmania in Iran were 750, 680 and 560 bp for L. tropica, Leishmanria infantum, and L. major, respectively. Deionized distilled water was used as negative control, while standard strains of Leishmania species in Iran, including Leishmanria infantum: MCAN/IR/96/Lon 49, L. tropica: MHOM/IR/89/ARD 2 and L. major: MHOM/IR/54/LV 39 were used as positive controls[7].

3. Results

During the study period, a total of 108 individual rodent species were caught, 63% of which were male and 37% female (Table 1). Three different rodent species were caught and morphologically identified. Libyan jird (M. libycus) was the most abundant species caught (n=87, 80.5%) and 5.7% of them were found smear-positive for Leishmania amastigote. The other species were R. rattus (n=16, 14.8%) and M. musculus (n=5, 4.7%), but none of them were found to be positive for Leishmania parasites. It is worth noting that in this study, rodents catch was simply performed from outside and marginalized rural areas. Thus only a few M. musculus were caught. The number and percentage of rodents and the prevalence of Leishmania infection in them is shown in Tables 1 and 2. Regarding the collected rodent samples, they are shown in Figure 3. Leishmania infection was observed in male and female rodent samples microscopically (Figure 4). The result of electrophoresis

of PCR products of M. libycus rodent’s liver and spleen specimens on agarose gel 1.5%, stained with ethidium bromide is shown (Figure 5). Rodents from the village of Lapooee showed the highest positivity of infection.Table 1 Number and percentage of rodents in rural area, Zarqan County, Fars Province, 2012.

SpeciesStudied villages No. of all of species [n (%)]

LapooeeBande-

amirRahmatabad Shahriyar Dormanezar Shool Male Female Total

M. libycus 53 7 3 15 4 5 58 (66.7) 29 (33.3) 87 (80.5)

R. rattus 5 1 3 2 3 2 7 (43.8) 9 (56.2) 16 (14.8)

M. musculus 0 0 1 3 1 0 3 (60.0) 2 (40.0) 5 (4.7)

All species 58 8 7 20 8 7 68 (63.0) 40 (37.0) 108 (100.0)

Table 2The prevalence of Leishmania infection in the rodents, as revealed by microscopy.Species Tested

numberSmear positive % of infected

casesMale Female TotalM. libycus 87 3 2 5 5.7R. rattus 16 0 0 0 0.0M. musculus 5 0 0 0 0.0All species 108 3 2 0 4.6

Figure 3. Collected samples.a: Dental formulation in sample collected from M. libycus; b: Triangular bone of ears in sample collected from M. libycus; c: Dental formulation in sample collected from R. rattus; d: Dental formulation in sample collected from M. musculus, Zarqan County, Fars Province, South of Iran, 2012.

Figure 4. Leishmania amastigotes in collected samples of the liver tissue from M. libycus, Zarqan County, Fars Province, South of Iran, 2012.

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Figure 5. The result of electrophoresis of PCR products of M. libycus rodent’s liver and spleen specimens on agarose gel 1.5%, stained with ethidium bromide. Lane 1: Size marker; Lane 2: L. major standard strain; Lane 3: L. tropica standard strain; Lane 4: Negative control; Lanes 5 and 6: M. libycus liver and spleen specimens.

4. Discussion

Outbreaks of human CL impose particularly serious burden of morbidity on people in rural areas of Iran. In this study, 108 rodents were caught among which M. libycus was the dominant species (80.5%) in which Leishmania infections were observed and 5.7% of them were found to be smear-positive for Leishmania amastigote. In this descriptive cross-sectional study, broom-tailed rodents’ fauna and reservoirs of ZCL in Zarqan County were studied using compound microscopes. In recent years, the incidence of CL has increased in Zarqan. The other captured species were R. rattus and M. musculus, but none of them were found to be positive. Sampling was conducted mainly in the border villages. Only a few M. musculus were caught. M. musculus has been found naturally infected with L. major in Fars Province, south of Iran[6,11]. Multiple numbers of host species are potentially involved in the transmission of Leishmania parasites, making its epidemiology highly complex[18]. A major challenge on studying the epidemiology of the Leishmania species that cause zoonotic disease in humans is identification of the

mammalian hosts that act as reservoirs of the involved pathogens. It is often difficult to collect large numbers of each putative wild rodent reservoir host, or to successfully detect Leishmania infections because the parasites may cause no or only minor skin lesions in these hosts. Thus it is difficult to verify that the detected parasites are the ones that cause human disease[19]. It is, however, necessary to identify the parasite reservoir hosts, as they are often fundamental factors in the epidemiology and control of human disease[20]. Numerous species of rodents have thus far been reported from Iran. Some 70 species were caught[21]. In the surrounding deserts and borders of northeast and eastern Iran, R. opimus is the main reservoir host of the disease. In Isfahan and Turkmen Sahra, occasionally infected M. libycus were found in the area that R. opimus was the main reservoir; therefore the former is the secondary reservoir in that area[4]. In the area that R. opimus is absent, M. libycus has been a primary reservoir. So far, few studies have been done on the rodents’ fauna and the ZCL reservoir host in Fars Provice, south of Iran. In rural areas of the cities Arsanjan and Neiriz, M. libycus species play the role of primary and proven reservoir of the infection[8,9]. The same species with 8.4% infection has been reported in Marvdasht of Fars Provice, south of Iran[9,10]. T. indica species infected with L. major has been reported in Kharameh[11]. Another report on Leishmania infection in Larestan region of Fars closest focus to the studied area found four cases of T. indica and two cases of infection in two rodent samples of Gerbillus spp[2,3,13]. One of the principal problems about trying to understand the epidemiology of the Leishmania species causing zoonotic disease in humans is the identification of the mammalian hosts that act as ‘reservoirs’ of the parasites. Although this information is often very important in the control of such disease, it is often difficult to collect large numbers of each potential ‘reservoirs’, and subsequently test each animal for Leishmania infection, and check that any parasites detected are the ones causing human disease[20]. This finding at Zarqan confirms the previous studies in Arsanjan, Neiriz and Marvdasht of Fars Province, Southern Iran, that M. libycus is the main reservoir host of ZCL in southern Iran. No other rodent species (e.g. R. opimus or T. indica) were captured in the study areas[2,9,10].

Conflict of interest statement

We declare that we have no conflict of interest.

Amin Masoumeh et al./Asian Pac J Trop Biomed 2014; 4(Suppl 2): S575-S580S580

Acknowledgements

The authors are grateful for the logistical and financial support given by the Vice-Chancellor for Research and Technology, Shiraz University of Medical Sciences, Shiraz, Iran. We appreciate the help with the field work done by Mr. M. Rezaee and Ms. M. Adnafi. We are indebted to Ms. P. Habibi and Ms. A. Vafafar for checking identification. This article was the result of a research project approved and it has been funded by the Research Council of Shiraz University of Medical Sciences No: 90-01-42-3034, 02/06/2012.

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