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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 10  |  Issue : 4  |  Page : 1032-1036
Acomys dimidiatus (Rodentia: Muridae): Probable reservoir host of Leishmania major, southern Iran


1 Research Centre for Health Sciences, Institute of Health, Shiraz University of Medical Sciences, Iran
2 Research Centre for Health Sciences, Institute of Health, Shiraz University of Medical Sciences; Department of Public Health, Mamasani Higher Education Complex for Health, Shiraz University of Medical Sciences, Shiraz, Iran
3 Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
4 Infectious and Tropical Diseases Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran

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Date of Web Publication5-Oct-2017
 

   Abstract 


Background: Rodents are the main reservoir hosts of zoonotic cutaneous leishmaniases (ZCLs) in different parts of Iran. Nonetheless, no evidence of natural infestation of Leishmania parasite has been reported from spiny mice Acomys dimidiatus (Rodentia: Muridae). Materials and Methods: Between March 2011 and March 2012, 62 wild rodents were captured and checked for leishmanial infestation of their livers and spleens using polymerase chain reaction in fourteen regions of Darab district in Fars province, southern Iran. Results: Overall, 62 rodents consisting of 42 Tatera indica, 6 Meriones libycus, 5 Meriones persicus, 5 Mus musculus, and 4 A. dimidiatus were identified. Infestation to Leishmania major was confirmed in all the captured species of rodents: T. indica (23.8%), M. libycus (50%), A. dimidiatus (50%), M. persicus (20%), and M. musculus (20%). Conclusions:T. indica was the reservoir host of L. major in this focus of ZCL. Moreover, M. libycus and M. persicus had a remarkable role in the maintenance of the parasite. A. dimidiatus was also introduced as a new probable reservoir host of L. major for the first time. Yet, the role of this species in the maintenance, transmission, and epidemiological cycle of ZCL needs more specific researches.

Keywords: Acomys dimidiatus, Iran, Leishmania, polymerase chain reaction, rodent

How to cite this article:
Azizi K, Askari MB, Kalantari M, Sarkari B, Turki H. Acomys dimidiatus (Rodentia: Muridae): Probable reservoir host of Leishmania major, southern Iran. Ann Trop Med Public Health 2017;10:1032-6

How to cite this URL:
Azizi K, Askari MB, Kalantari M, Sarkari B, Turki H. Acomys dimidiatus (Rodentia: Muridae): Probable reservoir host of Leishmania major, southern Iran. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Sep 21];10:1032-6. Available from: http://www.atmph.org/text.asp?2017/10/4/1032/215889



   Introduction Top


Leishmaniases are parasitic diseases with a wide range of clinical symptoms, infecting millions of individuals in numerous countries,[1] especially in tropical and subtropical areas, such as Iran.[2] Sand flies (Diptera: Phlebotominae) are considered to be the sole vectors of leishmaniasis and are naturally found to be infested with Leishmania.[3]

Gerbil rodents (Muridae: Gerbillinae) are one of the most important mammalian groups, which have a main role in the maintenance of various pathogens responsible for tropical zoonotic diseases, such as leishmaniases.[4] They are the main reservoir hosts and the main bases in the epidemiology and control strategy of zoonotic cutaneous leishmaniasis (ZCL) in Iran.[5] Moreover, variant causative agents of leishmaniases have been isolated from rodents in different parts of Iran.[2],[5]

Totally, 52 species of Murinae subfamily have been characterized in Iran.[6] Spiny mouse, Acomys dimidiatus, known as “the eastern spiny mouse” or “Arabian spiny mouse,” was described as Mus dimidiatus in 1826 for the first time.[7] It is a small rock-dwelling species of rodents in the family of Muridae with a wide morphological variety and is the only species of spiny mouse that may have black coloration.[8] Their diet is similar to that of other species of spiny mouse, consisting mostly of seeds.[9] They are distributed in middle eastern deserts and forests of Africa [10] as well as along the border of the Persian Gulf.[11] In Iran, they are reported from Kerman and Fars provinces.[12] There is a lot of misunderstanding and misinformation in the literature about this species living in Iran. Moreover, no evidence of natural infestation of Leishmania has been reported from A. dimidiatus.

Because of different species of Leishmania, various sources of Leishmania DNA have been used for molecular methods.[13] However, polymerase chain reaction (PCR)-based assays are characteristically used to detect Leishmania species in patients, vectors, and reservoir hosts.[14]

Considering the importance of rodents as the reservoir hosts of leishmaniasis, the present study aims to assess rodents' infestation with leishmaniasis in Darab district, south of Iran.


   Materials and Methods Top


Study area

Fars province is located in southern Iran, includes 23 counties, and covers an area of about 122,400 km2. Darab district is situated in southeast of Shiraz (capital city of the province) at 28° 45' 7” North, 54° 32' 40” East and about 1200 m above the sea level.[15] Because of favorable ecological conditions for vectors and reservoirs of leishmaniasis, Darab has been considered to be one of the most important foci of cutaneous leishmaniasis in southern Iran [Figure 1].
Figure 1: Map of Iran, showing the location of Darab district in Fars province, Southern Iran

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Collection and examination of rodents

Between March 2011 and March 2012, rodents were caught alive in wire traps on agricultural plantations surrounding and near the houses in fourteen regions of Darab district, including Arab-Ghanbari, Banooj, Badameh, Bakhtajerd, Behrooz-Abad, Bikhe-Deraz, Sange-Charak, Shamarz, Fathabad, Fadami, Ghaleh-Biaban, Khosuyeh, Nasrovan, and Nasirabad villages. Each trap was set in the evening and checked early in the next morning. At least 25 “trap-nights” were set per month. Each rodent was identified, euthanized, and dissected so that four specimens (two of the liver and two of the spleen) could be prepared.[16]

DNA extraction

All the specimens were checked for Leishmania kDNA using PCR assay. In doing so, each specimen was mixed with 195 μl of lysis buffer (1 mM EDTA, 50 mM Tris-HCl [pH 7.6, 1% (v/v] Tween 20) containing 5 μl of a proteinase K solution, which already had 23 mg/ml of the enzyme. The specimen was then incubated at 37°C for 12–16 h before 50 μl of a phenol: chloroform: isoamyl alcohol solution (25:24:1, by Vol.) was added.[17] After being shaken vigorously, micro tubes containing specimens were centrifuged at 12000 rpm for 5 min. Thereafter, DNA in the supernatant solution was precipitated with 200 μl of cold ethanol, resuspended in 50 μl of double distilled water, and stored at 4°C before being used in the PCR assay.[17]

Polymerase chain reaction assay

A specific PCR method was used to amplify the variable area of the minicircle kDNA of Leishmania spp. in the rodents' livers and spleens, as previously designed by Aransay et al. with minor modifications. The forward and reverse primers of LIN17 (5'-TTT GAA CGG GAT TTC TG-3') and LINR4 (5'-GGG GTT GGT GTA AAA TAG GG-3') were used in the PCR assay. Each 25-ml reaction mixture contained 1.5 mM of MgCl2, 2 mM of dntp, 2.5 μL of 10 × PCR buffer (Boehringer Mannheim, Mannheim, Germany), 1 unit of Taq DNA polymerase (Cinagene, Tehran), 10 picomol of each primer, and 5 μL of the DNA sample. These mixtures were amplified in a programmable thermocycler (Eppendorf AG (Mastercycler gradient), Germany) at 94°C for 5 min (1 cycle) followed by 30 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min followed by a final elongation step at 72°C for 10 min.[13] The World Health Organization's reference strains of Leishmania major (MHOM/IL/67/LV561) were used as the standard DNA. A band of 650 bp indicated that L. major kDNA was present in the sample.[14]

Electrophoresis

A 5-μl sample of each PCR product was exposed to electrophoresis in 1.2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet transillumination.[14]


   Results Top


Overall, the 62 rodents captured during the study belonged to five species as follows: 42 Tatera indica (67.7%), 6 Meriones libycus (9.7%), 5 Meriones persicus (8.1%), 5 Mus musculus (8.1%), and 4 A. dimidiatus (6.5%) [Table 1]. The PCR results revealed that 17 rodents (27.4%) (10 T. indica, three M. libycus, two A. dimidiatus, one M. persicus, and one M. musculus) were positive for kDNA of L. major [Figure 2].
Table 1: The distribution and infestation of Leishmania in the rodents, as revealed by polymerase chain reaction-based detection of Leishmania major kDNA in Darab district, Southern Iran

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Figure 2: The results of the polymerase chain reaction - based amplification of kDNA. Molecular-weight markers (Lane 1), reference specimens of Leishmania tropica (760 bp) (Lane 2), reference specimens of Leishmania infantum (720 bp) (Lane 3), reference specimens of Leishmania major (650 bp) (Lane 4), negative control (Lane 5), negative specimens of liver and/or spleen in the five species of the caught rodents (Lanes 6, 8, 12, 17, and 19), and positive specimens of liver and/or spleen in Tatera indica (Lanes 7, 9, and 10), Meriones libycus (Lanes 11 and 13), Meriones persicus (Lanes 14 and 15), Mus musculus (Lane 16), and Acomys dimidiatus (Lanes 18 and 20)

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The largest number of infested rodents was related to T. indica in several villages, particularly in Bakhtajerd in which the largest number of T. indica was captured (22.6%). M. libycus, M. persicus, and M. musculus were also infested with L. major. A. dimidiatus was found to be infested with L. major (3.2%), as well. However, the recent species was only caught in Sange-Charak village. Rodents' distribution and infestation with leishmaniasis in different parts of Darab district have been detailed in [Table 1].

Examination of the rodents' internal tissues demonstrated that 22.7% of their livers, and spleens were infested to leishmaniasis. However, the total infestation rate of the livers (27.4%) was higher than that of the spleens (19.4%) [Table 2].
Table 2: The prevalence of Leishmania infestation in the studied organs of the rodents, as revealed by polymerase chain reaction assay in Darab district, Southern Iran

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   Discussion Top


In the present study, five species of Gerbilinae rodents were caught as follows: T. indica, M. libycus, M. persicus, M. musculus, and A. dimidiatus. T. indica was the most common species and the results of the PCR assay showed that 23.8% of them were positive for L. major. In addition, M. libycus and M. persicus were the most important mammalian hosts of L. major in the rural areas of Darab district. It should be noted that this study included the first detection of L. major from Iranian A. dimidiatus caught for the first time in the area where ZCL has recently occurred.

In different endemic foci of Iran, Rhombomys opimus, T. indica, Meriones lybicus, and Meriones hurrianae have been reported as the major “reservoir” hosts of ZCL.[18],[19] Besides, Nesokia indica and Gerbillus nanus have been described as the accidental, main, or probable “reservoir” hosts in different parts of Iran.[20],[21]M. libycus, M. persicus, and T. indica have been found positive for Leishmania kDNA in different foci of Marvdasht,[22] Zarghan,[23] Fasa,[24] Estahban,[25] and Jahrom,[26] in Fars province. Moreover, M. musculus, Rattus rattus, and Rattus norvegicus have also been found to be naturally infested to L. major in this province, which may justify the remarkable increase in ZCL in some urban areas of Iran in the recent years.[26],[27],[28]

In the current study, A. dimidiatus was infested to L. major and was introduced as a new probable reservoir host of leishmaniasis for the first time. The only experimental infestation of this species to L. major refers to the study performed by Kamal et al. where the parasite was inoculated from the sand flies of Phlebotomus alexandri to the footpads of a laboratory colonized Acomys cahirinus dimidiatus.[29] This resulted in creation and development of leishmaniasis lesions on the site of the inoculation within 9 months, but the role of P. alexandri and A. dimidiatus in transmission of leishmaniasis in southern Sinai has remained discussable.[29] Although other studies have indicated no natural infestation of A. dimidiatus to leishmaniases in Iran, some findings have revealed the incrimination of Acomys albigena as the reservoir host of visceral leishmaniasis in Africa.[30],[31],[32],[33] On the other hand, other studies have considered the ectoparasites of A. dimidiatus. For instance, a tick of Rhipicephalus turanicus was separated from A. dimidiatus in Saudi Arabia.[34]

Up to now, different biochemical, molecular, and immunological methods, such as isoenzyme electrophoresis, PCR, and monoclonal antibodies, have been used to characterize the causative agents of leishmaniasis.[26] Yet, using a specific and sensitive PCR on the smears of rodent tissues would be beneficial to detect the Leishmania species accurately.


   Conclusion Top


The epidemiological patterns of ZCL depend on the relations among reservoir hosts, vectors, humans, and the species of parasites in control programs. Moreover, determination of “reservoir” hosts is unavoidable in the evaluation of the epidemiology of the disease.[35] Considering the role of rodents in these epidemiological patterns, the present study results revealed that T. indica was the main reservoir host of L. major in the ZCL focus of Darab. Moreover, M. libycus and M. persicus played significant roles in the maintenance of the leishmanial agent. On the other hand, A. dimidiatus was presented as a new probable reservoir host of L. major for the first time. Thus, further studies are required to be conducted on the role of this species in the maintenance, transmission, and epidemiologic cycle of ZCL.

Acknowledgment

This study was financially supported by the Research Vice-chancellor of Shiraz University of Medical Sciences and extracted from the results of an approved MSc student thesis (No: 91-6204) conducted by the author, Mr. Mohammad Bagher Askari. Hereby, the authors would like to thank Ms. A. Keivanshekouh at the Research Improvement Center of Shiraz University of Medical Sciences for improving the use of English in the manuscript.

Financial support and sponsorship

Research Vice-chancellor of Shiraz University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Mohsen Kalantari
Department of Medical Entomology and Vector Control, School of Health, Shiraz University of Medical Sciences, Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ATMPH.ATMPH_412_16

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