MOLECULAR STUDY OF CUTANEOUS LEISHMANIASIS HUMAN RESERVOIRS AND INFECTIONS IN BASTAK

Leishmaniasis is one the six most important diseases in tropical areas. Study and research on various aspects of it is recommended and supported by the World Health Organization. Broom tail rodents (Rodentia: Muridae: Gerbillinae) are the most important reservoir hosts of parasite causes wet cutaneous leishmaniasis (or Zoonotic Cutaneous Leishmaniasis (ZCL) disease) means Leishmania major. Different species of rodents from different regions of Iran play role in maintenance of the parasite. The purpose of this research is molecular study of cutaneous leishmaniasis human reservoirs and infections in Bastak County, Hormozgan Province, Iran. Live traps were used to catch rodents. The rodents were anesthetized with chloroform in the field and four slides were prepared from earlobe of each animal by sanding method and their morphometric characteristics were completely measured and recorded. Rodent abdomen was opened with blade and the liver and the spleen of each sample were kept in a numbered container containing 70% ethanol. The prepared slides were examined with microscopic (Giemsa staining) and molecular methods. Finally, using Nested-PCR method, they were evaluated using species-specific primers (LIN R4, LIN 17, LIN 19). A total of 108 rodents from five species of three different genera were collected. Meriones persicus species with 26.9% was the predominant species. A contamination in T.indica species and two cases of contamination in two samples of female M.hurrianae were observed. L. major species was also identified. Broom tail rodents are abundance and have relatively high diversity in Bastak County. They live in proximity to human dwellings. Tatera indica and Meriones hurrianae species are introduced as the disease reservoir hosts in this area and use of molecular and PCR methods provides the possibility of faster diagnosis of parasite species.


INTRODUCTION
Leishmaniasis (Cutaneous leishmaniasis (CL)) has long been known in Iran. In Iranian ancient books, including Canon of Medicine compiled by Avicenna (IbnSina), a wound called "khyroniyeh" was mentioned. Its treatment was difficult and lasted a long time and was resistant to different medications. By referring to signs and its symptoms mentioned about the wound, it is thought to be lesion of leishmaniasis (Nadim et al., 2008). Cutaneous leishmaniasis is a chronic disease of skin and named differently in various areas (e.g., it is known to Salak in Shiraz and Lakehye Sal in Mashhad). The disease is especially observed in warm seasons and in tropical regions. It is caused by a protozoan of genus leishmania and Trypanosoma tidae family. It can be transferred between human and some animals, including rodents and dogs and it can also transfer by a sand fly (Phlebtomus) from a patient to others. Leishmaniasis arises at first as a small papule and then becomes larger (nodule), and finally converts to wound. It is possible that the wounds recover spontaneously in a few weeks or even several years (Dedet and Pratlong, 2003). Leishmaniasis is one of the most common parasitic diseases in Iran that is called urban (dry) and rural (wet) Leishmaniasis. Leishmania tropica and L. major species are agents of urban (dry) leishmaniasis and rural (wet) leishmaniasis, respectively (Parvizi et al., 2009). The presence of parasite can be identified by microscopic observation of slides stained with Giemsa. However, its type is not recognizable (Aransay et al., 2000). Molecular techniques are widespread in recent years to detect leishmaniasis. One of these techniques is determination of parasite type by PCR method. This method can be done even with small number of parasites in a sample (Faiman et al., 2013). Leishmaniasis is not so problematic in comparison with other diseases. However, the wounds caused by it due to various reasons may leave some traces as improper and disfiguring scars. Identification of parasite species is effective in adoption of the disease prevention and control programs. Its classification to different species and strains is very difficult due to several reasons and similarity in appearance of parasite species. Epidemiological and clinical evidence are not alone effective in differentiating between various species (Killick-Kendrick, 1990). Since the first step in planning for control and fighting a disease is determination of exact specification of pathogens, molecular studies that in addition to detection of genetic diversity among species and within species, diagnose the genus of Leishmania, are of great importance (Killick-Kendrick, 1990;Killick-Kendrick and Ward, 1981). So, study and investigation of cutaneous leishmaniasis molecular reservoirs and human infections is necessary to control the parasite. Since the evaluation process of the disease is greater in warm climates, Bastak County with a warm climate is a suitable area to evaluate the subjects.

Methods
To determine reservoirs of cutaneous leishmaniasis and parasites cause the disease in the patient, rodents' hunting was performed in spring, autumn and winter of 2014 in selected villages of Bastak County (the city of Bastak location; longitude: 54 degrees and 23 minutes; Latitude: 27degrees and 14 minutes; height 2120 m above sea level), which were selected from different climatic regions of the County. Once in a month and each time 10 Sherman live traps were installed at sun sets around active nests and they were collected in morning of the next day. Date palm, cucumber and walnuts were used as bait. After collecting the traps containing rodent, they were placed in a plastic bag and were anesthetized with chloroform-soaked cotton. When the researchers became sure of the animal death, its abdomen was opened using a sterile surgical blade and the liver and spleen were placed in a numbered container containing 70% ethanol. After clearing the abdomens, rodent bodies in containers containing 10% formalin were sent for taxidermy and identification to Zoology Laboratory of Shiraz University. Rodents' identification was conducted in Zoology Laboratory of Natural History Museum of Shiraz University and by the help of Iranian rodent diagnostic key (Nadim et al., 2008). To confirm the suspicious samples, they were sent to Rodent Research Center of Mashhad University. Two slides were prepared from Serosity of the ear and the tail of each animal using sanding method. The prepared slides from the ears and the tails of the rodents were examined using microscopic method and Giemsa staining for observation of amastigotes. In case of observation of Leishman body, DNA extraction process began for the positive slides using Motazedian et al., method (2002). DNA extraction from the liver and the spleen was also carried out using Azizi et al., method (Rassi et al., 2007;Azizi et al., 2008;Yaghoobi-ershadi et al., 1994). Nested-PCR-based approach was used for proliferation of variable region of Leishmania kinetoplast DNA (k DNA) mini circle. LINR4, LIN17 and LIN19 were used in this approach as a forward primer in both stages, reverse primer of the first stage and reverse primer of the second stage, respectively.
This technique was designed by Aransay et al., (2000) and it was used by the author in numerous studies for diagnosis of Leishmania parasite in carriers' body and Leishmania reservoirs (Azizi et al., 2010). The reference strains of the parasite were used as follows to set up PCR reaction: the strains Infantum: MCAN/IR/96/Lon49Le. Le. Tropica: MHOM/IR/89/ARD2 Major: MHOM/IR/54/LV39Le. The above strains were used for comparison. These strains were taken from Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences. Negative control, which was the extracted DNA from the liver of healthy rodents, was used for all reactions to ensure of the accuracy of performance. Thermal profile at the first stage was as follows: after a preliminary heat at 94˚C for 5 min (Initial Denaturation), proliferation continued for 30 seconds at 94˚C (Denaturation), 30 seconds at 52˚C (Annealing) and 1 minute at 72˚C (Extension) and finally, 10 minutes at 72˚C (Final Extension) (the first stage was repeated in 30 cycles). The second phase of thermal profile was also similar to the first stage, but without preliminary heat and the heat related to Annealing stage was 58˚C. The second thermal profile was repeated 33 cycles. After proliferation of DNA, 5 microliter of PCR product mixed with loading buffer was electrophoresed for 40 min on 1.5% agarose gel containing ethidium bromide. By detection in UV Transilluminator device, the obtained bonds from the samples were compared with each other and studied. They were also compared with the obtained bonds from the standard strains and were recognized. The results were analyzed using SPSS statistics 19 and Post Hoc test, Anova, paired t-test and chi-square tests.

Results
108 rodents of 5 different species were caught in this study. Rodents' hunting was done completely out of rural places and borders and as a result, no house mice (Mus Musculus) commonly hunted in the studies for determination of rodent fauna, were caught. Species, frequency and percentage of rodent hunting in the studied areas of Bastak County are listed in Table 1 and Table 2. Frequency percentage of species in the city of Kukherd showed that the highest frequencies with 57.1% and 42.9% were related to Meriones hurrianae and Gerbillus nanus species, respectively (Table 1). Frequency percentage of species in Gachuyeh Village showed that the highest frequencies with 56.3%, 37.5% and 6.3% were related to Meriones hurrianae and Gerbillus nanus and Meriones lybicus species, respectively (Table 1). Frequency percentage of species in Hanguyeh Village showed that the highest frequencies with 55%, 40% and 5% were related to Merioneshurrianae and Gerbillusnanus and Merionespersicus species, respectively (Table 1). Frequency percentage of species in the city of Bastak showed that the highest frequency with 100% was related to Tateraindica species (Table 1). Frequency percentage of species in Guwdah Village showed that the highest frequencies with 50%, 27.8%, 11.1% and 11.1% were related to Meriones persicus, Tatera indica, Meriones hurrianae and Meriones lybicus species, respectively (Table 2). Frequency percentage of species in Deh Tall Village showed that the highest frequencies with 36.4%, 36.4% and 27.3% were related to Meriones persicus, Tatera indica and Meriones lybicus species, respectively (Table 2). Frequency percentage of species in Dehang Village showed that the highest frequencies with 47.8% and 52.2% were related to Meriones persicus and Tatera indica species, respectively (Table 2). -Using Fisher's Exact Test, it was found that there was a significant relationship at level of 1% between studied location and species (P-Value=0.000). Sampling process using microscopic method was done from all hunted rodents to recognize Leishmania contamination. They were also studied in terms of DNA detection of Leishmania parasite in their liver and spleen. The results are separately specified in Table 3. Using Fisher's Exact Test, it was found that there was no significant relationship between contamination and species (P-Value=0.707). The approved contamination in all samples was related to female Tateraindica and female Merioneshurrianae species (Figure 1).

Research Article
Centre for Info Bio Technology (CIBTech) 76

Discussion
Five species of rodents (Gerbillinae subfamily) were identified as broom tail rodents in this study in Bastak County. The highest frequencies among the collected samples were related to Meriones persicus (26.9%), Tateraindica (25%), Meriones hurrianae (24.1%), Meriones lybicus (8.3%) and Gerbillusnanus (15.7%), respectively. Since the sampling was done in the burden of rural areas, no house mice (MusMusculus) commonly reported in the studies for determination of rodent fauna, were caught. So far, a few studies have been done related to broom tail rodents in Hormozgan Province. In the study conducted by Solymani-Ahmadi et al., (1998) only Rhombo-mysopimus species were collected from Kahorestan region in Bandar Abbas. The remarkable point in the present study was that Rhombomysopimus species, the main reservoir host of the disease in most central areas and north eastern regions of Iran, did not collected. Although, animal fauna may be different in various geographical conditions, the presence of high similarity of climatic conditions in Bastak and Kahorestan areas makes necessary revision and repeat of a similar study in Kahorestan area. Hanafi-Bojd et al., (2006) reported two species of Meriones persicus and Tatera indica in Haji Abad County. These two was also the dominant species in the current study. But no Leishmania contamination was observed by the researchers in the collected samples.
Leishmania contamination was observed with microscopic and molecular methods in the two rodent species of Meriones persicus and Tatera indica. Only one case of contamination to L. major was observed in Tatera indica species with both identification methods. One case of contamination in one female sample and two cases of contamination in two female samples were proved in Meriones hurrianae specie, using microscopic and specific PCR methods, respectively. It is worth noting that the microscopic method was only performed by investigation of prepared slides from rodents' ears. However, the molecular method was taken both on positive skin smears and the liver and spleen samples. So, the molecular method was able to identify positive the two slides prepared from ears which had been diagnosed negative by microscopic method. This reveals more sensitivity of the molecular method in detection of parasite.
Indian gerbil (Tatera indica) infected to L. major has been reported in several studies, especially in the west and the southwest regions of Iran. The species is reported by Javadian et al., (1998) as the main reservoir host of wet Leishmaniasis in Ilam and Khozestan Provinces. Nesokia indica and Meriones libycus species are considered as the second reservoir hosts of the disease. The species were reported by Asgari et al., (2006) as Leishmania reservoir in Kherameh region, Fars Province. Contamination to the similar species was also detected using microscopic and molecular methods in Larestan County, Fars Province located in vicinity of Hormozgan Province. This was reported by Mehrabani et al., (2007). Indian desert jird species, Meriones hurrianae, is considered as the main rural reservoir host in the southeast of Iran, where Tatera indica acts usually as the second reservoir (Seyedi-Rashti et al., 1984). The infection of this species to L. major was identified by Mohebali et al., (2004) using microscopic and RAPD-PCR methods and iso-enzymic analysis in the southeastern of Iran (e.g. the south of Baluchestan, and Dashtyari regions, Konarak and Chabahar). The contamination to this species has been limited in Iran to the southeastern areas and this contamination was also reported in the west of Hormozgan province in the present study. Meriones hurrianae species infected by L. major was also reported in Rajasthan Province in India (Morsy et al., 1987). Based on the extensive studies of researches, the contamination of Gerbillus nanus to L. major has not been mentioned in any scientific sources. For the first time and using microscopic and molecular methods, Mehrabani et al., (2007) reported two rodent samples of Gerbillus spp. genus infected to L. major in Larestan region, Fars Province. They did not report the rodent species. However, based on the size of the samples (two cases of contaminations) which were big, the two samples were not certainly from G. nanus species. Of course, some cases of contamination to Gerbillus species, including Gerbillus pyramidum and Gerbillus dasyurus to L. major have been reported in some countries of Middle East, including Egypt, Jordan and Palestine. However, Gerbillus nanus was found in the present study but did not infect to L. major, parasite (Peters, 1981;Wesserberg et al., 2002). Two species of rodents' contaminations mentioned above were observed with both the microscopic and specific PCR methods. The molecular method was more successful in detection of contaminations, so that and recognition ability of microscopist. This issue has been highlighted by studies of other researchers (Asgari et al., 2006;.

Conclusion
Based on the above findings, leishmaniasis in this emerging focus was of rural or wet type and was caused by L. major. Two Meriones hurrianae and Tatera indica species of rodent were introduced as the disease reservoir hosts. So, considering the type of primers and used sequence in this study to differentiate species of cutaneous leishmaniasis pathogens in the city of Bastak, finding a practical method with high sensitivity and performance is essential to enhance accuracy and to provide possibility of early diagnosis.