Microsoft Word - Dr Rassi RTL.doc Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 21 Original Article Molecular Detection of Leishmania major in the Vectors and Reservoir Hosts of Cutaneous Leishmaniasis in Kalaleh District, Golestan Province, Iran *Y Rassi 1, A Sofizadeh1, MR Abai1, MA Oshaghi1, S Rafizadeh 2, M Mohebail 3, F Mohtarami1, R Salahi 4 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Iran 2 Department of Emergency, Ministry of Health and Medical Education, Iran 3 Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Iran 4 Healh Center of Kalaleh, Golestan Province, Iran (Received 18 Nov 2008; accepted 5 April 2009) Abstract Background: An epidemiological study was carried out on the vector(s) and reservoir(s) of cutaneous leishmaniasis in rural areas of Kalaleh District, Golestan Province during 2006 - 2007. Methods: Totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for detection of leishmanial parasite. Results: Phlebotomus papatasi was the common species in outdoor and indoor resting places. Employing PCR technique showed only 1 out of 372 P. papatasi (0.3%) was positive to parasite due Leishmania major. Sixteen ro- dent reservoir hosts were captured by Sherman traps and identified as Rhombomys opimus. Microscopic investiga- tion on blood smear of the animals for amastigote parasites revealed 6(37.5%) infected rodents. Infection of these animals to L. major was then confirmed by PCR against rDNA loci of the parasite. Conclusion: This is the first molecular report of parasite infection of both vector (P. papatas) and reservoir (R. opimus) to L. major. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and R. opimus was the most important host reservoir for maintenance of the parasite source in the area. Keywords: Cutaneous leishmaniasis, Vector, Reservoir, Iran Introduction There are several reports indicating oc- currence of Cutaneaous Leishmaniasis due to Leishmania major (CLM) in Iran (Yaghoobi- Ershadi et al.1996, Rassi et al. 2006). Based on animal reservoir host, there are four foci of disease in our country (Rassi et al. 2006) The first one has been located in central and northeast of Iran, where Rhombomys opimus and Phlebotomus papatasi play important roles as reservoir and vector of the disease (Seyedi-Rashti et al. 1967, Javadian et al. 1976, Yaghoobi-Ershadi et al. 2001). The second focus of Zoonotic cutane- ous leishmaniasis is located in the west and southwest of Iran, where Tatera indica re- placed with R. opymus as a reservoir and P. papatasi as a vector. (Javadian et al. 1988). Baluchistan Pprovince, in the southeast of Iran is considered as the third focus of ZCL. In this region Meriones hurrianae has been approved as a natural reservoir host (Seyedi-Rashti and Nadim 1984). From the reported evidences, *Corresponding Author: E-mail: rassiy@sina.tums. ac.ir Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 22 it is apparent that the most rural areas of Fars Province in southern Iran can be considered as the ZCL focus where M. libycus is the primary and main reservoir host of the disease, while R. opimus and T. indica were absent and P. papatasi is considered as the proven vector of ZCL (Rassi et al. 2006, Rassi et al. 2007). Kalaleh district from Golestan Province in northern Iran is a ZCL focus and this study was performed to put light through the epide- miology of the disease in the region. The main objectives were to determine the sand flies spe- cies responsible for most transmission of L. major to human, as well as to determine the main reservoir hosts of the disease in the study area. Material and Methods Study area The study was carried out in 3 villages (Ghare ghol-e-gharbi, Sozesh and Allah nour) of Maraveh tapeh district (55° 57 E, 37° 54 N) at an altitude 228 m above sea level and 90-100 km far from Kalaleh City. The weather is hot in the summer and cold in the winter. It receives an average of 311.3 mm of rain per year. The temperature ranges between 2.5-36.6° C and the ratio humidity ranges be- tween 25- 85%. The total population of the district was about 156939 people in 2007 (Un- published data). The major activities of the po- pulation are agriculture and animal farming. Collection of sand flies Sand flies were collected from indoors (bedroom, guestroom, toilet) as well as out- doors (rodent burrows, wall cracks) biweekly using sticky traps. Three above mentioned vil- lages were selected and 180 sticky traps were set up during the sand flies activity period (May- November). Sand flies were rinsed from the sticky traps and mounted in a drop of Puri’s medium and identified after 24 h using a valid key (Nadim and Javadian 1976, Rassi et al. 2006 a). Sand flies DNA Extraction DNA was extracted through ISH-Horo- wicz (Ready et al., 1991). Individual female sand flies were homogenized with a sealed pasture pipette in 1.5 ml tubes. Then 100 µL lysis buffer [0.1M Tris-HCl Ph 7.5; 0.6M NaCl; 0.1 EDTA] and 10 µL [0.8M Tris- HCl Ph 9.0: 0.27M EDTA] were added and incubated at 65 °C for half an hour before 30 µL acetate potassium (8 mol) were added. Af- ter short centrifuge it was cooled for 45 min and transferred to new tube and added 350 µL cold pure ethanol and stored at -20°C for 24 h. The tube was centrifuged at 13000 rpm for 30 min then emptied the solution until dried and washed by 500 µL ethanol (75 °C) and the TE buffer was added. Semi-Nested PCR for detection of Lesh- mania infection in sand fly Semi-Nested PCR was employed for detec- tion of kinetoplast DNA (kDNA) of L. ma- jor in sand fly specimens. The primers were: LINR4 (Forward):5´-GGGGTTGGTGTAA- AATAGGG-3´ (20bp); LIN19 (Reverse): 5´- CAGAACGCCCCTACCCG-3´ (17bp), and LIN17 (reverse): 5´-TTTGAACGGGATTT- CTG-3´ (17bp). Positive samples were tested by PCR method against ITS gene using the primers of ITS1 (Forward): 5´- TCCGTAG- GTGAACCTGCGG-3´ and ITS2 (Reverse): 5´- GCTGCGTTCTTCATCGATGC -3´. PCR amplification was followed by RFLP tech- nique using HaeIII enzymes for final species identification of the parasite. Collection of rodents Rodents were captured by setting the Sherman live traps. Traps were baited with roa- sted walnut, cucumber, tomato and placed in the active burrows. The traps were set up early morning and evening in December, February, May, and July. In order to approve the infec- tivity of rodents by the parasites, their ears were examined and an impression smear was taken and stained by Geimsa staining method. The Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 23 presence of the parasite was checked under microscope. Samples from infected rodents were inoculated subcutaneously at the base of tail of BALB/c. Procedure of species identification by PCR method is described previously. DNA Extraction from positive smears of rodents Total DNA was extracted from posi- tive smears by digestion in 100 µL PBS buffer and the tube was centrifuged at 10000 rpm for 10 min, then 300 µL lysis buffer and 30 µL proteinase K added. The tube was incubated for 24 h at 37 °C before adding 300 µL sacharin phenol. After adding this so- lution the tube was centrifuged at 9300 rpm for 5 min. After transferring upper phase to new tube, 300 µL phenol- chloroform should be added and was centrifuged at 10000 rpm for 5 min. Again transferred the upper phase to new tube and washed by pure chloroform. Thirty µL MgCl2 and 1000 µL ethanol were added to upper phase and stored at -20° C for 2 h before was centrifuged at 10000 rpm for 10 min and washed down phase by 70% ethanol with TE and was centrifuged at 10000 rpm for 10 min and the TE buffer was added. PCR-RFLP for detection of Leishmania infection in Rodents PCR-RFLP was employed for detec- tion and identification of L. major in rodent specimens by the method explained by Dweik et al. (2007). The primers were: IR1 5´-GCT GTA GGT GAA CCT GCA GCA GCT GGA TCA TT-3´ and IR2 5´-GCG GGT AGT CCT GCC AAA CAC TCA GGT CTG -3´ (Cupolillo et al. 1995). Reference strains of L. infantum (MCAN/IR/96/Lon49), L. tropica (MHOM/ IR/89/ARD2) and L. major (MHOM/IR/54/ LV39) were used as positive controls. All were obtained from the Medical Parasitology Labo- ratory, the School of Public Health, Tehran University of Medical Sciences, Iran. PCR pro- duction was followed by RFLP technique using HaeIII enzymes for final identification of the parasite. Results Sand flies Totally 4900 sand flies were collected, including P. papatasi (41.2%), P. mongolensis (5.8%), P. caucasicus (3.6%), P. caucasicus group (7.3%), P. sergenti (2.2%), P. alexan- dri (2%), P. (adlerius) sp. (0.02%), P. brevis (0.02%), P. kazeruni (1.5%), S. sintoni (36%), S. clydei (3%) and S. sogdiana (0.02%). Three hundred and seventy two P. pa- patasi specimens were examined by Semi- Nested PCR for Leishmania infection. Spe- cies-specific amplification of L. major DNA was found in one (0.3%) of the P. papatasi Giemsa stained prepared for detection of pro- mastigote (Fig.1). Furthermore ITS amplifi- cation by Nested PCR primers followed by RFLP technique confirmed the DNA of para- site in the infected P. papatasi sample (Fig. 2 & 4). The infected P. papatasi was collected from bed room with parous and empty abdo- men position. Rodents During this study 16 rodents were cap- tured and identified. All of them were R. opi- mus. Although all collected animals were ex- amined for parasite infection under light mi- croscope, amastigotes were only found in sme- ars of 6(37.5%) of them. Each sample from infected rodents was inoculated subcutaneously at the base of tail of one BALB/c. Results from inoculation of parasite from infected ro- dents, revealed the presence of amastigotes in the nodules and ulcers of the experimen- tally mice after 35 d of inoculation period. Parasites infection was observed in both males and females animals. Isolated parasites from infected rodents were identified as L. major using PCR followed by RFLP technique (Figs. 3, 4). Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 24 Fig. 1. kDNA PCR amplification of L.major in P.papatasi using Semi-nested PCR M: Molecular size Marker, 1: Negative control, 2: Sample of P.papatasi, 3: positive control L.major Fig. 2. ITS amplification of L.major in P.papatasi using Nested PCR primers M(Marker) , 1(L.major standard), 2(blank), 3 (sample of P.papatasi) Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 25 Fig. 3. ITS-rDNA amplification of Leishmania parasite in R.opimus 1-6(infected R.opimus), 7(L.major standard), 8(Negative Control), M (Marker) Fig. 4. PCR-RFLP analysis of ITS region for identification of Leishmania species using HaeIII L.major standard (1), P.papatasi (2), L.tropica standard (3), L.infantum standard (4), and Rhombomys opimus (5) Iranian J Arthropod-Borne Dis, (2008), 2(2): 21-27 Y Rassi et al: Molecular Detection of…. 26 Discussion Ecology and epidemiology of leishma- niasis are important measures for manage- ment and planning of the disease control. The entomological survey accompanied by epide- miological data is a major component for com- bating against disease. Several epidemiological and entomological finding including anthro- pophily, common infection of the sand-flies with the same Leishmania parasite that found in man in the same places, suggested the ca- pacity of sand-fly as a vector (Killick-ken- drick et al. 1990). For further confirmation, molecular tech- niques (PCR) have been employed too. The highly sensitive technique of PCR has been used for detecting Leishmaniain sandflies in the world (Mukherjee et al. 1997), Iran (Azizi et al. 2006, Rassi et al. 2006, Rassi et al. 2007) and India (De Bruijn et al. 1992). Re- sults of our study revealed that the high den- sity of P. patasti in indoor resting places and infectivity with L. major is attributed that this species can play a major role as a principle vector in the region. Another important finding of this sur- vey was confirmation of R. opimus as the principal reservoir of ZCL in rural regions of Kalaleh district. This rodent has been also reported as a main reservoir in the other foci of disease (such as Isfahan and Khorassan provinces) in Iran (Yaghoobi-Ershadi et al. 1996, Javadian et al. 1976, Seyyedi-Rashti et al. 1967). This great gerbil, a colonial, bur- rowing rodent, is a common species in the arid desert and steppe regions of central Asia. This species also exist in the southern territories of the former U.S.S.R. (i.e. Turkmenistan, Uz- bekistan, Kazakhstan, Tajikistan) and neigh- boring countries where ZCL caused by L. major is endemic and considered as an impor- tant public health problem, therefore R. opi- mus is considered the principal mammalian host of the parasite (Strekova et al. 2001). Human activities close to R. opimus bur- rows, the presence of high density of P. pa- patas in the rodent burrows and indoors as well as proximity of human habitat to R. opi- mus colonies lead to a rise in human contact with the disease agents and possible appearance of a new focus of leishmaniasis in the region. Acknowledgements The author would like to appreciate the kind collaboration of Health Center staff of Kalaleh district. This study was financially sup- port by the School of Public Health, Tehran University of Medical Sciences: Project No.: 5416. 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