J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 403 http://jad.tums.ac.ir Published Online: September 08, 2017 Original Article Epidemiological Study on Cutaneous Leishmaniasis in an Endemic Area, of Qom Province, Central Iran Abedin Saghafipour 1, *Hassan Vatandoost 2,3, *Ali Reza Zahraei-Ramazani 2, Mohammad Reza Yaghoobi-Ershadi 2, Moharram Karami Jooshin 4 , Yavar Rassi 2, Mohammad Reza Shirzadi 5, Amir Ahmad Akhavan 2, Ahmad Ali Hanafi-Bojd 2 1 Department of Medical Entomology and Vector Control, School of Public Health, International Campus (IC-TUMS), Tehran University of Medical Sciences, Tehran, Iran 2 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4 Qom Provincial Health Center, Qom University of Medical Sciences, Qom, Iran 5 Communicable Diseases Management Center, Ministry of Health and Medical Education, Tehran, Iran (Received 25 Jan 2016; accepted 21 Aug 2017) Abstract Background: Cutaneous leishmaniasis (CL) is one of the most important health problems in many areas of Iran. There are two forms of the disease in Iran, anthroponotic and zoonotic CL. This study conducted to assess the epidemiological situation of CL in an endemic area of Qom Province, central Iran from Apr to Nov 2015. Methods: The sticky paper traps and aspirating tubes were used for collecting adult sand flies. Sherman traps and small insect nets were used to capture rodents and small mammals. Giemsa staining was used for preparing the expanded smear and followed by PCR for identifying the causative agent in human, vectors, and reservoirs. In this study, relative frequency of CL was also calculated. Results: Fourteen species of Phlebotomine sand flies were collected. Phlebotomus papatasi (61.74%) was the predom- inant species through the period of activity. Overall, 62 Meriones libycus, 8 Nesokia indica, 4 Mus musculus, 16 Al- lactaga elater and 2 Hemiechinus auritis were caught. PCR technique showed 6 out of 150 P. papatasi (2%), two out of 62 M. libycus (3.23%) and all of suspected human's skin tissue samples (100%) were infected with Leishmania major. The relative frequency of CL was 0.30%. Conclusion: This is the first detection of L. major within P. papatasi, M. libycus and human in Kahak District in Qom Province of Iran. Zoonotic cycle of CL exists in this area, L. major is the causative agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease. Keywords: Zoonotic cutaneous leishmaniasis, Leishmania major, Phlebotomus papatasi, ITS1- PCR, Iran Introduction Currently, cutaneous leishmaniasis (CL) is one of the most important vector borne dis- eases in Iran (1) and still the leading cause of considerable morbidity of a large number of people in the endemic foci characterized by chronic skin lesions followed by scars and de- formation of the infected tissue (2). Phelebotomus papatasi is the main and proven vector and Leishmania (Leishmania) major is the causative agent of Zoonotic CL in Iran. Leishmania major, L. (L.) tropica and L. (L.) aethiopica cause CL in the Old World (3). The parasite has been isolated and identi- fied from naturally infected P. papatasi, P. caucasicus, Rhombomys opimus, Meriones libycus and human in such endemic areas (4- 8). The P. papatasi and M. libycus were as proven vector and reservoir in Qomrood, Iran *Corresponding authors: Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, Dr Ali Reza Zahraei-Ramazani, E-mail: azahraei@tums.ac.ir mailto:hvatandoost1@yahoo.com mailto:azahraei@tums.ac.ir J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 404 http://jad.tums.ac.ir Published Online: September 08, 2017 district (9) and as probable vector and reser- voir in Ghanavat district (10). In addition, a big part of the lowland areas of Qom Province provides good ecological niches for P. papatasi and therefore it has higher transmis- sion potential (11). In the recent years, molecular methods have been employed for identification of cer- tain species of Leishmania, either isolated from cultures or from patients (3) as well as in the detection of the parasite in individual or pooled Phlebotomine specimens (12). Suc- cessful establishment of the disease in an en- demic area is the outcome of a close associa- tion between the Leishmania parasite and its natural sand fly vector (13). Thus, vector and parasite identification have great impact on predicting expansions of the disease in en- demic area, also help authorities to design new strategic programs to limit spreading vectors and disease (14, 15). Molecular methods are increasingly em- ployed for diagnostic and epidemiological purposes in order to confirm Leishmania in- fection and to characterize the parasites at the species or genotype level in hosts and vectors (16, 17). The detection of Leishmania para- sites by PCR methods is highly specific and sensitive, with values reaching up to 100%. Accurate and sensitive diagnostic and identi- fication procedures are required to distinguish Leishmania species/strains whose geographic distribution can overlap, which is crucial for adequate treatment and appropriate public health control measures (18). Based on a rapid increase in incidence of CL reported in an endemic area of Kahak Dis- trict of Qom Province in central Iran (19), and due to lack of knowledge on the epidemiolog- ical situation of CL, this study was conducted to determine the epidemiological features of CL including human infection and the reser- voir hosts and their putative vector species in this endemic area during 2015. Materials and Methods Study area The Qom Province is bounded by Tehran Province in the north, Esfahan Province in the south, Semnan Province in the east, and Markazi Province in the west with an area of approxi- mately 11240 square kilometers (0.68% total area of Iran) (Fig. 1). This study was per- formed from Apr to Nov 2015 in 3 villages (Khor Abad, Sarm, and Ghobadbezan) of Kahak rural district (34o09'–35o11' N latitude and 50o06'–51o58' E longitude) of Qom Prov- ince with the elevation of almost 1500m above sea level (20). The average annual min- imum and maximum temperatures were 16.5 °C and 49 °C in Jan and Jul, respectively. The total annual rainfall was about 150mm and the average Max and Min monthly Relative humid- ity were 84% and 28% in Dec and Jun, re- spectively (21). Sand fly collection Sand flies were collected from indoor (bed- room, bathroom, toilets, hall, and stables) and outdoor (rodent burrows) fixed places from the first half of Apr 2015 to the first half of Nov 2015. To capture the sand flies, 30 Sticky Paper Traps (castor oil coated white paper 20×32cm) was used twice a month from sun- set to sunrise. The caught sand flies were transferred to the laboratory in Qom Health Center. To species identification, the head and the last two abdominal segments of the sand flies were mounted in Puris’ medium (24) and identified after 24–72h, using the morpholog- ical characters (25). Then, they were counted and segregated by sex. The rest of the abdo- men, the wings and the legs used for DNA ex- traction were stored in 1.5ml sterile micro tubes containing 96% ethanol. The females were examined for abdominal status and the numbers of unfed, fresh-blood fed, and gravid and semi-gravid sand flies were recorded. The parous females were distinguished from nulliparous sand flies by observation of the J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 405 http://jad.tums.ac.ir Published Online: September 08, 2017 appearance of the accessory glands (26). In order to determine the natural promastigote infections of female sand flies, some unfed, blood fed, semi-gravid and gravid female sand flies (captured from rodent burrows) dissected in a fresh drop of sterile saline (9/1000) for the presence of promastigotes in alimentary canal during Jun and Sep 2015. Reservoirs collection and smear prepara- tion The rodents were captured by Sherman Live Traps (Fig. 2). Collection of dipodids (Rodentia: Dipodidae) was done by using small insect nets. Then the impression smears of ears were fixed with absolute methanol and examined by Giemsa staining method. The Leishman bodies were observed using light microscope and then were subjected to molecular technique for identification. Tissue samples were also taken from the edge of the lesions in 45-suspected CL patients. Lesion smears were fixed with absolute methanol and then stained with Giemsa for CL diagnosis. In this present study, we also calculated the rel- ative frequency of CL. DNA extraction DNA extractions were carried out by crash- ing of the contents on the slides prepared from the digestive system of the 180 dissected fe- male sand flies, the 78 rodent's ear lobes se- rous fluids and the 45 patient's lesion tissues, using the Bioneer Genomic DNA Extraction Kit. The extracted DNA was stored -20 °C for long storing and at 4 °C for daily working. DNA from L. major (MRHO/IR/75/ER) that provided by Department of Medical Parasit- ology, School of Public Health, Tehran Uni- versity of Medical Sciences (TUMS), Iran was used as positive control. Total DNA was extracted from the smears by digestion in 100 μl PBS buffer and the tube was centrifuged at 10000rpm for 10min, then 300μl lysis buffers and 30μl proteinase K added. The tube was incubated for 24h at 37 °C before adding 300μl saccharin phenol. After adding this so- lution, the tube was centrifuged at 9300 rpm for 5min. After transferring upper phase to new tube, 300μl phenol- chloroform should be added and was centrifuged at 10000rpm for 5min. Again transferred the upper phase to new tube and washed with pure chloro- form. Thirty μl MgCl2 and 1000μl ethanol were added to upper phase and stored at -20 °C for 2h before was centrifuged at 10000rpm for 10min and washed down phase by 70% ethanol with TE and was centrifuged at 10000 rpm for 10min and the TE buffer was added. Polymerase chain reaction-restriction frag- ment length polymorphism (PCR-RFLP) for detection of Leishmania infection The DNA samples were examined for the Leishmania species ITS1 by PCR amplifica- tion using the universal primer pair L5 8S (5´- TgA-TaC-CAC-TTA-TCg-CAC-T--3´) and LITSR (5´-CTg-gAT-CAT-TTT-CCg- AT--3´) (Table 1). Molecular study PCR production was followed by RFLP technique (27). The cycling conditions were 95 °C for 5min., followed by 35 amplification cycles, each consisting of three steps: dena- turation at 94 °C for 30sec, annealing 48 °C for 30sec, and extension at 72 °C for one min, followed by a final extension at 72 °C for 7min in thermocycler. PCR productions were digested with the restriction endonuclease Hae III for 2 h at 37 °C. The restriction fragments were separated by electrophoresis on agarose gel and com- pared with those of standard reference strain of L. major and negative control (distilled wa- ter). Results A total of 4164 sand flies (68.90% from outdoors and 31.10% from indoor resting J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 406 http://jad.tums.ac.ir Published Online: September 08, 2017 places) were collected using sticky paper traps. Two genera (Phlebotomus and Sergentomyia) and 14 species were identified, including Phle- botomus (Phlebotomus) papatasi (61.74%), P. (Paraphlebotomus) sergenti (6.87%), P. (Paraphlebotomus) alexandri (1.00%), P. (Paraphlebotomus) caucasicus (2.55%), P. (Paraphlebotomus) caucasicus group (4.13%), P. (Larroussius) kandelakii (5.72%), P. (Larroussius) tobbi (0.10%), P. (Larroussius) major (5.62%), P. (Adlerius) halepensis (0.10%), P. (Adlerius) brevis (0.05%), P. adlerius group (0.31%), Sergentomyia (Ser-gentomyia) sintoni (10.83%), S. (Sergentomyia) theodori (0.91%) and S. (Rondanomyia) pawlowskii (0.07%) (Table 2). Two species of P. halepensis and the S. pawlowskii were not captured from outdoor and indoor respectively. In the rodent burrows, P. papatasi appeared nearly May and dis- appeared in the late Oct and in indoors, it appeared in the late Jun and disappeared in the early Oct. The month wise density of P. papatasi in rodent burrows and indoors are shown in Fig. 3. The peak of activity was detected for P. papatasi first in the late Jun and the second in the early Aug (Fig. 3). The sex ratio calculates at 83.71 and 62.25 in outdoors and indoors for P. papatasi res- pectively as well as 101.5 in outdoors for S. sintoni. In Aug and Sep 2015 a total of 211 sand flies were collected in the vicinity of rodent burrows including P. papatasi (51.66 %), P. caucasicus (10.9%), P. sergenti (18%), and S. sintoni (19.44%). Two percent of P. papatasi species had promastigote infections. The total of 300 P. papatasi specimens was selected and dissected for Leishmania infection, the result showed 67.33%, and 32.67% dissected sand flies were parous and nulli parous, respectively (Table 3). The analysis of physiological status of dissected P. papatasi revealed (60%) unfed, (20%) blood-fed, (6.67%) semi-gravid and (13.33%) gravid (Table 3). The used Leish- mania primers of the PCR technique suc- cessfully amplified the ITS1 region of the strains L. major and L. tropica revealed that among 360 specimens sand flies examined by PCR, 6 parous specimens (2%) were infected by L. major (Fig. 4, 5). Among 92 collected rodents (67.39%), (8.70%), (4.35%), (17.39%) and (2.17%) were identified as M. libycus, Nesokia indica, Mus musculus, Allactaga elater and Hemiechinus auritis respectively (Table 4). Leishmania major has been detected in 2 (3.23%) out of 62 M. libycus by PCR (Fig. 6). Both of the infected M. libycus were females. All of the 45 human cases, which examined through observation passively, confirmed by PCR but only 15 numbers of them were posi- tive by light microscope. In this study, the rel- ative frequency of CL patients was 0.30%. Twenty-eight (62.22%) of 45 confirmed cases were males and 17 (37.78%) were fe- males. The mean age of patients was 30.78± 16.91. The most of the cases 34 (75.60%) oc- curred in autumn. Eighteen cases (40%) of patients had no positive history of traveling to leishmaniasis endemic areas during the past year. Twenty-two cases (48.89 %) of patients had one lesion. The most common location of lesion was on hands (46%). The results of PCR-RFLP indicated that 45 (100%) cases were infected as L. major (Fig. 7). Table 1. The sequence of primers used in PCR amplification the first internal transcribed spacer (ITS1) Primers The sequence Response function LITSR 5´CTGGATCATTTTCCGATG3´ Forward 18bp 20bp L5 8S 5´TGATACCACTTATCGCACTT3´ Reverse J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 407 http://jad.tums.ac.ir Published Online: September 08, 2017 Table 2. The fauna and the number of collected sand flies from the endemic area in Kahak District, Qom Province, 2015 Site Outdoor Indoor Total Each species of the total Species Male Female Male Female Outdoor Indoor Male Female Total % No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) P. papatasi 915 (45.57) 1093 (54.43) 216 (38.37) 347 (61.63) 2008 (78.10) 563 (21.90) 1131 (43.99) 1440 (56.1) 2571 (100) 61.74 P. sergenti 41 (40.20) 61 (59.80) 131 (71.20) 53 (28.80) 102 (35.66) 184 (64.34) 172 (60.14) 114 (39.86) 286 (100) 6.87 P. alexandri 7 (58.33) 5 (41.67) 11 (36.67) 19 (63.33) 12 (28.57) 30 (71.43) 18 (42.86) 24 (57.14) 42 (100) 1.00 P. caucasicus 45 (100) … 61 (100) … 45 (42.45) 61 (57.55) 106 (100) … 106 (100) 2.55 P. caucasicus group … 157 (100) … 15 (100) 157 (91.28) 15 (8.72) … 172 (100) 172 (100) 4.13 P. kandelakii 23 (31.51) 50 (68.49) 35 (21.21) 130 (78.79) 73 (30.67) 165 (69.33) 58 (24.37) 180 (75.63) 238 (100) 5.72 P. tobbi 1 (100) 0 (0) 0 (0) 3 (100) 1 (25.00) 3 (75.00) 1 (25.00) 3 (75.00) 4 (100) 0.10 P. major 6 (24.00) 19 (76.00) 60 (28.71) 149 (71.29) 25 (10.68) 209 (89.32) 66 (28.21) 168 (71.79) 234 (100) 5.62 P. halepensis 0 (0) … 4 (100) … 0 (0) 4 (100) 4 (100) … 4 (100) 0.10 P. brevis 1 (100) … 1 (100) … 1 (50.00) 1 (50.00) 2 (100) … 2 (100) 0.05 P. adlerius group … 2 (100) … 11 (100) 2 (15.38) 11 (84.62) … 13 (100) 13 (100) 0.31 S. sintoni 207 (50.36) 204 (49.64) 18 (45.00) 22 (55.00) 411 (91.13) 40 (8.87) 225 (49.89) 226 (50.11) 451 (100) 10.83 S. theodori 15 (51.72) 14 (48.28) 6 (66.67) 3 (33.33) 29 (76.32) 9 (23.68) 21 (55.26) 17 (44.74) 38 (100) 0.91 S. pawlowski 0 (0) 3 (100) 0 (0) 0 (0) 3 (100) 0 (0) 0 (0) 3 (100) 3 (100) 0.07 Total 1261 (43.93) 1608 56.07) 543 (41.92) 752 (58.08) 2869 (68.90) 1295 (31.10) 1813 (43.54) 2351 (56.46) 4164 (100) 100 Fig. 1. Map of Iran and Qom Province, highlighting the location of Kahak District, (2) within Qom Province (22, 23) J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 408 http://jad.tums.ac.ir Published Online: September 08, 2017 Table 3. Natural infection of sand flies with promastigote, Kahak District, Qom Province, 2015 S p e c ie s Catch place Physiological status Age Group Total Infected specimens I* O** Unfed Blood fed Semi gravid Gravid Parous Nuli parous No % No % No % No % No % No. % No. % No. % No. % P . p a p a ta si 150 50 150 50 180 60 60 20 20 6.67 40 13.33 202 67.33 98 32.67 300 (100) 6 2% P . se rg e n ti 20 50 20 50 20 50 20 50 0 0 0 0 31 77.50 9 22.50 40 (100) 0 0 P .c a u c a sic u s 10 50 10 50 20 100 0 0 0 0 0 0 18 90 2 10 20 (100) 0 0 *: Indoor, **: Outdoor Fig. 2. Specimens of some collected rodents in Kahak district, Qom Province, 2015 Fig. 3. The month wise density of Phlebotomus papatasi in Kahak District, Qom Province, 2015 Fig. 4. Agarose gel electrophoresis of the first inter- nal transcribed spacer (ITS1) - Polymerase chain re- action (PCR) products. M, 50bp ladder: lane1: Leish- mania major (MRHO/IR/75/ER), 2, 3: Leishmania major in human skin samples, lane 4, 5, 6 Leishmania major in P. papatasi, N: negative control (distilled water), M: Marker Fig. 5. Agarose gel electrophoresis of Polymerase chain reaction-restriction fragment length polymor- phism (HaeIII) products. M, 50bp ladder: lane 1, 2, 3, 4, 5, 6 Leishmania major in P. papatasi, lane 7 posi- tive Leishmania major (MRHO/IR/75/ER), M: Marker J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 409 http://jad.tums.ac.ir Published Online: September 08, 2017 Fig. 6. Agarose gel electrophoresis of the first inter- nal transcribed spacer (ITS1) - Polymerase chain re- action (PCR) products. M, 50bp ladder: lane 1, nega- tive control (distilled water), lane 2, Leishmania ma- jor (MRHO/IR/75/ER), lane 3 Leishmania major iso- lated from Meriones libycus, M: Marker Fig. 7. Agarose gel electrophoresis of Polymerase chain reaction-restriction fragment length polymor- phism (HaeIII) analysis of ITS region for identifica- tion of Leishmania species using M, 50bp ladder: lane1, 2, 3, 4 Leishmania major in human skin sam- ples, lane 5, 6 Leishmania major isolated from Meri- ones libycus, lane 7, positive Leishmania major (MRHO/IR/75/ER), M: Marker Table 4. Molecular detection of Leishmania infection in rodents, Kahak District, Qom Province, 2015 Species No. Rodents No. exanimated No. infected Infected rate,% (ITS1-PCR) N0 % No % Meriones libycus 62 67.39 62 100 2 3.23 Nesokia indica 8 8.70 8 100 0 0 Mus musculus 4 4.35 4 100 0 0 Allactaga elater 16 17.39 4 25 0 0 Hemiechinus auritis 2 2.17 0 0 0 0 Total 92 100 78 84.78 2 2.17 Discussion We identified L. major in P. papatasi, M. libycus and human by PCR technique. Previ- ous surveys carried out in Qomrood and Gha- navat districts of Qom Province, Iran showed that P. papatasi and M. libycus were proven (in Qomrood) and probable (in Ghanavat) vector and reservoir of ZCL (9, 10). Aware- ness of epidemiology of ZCL is an important measure for management and planning of control (28). The entomological and reser- voirs (rodents and human) fields and labora- tory surveys accompanied by correct identifica- tion the common agent in their target part of their bodies is a major component for combat- ing against disease. Recently, genetic analyses and genotyp- ing using PCR-RFLP have been applied and performed on vector (s) and reservoir hosts of ZCL (29). This paper reviews recent advanc- es on sand flies vectors, M. libycus and human of ZCL, using molecular biological approaches. P. papatasi captured in this study is the main vector of L. major from animals to man in central Asia and appears to be the vector of L. major in Iran and 21 other countries in old world (30). In this study, P. alexandri was also cap- tured. It has been found naturally infected with promastigotes and is suspected vector of VL in Iran (31). Phlebotomus caucasicus group, P. kandelaki and P. major were also collected. Phlebotomus caucasicus was naturally infect- ed with promastigotes in a new focus of VL in north-west of Iran (32). Phlebotomus kan- delaki is naturally infected with Leishmania sp. promastigotes in northwest Iran and is sus- J Arthropod-Borne Dis, September 2017, 11(3): 403–413 A Saghafipour et al.: Epidemiological Study on … 410 http://jad.tums.ac.ir Published Online: September 08, 2017 pected as probable vector of VL in the region (33). In Iran, P. major has been found in all area where human cases of VL have been re- ported and natural promastigote infection of this species has reported in endemic focus VL in Ghir County (Fars Province) South of Iran (34). Moreover, in this area, we captured S. povlovskyi in indoor. This result has been confirmed with the previous study in Esfahan (35). The identified species were consistent with previous study in other parts of Iran (1). Low percentage of collected sand flies in indoor shows people prevent sand flies to en- ter their homes. P. papatasi was the predom- inant species of the genus Phlebotomus in in- doors (31.10%) and outdoors (68. 90%). This suggests that P. papatasi is the anthropophilic species in this area. In addition, only P. papa- tasi is able to develop L. major in its mid gut and transfer the parasite to its proboscis to cause ZCL (36). S. sintoni enters the indoors because of nearby homes to rodent burrows (37). P. major and P. kandelakii are more dominant sand flies were collected from in- door after P. papatasi but due to the low fre- quency. These species do not involve in leish- maniasis transmission. In the present study, we distinguished par- ity of sand flies by observation of the appear- ance of the accessory glands (26). Accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of P. papatasi females (38). In this study, the parous rate of P. papatasi was 67.33%, so it is not surprising to see high level of pro- mastigote infection among this sand fly spe- cies. The same trend of infection is reported from north Siani in Egypt (39, 40). The results of dissection showed that 2% of P. papatasi species were naturally infected with pro- mastigote infection. The Leishmania infec- tion rate in sand flies is usually very low even in endemic areas (41). The low density of Leishmania parasites in M. libycus in this area may be due to low sensitivity of this rodent compared to R. opimus (4). In the present study, based on molecular results, L. major was found in M. libycus as a reservoir, P. papatasi as a vector and sus- pected patients as incidental host. M. libycus as the main reservoir host of ZCL has been distributed in the northeastern, central and southwestern regions in Iran (42, 43). For the first time in 1996, isolation and characteriza- tion of L. major from M. libycus in Iran, has been reported (4). Meriones libycus has pre- viously been infected with L. major parasites from Golestan, Esfahan and Yazd Provinces (7, 42, 44-46). Leishmania major was firmly identified in M. libycus that indicates this ro- dent species can be incriminated as reservoir host of ZCL in this location (47). The results of this study have shown, M. libycus is the main and potential reservoir host of ZCL in Kahak district of Qom Province and it has an im- portant role in stable ZCL in this area, which has pay attention to future rodent control pro- grams with administrative health center. Conclusion This study confirms the existence of local transmission of zoonotic cycle of CL in Kahak District of Qom Province, and pro- vides that in this cycle, L. major is the causa- tive agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease. 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