J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 134 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Emerging of Cutaneous Leishmaniais due to Leishmania major in a New Focus in Esfahan Province, Central Iran Reza Jafari1; Hamid Abdoli1; Mohammad Hossein Arandian1; Nilofar Shareghi1; Maryam Ghanei1; Nilofar Jalali-Zand1; Shahram Nekoeian2; Arshad Veysi3, Ahmad Montazeri4; Amirabdollah Ghasemi4; Javad Ramazanpour2; Reza Fadaei2; *Amir Ahmad Akhavan5 1 Esfahan Health Research Station, School of Public Health, Tehran University of Medical Sciences, Esfahan, Iran 2 Esfahan Province Health Center, Isfahan University of Medical Sciences, Esfahan, Iran 3 Zoonoses Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran 4 Naein Health Care Network, Isfahan University of Medical Sciences, Esfahan, Iran 5 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 06 Dec 2018; accepted 04 Feb 2020) Abstract Background: Esfahan Province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Recently, ZCL distribution is expanding through this province leading to report of new cases in non-endemic areas. In the current study epidemiological aspects of ZCL has been investigated in Naein County in Esfahan Prov- ince. Methods: Adult sand flies were collected from beginning to the end of their seasonal activity. Rodents were caught by Sherman live traps once a month for one year. To active case detection, a hundred households in each selected village were visited in November and December 2016. Nested-PCR was employed to detect Leishmania parasite in the vector, reservoir and human. Results: Totally 1562 sand flies including Phlebotomus sergenti, Phlebotomus papatasi, Sergentomyia sintoni and Sergentomyia mervinae were collected and identified. No Leishmania infection was detected in the collected sand flies. All of the 30 collected rodents were identified as Rhombomys opimus, and of these 3.3% and 26.7% were in- fected by Leishmania major using microscopic and molecular technique respectively. Totally, 914 individuals were investigated and the ulcer and scar rates of ZCL calculated to be at 1.1 and 15.3 per 1000 population, respectively. Molecular results confirmed L. major infection in human and reservoir samples. Conclusion: It is concluded that ZCL is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in the near future in this region. Keywords: Cutaneous leishmaniais; Leishmania major; Epidemiology; Central iran Introduction Although estimated to cause ninth largest disease burden among infectious diseases, leishmaniasis is mostly neglected among tropical diseases. Based on a recent report, it is estimated that, annually 0.2 to 0.4 and 0.7 to 1.2 million visceral leishmaniasis (VL) and cautaneus leishmaniasis (CL) new cases, oc- cur respectively in the world (1). High level of reported cases listed Iran in ten countries which together account for 70 to 75% esti- mated cases of CL globally (1). More than 20000 cases of CL cases are reported in Iran annually, although the actual figures are es- timated to be 4 to 5 folds (2). Cutaneous leishmaniasis manifest in two forms in the country, zoonotic (ZCL) and anthroponotic CL (ACL), and about 80% of reported cases has been ZCL form; especially in the rural *Corresponding author: Dr Amir Ahmad Akha- van, E-mail: aaakhavan@tums.ac.ir http://jad.tums.ac.ir/ https://www.google.com/search?q=cutaneous+leishmaniasis&spell=1&sa=X&ved=0ahUKEwi_3-_dkLXZAhXK66QKHaswBoAQkeECCCQoAA https://www.google.com/search?q=cutaneous+leishmaniasis&spell=1&sa=X&ved=0ahUKEwi_3-_dkLXZAhXK66QKHaswBoAQkeECCCQoAA mailto:aaakhavan@tums.ac.ir J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 135 http://jad.tums.ac.ir Published Online: June 30, 2020 areas of the country (3). Zoonotic cutaneous leishmaniasis is caused by a protozoan para- site Leismania major, and Phlebotomus pa- patasi plays as the main vector in the disease foci inside the country. Seventeen out of 31 province deal with the disease (4). Rodents belonging to Gerbillinae subfamily play as reservoir host of ZCL in different part of the country, as well as some other parts of the world. Rhombomys opimus (great gerbil) known as the main reservoir host of ZCL in central and the northeast of Iran (5). Esfa- han Province known as one of the most im- portant foci of ZCL, and in 2012 this prov- ince was categorized as one of the high re- ported cases of ZCL (6). Since the last two decades, Iranian scientists along with health authorities have employed and conducted several measures such as spraying of rodent burrows with pesticides (7), baiting the res- ervoir hosts with rodenticides (8-13) and using deltamethrin-impregnated bed nets and curtains (14, 15) to control ZCL in the coun- try. In addition, despite extensive research in the field of vaccine, still there is no licensed vaccine against any form of leishmaniasis (16). Iranian researchers have conducted a successful leishmanisis prevention through leishmanization, unfortunately it was banned due to very rare case of un-healing lesions, and since then it was recommended just for military personnel in the high risk areas (17). From 2012 in Naein County the number of indigenous cases which were visited in the Health Centers with signs of cutaneous leish- maniais gradually increased and based on local health authorities report, surprisingly the confirmed recorded cases increased to 26 cases in 2015. Considering the importance of this county as a main roads intersection of the country and passengers commuting, it is assumed, without taking serious measures to conduct research and control of the disease in this region, it might become a potential fo- cus to spread ZCL to other free areas. Regard- ing this issue as well as lacking any compre- hensive study on ZCL in this county, con- ducting an epidemiological research seemed to be necessary. Hence, this survey is the first epidemiological study that has ever been conducted in these areas. The current study aimed to study the different epidemiological aspects of the disease in this new emerging focus of ZCL. Materials and Methods Study area The present study was carried out in Naein County (32°51'49.1"N 53°05'04.5"E), Esfahan Province, Iran from October 2015 to February 2016. Naein is located at 170km from north of Yazd and 140km from east of Esfahan with an area about 22,570km2 and it is considered as the widest County in Es- fahan Province (Fig. 1). The selected areas have desert climate, hot in summer and cold in winter. In 2015 the maximum and minimum mean monthly temperature was 40.8 and -10.6 °C in July and December, respectively; with the total rainfall of 40.8mm. Entomological survey Adult sand flies were collected from the beginning (April) to the end (November) of their active season, once a month using sticky traps in the rural selected areas (Bafran, Mo- hamadieh and Mazraee Emam) of Naein. The sticky traps (15 in outdoor resting places and 15 in indoors in each selected point) were in- stalled before sunset and collected next morn- ing before sunrise in three selected villages. The captured sand flies were preserved in eth- anol until the time of use. Head and the last two abdomen segments of sand flies detached, mounted in Puri’s media and the rest of the sand flies’ body was kept in 96% alcohol to examine for Leishmania infection using mo- lecular techniques. The species identified us- ing valid keys based on morphological char- acters (18, 19). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 136 http://jad.tums.ac.ir Published Online: June 30, 2020 Rodent collection and parasitological ex- amination Active colonies of gerbils have been found around Bafran, Mohamadieh and Mazraee Emam villages of Naein. Rodents were caught by 40 Sherman live traps, baited with cu- cumber and/or carrot, once a month along a year. During spring to fall, the traps were in- stalled in the vicinity of the gerbil holes in the afternoon and collected at the following morning, while during winter traps were in- stalled at early mornings and collected at the afternoons. The collected gerbils were trans- ferred to the Esfahan Health Research Station to further examination. The captured rodents were anaesthetized using Ketamine hydrochlo- ride (60mg/kg), and Xylazine (5mg/kg) and were identified by morphological characters (20). Then impression smears were prepared from the ear lobes of the anaesthetized ani- mals, fixed by absolute methanol and stained by Giemsa, and then the slides were micro- scopically examined at high magnification (1,000x). Afterward the ear lobes were cut and transferred into 500μl cold PBS (pH= 7.4), and then disrupted by grinding using a pestle and stored in -20 °C until use. Animal ethics was considered in treating with animals during this study. Human infection To determine the prevalence and incidence of the disease in the selected village areas (Bafran, Mohamadieh and Mazraee Emam) of Naein, 100 households in each village were visited in November and December 2016. Ep- idemiologic data including ID, presence or ab- sence of scar (s) or active lesion (s), number of the lesion (s) or scar (s), and history of travelling to the other ZCL foci were filled out in a questionnaire for all households. In- dividuals who had travelled to other foci of ZCL were excluded from the study. New cas- es of the disease and the number of active lesions were recorded on each visit. Samples were prepared from the active lesions; be- fore sampling, lesions were disinfected with alcohol 70% then the margin of ulcer was scratched using vaccinostyle. The prepared se- rosity was allocated in two parts, one part transferred into alcohol 96% for molecular parasite detection and another part was smeared on a slide, fixed with methanol, stained by Giemsa and examined under microscope at high magnification (1,000x). Molecular detection of Leishmania infection Genomic DNA from samples of rodents, human and sand flies was extracted by Ge- neAll® Exgene TM Tissue kit (Cat No: 101– 109) following the protocol of cultured ani- mal cell or lymphocytes. The ITS2 region of Leishmania parasite was amplified by Nest- ed-PCR, using the following primers (21) leish out F (5′-AAA CTC CTC TCT GGT GCT TGC-3′), Leish out R (5′-AAA CAA AGG TTG TCG GGG G-3′), Leish in F (5′- AAT TCA ACT TCG CGT TGG CC-3′) and Leish in R (5′-CCT CTCTTT TTT CTC TGT GC-3′). The targeted gene of ribosomal DNA was amplified by a Biosystems thermocycler. The volume of first reaction micro-tube of PCR was 25μl containing 0.6μM of both for- ward (Leish out F) and reverse (Leish out R) external primers, 12.5μl Taq DNA polymer- ase enzyme, 2X Master Mix (Amplicon, Den- mark) and sterile distilled water. The first de- naturation step was carried out at 95 °C for 5min and was followed by 30 cycles of de- naturation at 94 °C for 30s, annealing at 60 °C for 45s and extension at 72 °C for 1min, finally ended with a final extension step of 72 °C for 5min. The second step of nested- PCR was carried out in a volume of 20μl containing 1μl of a 1:25 dilution of distilled water and the first-round PCR product as a template, 0.3μM of each forward and reverse internal primers, 10μl of Taq DNA polymerase and 2X Master mix. The amplification was performed as initial denaturation at 95 °C for 2min, 25 cycles of 94 °C for 15s, 62 °C for 30s, 72 °C for 45s followed by the final ex- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 137 http://jad.tums.ac.ir Published Online: June 30, 2020 tension at 72 °C for 5min. Finally, PCR prod- ucts were loaded on 1.5% (w/v) agarose gel electrophoresis in TBE buffer (0.09mM Tris, 0.09mM boric acid, and 20mM EDTA, pH= 8.3); ethidium bromide (0.5μg/ml) was used to visualize the amplified DNA bands on the gel and photographs were taken. Leishmania major (MRHO/IR/75/ER), and distilled water were used as positive and neg- ative controls respectively. To prevent any ac- cidental contamination, primary precautions such as using filter pipette tips, and steriliz- ing equipment by 10% sodium hypochlorite solution were taken. Polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) To distinguish common species of Leish- mania genus, the products of Nested-PCR were subjected to RFLP using Rapid Digest Mnl1 (Cat. No: RD 1191) restriction enzyme. To this purpose, 10µl PCR products of the nest- ed-PCR, 2µl Buffer, 1µl Mnl1 restriction en- zyme, were added to a micro tube and reached to final volume of 30μl by distilled water. To activate the restriction enzyme, the mixture was kept in 37 °C for 1 hour, and then the products were loaded onto 2.5% (w/v) agarose gel elec- trophoresis in TBE buffer. Finally loaded DNA was visualized by ethidiumbromide (0.5µg/ ml), and photographed. Results Entomological survey From April to November 2016, totally 1562 sand flies (1451 from outdoors, 111 from in- doors) were collected and identified based on the valid morphological keys. Four species of sand flies including two species of Phlebotomus genus and two species of Ser- gentomyia genus were identified as follows: Phlebotomus (Paraphlebotomus) sergenti: This sand fly was the dominant species in indoors, which 70 (63.1%) and 8 (0.6%) of collected sand flies in indoors and outdoors were belonged to this species respectively. Phlebotomus sergenti was caught in all months of sand flies active season in indoor places. The maximum relative frequency of this phlebotomine in indoor places was recorded in June (Fig. 2). Phlebotomus (Phlebotomus) papatasi: The number of caught sand flies belonging to this species were 34 (30.6%) and 48 (3.3%) in in- doors and outdoors respectively. Phlebotomus papatasi was caught in all months of sand flies active season in either indoors or outdoors. The monthly activity of this species recorded a re- markable pick in June, both in indoor and outdoor places (Fig. 3). Sergentomyia (Sergentomyia) sintoni: The results showed Se. Sintoni was the most dom- inant species in outdoor places. Of all col- lected sand flies, 4 (3.6%) from indoor and 1002 (69%) from outdoors places were iden- tified as Se. sintoni. Similarly, the maximum activity of this species occurred in June (Fig. 4). Sergentomyia (Sergentomyia) mervinae: This species comprised 3 (2.7%) of the sand flies from indoors, and 393 (27.1%) from outdoor resting places. Similarly, the maxi- mum number of this species was caught in June (Fig. 4). To find Leishmania infection among Ph. papatasi randomly 30 females were examined by nested-PCR technique. Results of molec- ular experiment showed none of the sand flies were infected by Leishmania parasite. Sufficient numbers of Ph. sergenti from outdoors and Se. sintoni and Se. mervinae from indoors were not collected to demon- strate monthly activity. Reservoir host investigation Totally 30 desert rodent were collected by Sherman live-tarps during a year, and all identified as R. opimus based on the mor- phological characteristics. Out of 30 exam- ined rodents, only one (3.3%) was infected http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 138 http://jad.tums.ac.ir Published Online: June 30, 2020 by amastigote form of Leishmania through microscopic examination. In addition, by us- ing molecular examination eight (26.7%) of the captured rodents were found to be infect- ed by L. major (Fig. 5). Human infection survey To determine scar rate and ulcer rate of ZCL among inhabitants, active case finding was carried out at the end of fall in Moham- adieh, Bafran and Mazraee Emam villages. Taken together, 914 individuals including 449 males (49.1%) and 465 females (50.9%) were investigated and demographic data were rec- orded as well. Among all visited individuals only one woman (0.1%) in ≥25 age-group had one active lesion of ZCL on her hand. Out of 914 examined persons, 14 individuals (1.5%) had scar which eight (1.8%) were man and six (1.3%) were woman belonging to ≥ 25 age-group. The distribution rate of scar site/s on the body of examined people is shown in Fig. 6. The ulcer and scar rates of ZCL calculat- ed to be at 1.1 and 15.3per 1000 population, respectively (Table 1). Molecular experi- ments on one sample of active lesion con- firmed L. major infection. Table 1. The prevalence of scar rate of the examined population in the studied villages, Naein County, Esfahan Province, Iran, 2011 Age groups (yr) Male Female Total No. ob- served No. of scar/s No. ob- served No. of scar/s No. ob- served No. of scar/s No % No % No % 0–4 22 0 0 25 0 0 47 0 0 5–9 29 0 0 24 0 0 53 0 0 10–14 25 0 0 26 0 0 51 0 0 15–19 26 0 0 25 0 0 51 0 0 20–24 49 0 0 42 0 0 91 0 0 ≥ 25 298 8 2.7 323 6 1.9 621 14 2.3 Total 449 8 1.8 465 6 1.3 914 14 1.5 Fig. 1. Map of the studied areas, Naein County, Es- fahan Province, Iran M a y J u n J u l A u g S e p O c t 0 1 0 2 0 3 0 4 0 5 0 M o n t h N o . p e r 3 0 t r a p s Fig. 2. Monthly fluctuation of Phlebotomus sergenti in indoors of the studied areas, Naein County, Esfa- han Province, Iran, 2011 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 139 http://jad.tums.ac.ir Published Online: June 30, 2020 M a y J u n J u l A u g S e p O c t 0 1 0 2 0 3 0 M o n t h s N o p e r 3 0 t r a p s I n d o o r O u t d o o r Fig. 3. Monthly fluctuation of Phlebotomus papa- tasi in studied areas, Naein County, Esfahan Prov- ince, Iran, 2011 M a y J u n J u l A u g S e p O c t 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 M o n t h s N o p e r t r a p S e . s i n t o n i S e . m e r v i n a e Fig. 4. Monthly fluctuation of Sergentomyia sintoni and Sergentomyia mervinae in outdoors of the stud- ied areas, Neain County, Esfahan Province, Iran, 2011 Fig. 5. Gel electrophoresis of nested-PCR targeted sequences of DNA extracted from the smears of the captured rodents. M; 100bp DNA ladder (Fermentas), 2, 7, 8, 9 and 12 showing the samples were positive for Leishmania major. Pos; positive L. major reference. Neg; negative control 7 7 .0 8 % H a n d 1 8 . 7 5 % L e g 4 . 1 7 % F a c e Fig. 6. The percent of scars on the body of visited inhabitant in the studied areas, Neain County, Esfa- han Province, Iran, 2011 Discussion The current study is the first epidemio- logical survey on zoonotic cutaneous leish- maniasis in Naein County, Esfahan Province. In the current study four species of sand flies including two species of Phlebotomus and two species of Sergentomiya genus were collected and identified. In indoor places Ph. sergenti with the 63.1% density was the most abundant species and Ph. papatasi with 30.6 % density ranked in the second place. These two species were caught throughout the sand flies’ active season. Generally, Sergentomy- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 140 http://jad.tums.ac.ir Published Online: June 30, 2020 ia species prefer to feed on cold-blooded vertebrates, thus they are known as vectors of reptile Leishmania species. It is widely believed, they cannot transmit Leishmania to human, although promastigote infection of Se. sintoni have shown by direct examination previously (22). However, they are usually occurred sympatrically with Phlebotomus spe- cies, even though in some cases, same to this study, they have higher density comparing to Phlebotomus genus. Hence, the role of these sand flies in transmitting of human leish- maniaisis needs more investigation. Majori- ty of studies conducted in central Iran have shown Ph. papatasi as the predominant spe- cies in either indoors and outdoors places (23, 24). In agreement with our result, gen- erally it has been shown there were two peaks in the density curve of the most spe- cies in the hot and arid areas of central part of the country; one in June or July and the second in August or September (23, 24). Of 30 Ph. papatasi were passed to nested-PCR experiment none of them were infected by Leishmania parasite. Natuaral Leishmania infection has been reported from Ph. papa- tasi, Phlebotomus caucasicus, Phlebotomus ansarii and Se. sintoni (22-24), and also L. major was detected in Ph. papatasi using molecular methods (25-27). In the current study out of 30 captured R. opimus, 8 (26.7%) were infected by L. major using nested-PCR, and reconfirmed by PCR- RFLP technique. Till now, several studies have conducted in the country showed Leish- mania infection in R. opimus by either direct examination or molecular methods (5, 28-30). As the results showed, the detected Leishma- nia species from R. opimus and human le- sion was the same. In the previous studies L. major has been isolated from naturally infect- ed Ph. papatasi, Ph. caucasicus, R. opimus, Meriones libycus and human in endemic ar- ea of ZCL in the country (5). Ulcer and scar rates among surveyed pop- ulation were 0.1% and 1.5% respectively, these rates were reported as 1.3% and 3.26% in other county of Esfahan Province (31). The most infected age-group was ≥ 25, while in the previous study was 10–14 age-group (31). In an epidemic situation, the rates of ul- cer and scar were reported as 3% and 10.4% respectively, and also the most infected age- group was 0–4 years old (22). This finding showed high level of en- dophilic behavior for Ph. sergenti. It is worth to mention that high density of Ph. sergenti, the main vector of ACL, in indoors com- panioning to a case of ACL or dogs infected by L. tropica in the areas, pose a potential threat of ACL establishment in the region. On the other hand, despite the high density of Ph. sergenti and its refractory to L. major, so it cannot play a role in transmitting of L. major in the areas. Considering the results of the current study, this area is not categorized as a high risk area for ZCL transmission due to L. major. Due to low density and no Leishmania infection in potential vector of ZCL, low population of reservoir host, spo- radic cases and lack of history of the disease below 25 years old group, this area could be considered as a low risk focus. Conclusion It is concluded that ZCL has established in low endemicity in this area. It is extrapo- lated that, the disease will not be a serious health problem in the near future in this re- gion. Based on the results of this study, pres- ently no vector and/or reservoir host control is recommended. Only treatment of rare new cases with active lesion following the na- tional protocol is recommended. Considering climate changes and ecological status, con- sequent probable epidemiological change of ZCL is not unexpected, so that continuing of disease surveillance to take urgent action in special condition is recommended. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2020, 14(2): 134–143 R Jafari et al.: Emerging of Cutaneous … 141 http://jad.tums.ac.ir Published Online: June 30, 2020 Acknowledgements Authors wish to appreciate the personnel of Naein County Health Centre, Esfahan Uni- versity of Medical Sciences, for their kind collaboration in the field operation. This re- search was financially supported by Nation- al Institute of Health Research, Tehran Uni- versity of Medical Sciences (No. 93216/M/ 241). The authors declare that, there is no con- flict of interest. References 1. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, de Boer M (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7(5): e35671. 2. 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