J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 233 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Original Article Molecular Detection of Leishmania Infection in Phlebotomine Sand Flies from an Endemic Focus of Zoonotic Cutaneous Leishmaniasis in Iran Sadegh Mohammadi-Azni1, Mohsen Kalantari2, *Behrad Pourmohammadi3,4 1Damghan Health Center, Semnan University of Medical Sciences, Semnan, Iran 2Research Center for Health Sciences, Institute of Health, Department of Medical Entomology and Vector Control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran 3Department of Parasitology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran 4Research Center for Health Sciences and Technologies, School of Health, Semnan University of Medical Sciences, Semnan, Iran *Corresponding author: Dr Behrad Pourmohammadi, E-mail: behrad89@gmail.com (Received 08 Oct 2020; accepted 25 July 2022) Abstract Background: Due to the outbreak of zoonotic cutaneous leishmaniasis (ZCL), a disease caused by Leishmania major and mainly transmitted by Phlebotomus papatasi, in Damghan City, Semnan Province, the probable vectors of the dis- ease were investigated in the city from 20 March 2016 to 20 January 2018. Methods: Sand flies were collected from indoors and outdoors biweekly by sticky traps in different parts of the city. The trapped sand flies were stored in 70% ethanol. They were identified and checked for Leishmania infections using nested-PCR method and specific primers; CSB1XR, CSB2XF, LiR, and 13Z. Results: Overall, 1862 phlebotomine sand flies of Ph. papatasi (48.8%), Ph. andrejevi (8.3%), Ph. caucasicus (7.7), Ph. mongolensis (2%), Ph. sergenti (1.2%), Ph. alexandri (0.7%), Sergentomyia murgabiensis sintoni (29.3%), and Se. sumbarica (2%) were collected indoors (31.1%) and outdoors (68.9%). The highest and lowest numbers of collected sand flies were belonging to Ph. papatasi (48.8%) and Ph. alexandri (0.7%) respectively. 2.2% of the examined sand flies were shown to be infected with L. major and all were belonging to Ph. papatasi. Conclusion: This study confirms the report of Ph. papatasi infection with L. major and also the existence of Ph. ser- genti and Ph. alexandri, the potential vectors of L. tropica and L. infantum respectively, in Damghan City. According to the findings, it is necessary for health officials to plan and take action to prevent the occurrence of ZCL epidemic in the city as well as the occurrence of other forms of leishmaniasis. Keywords: Molecular survey; Leishmania major; Sand fly; Nested PCR; Damghan Introduction Leishmaniasis, a protozoan parasitic infec- tious disease transmissible by the bite of the subfamily phlebotomine (Diptera: Psychodidae) sand flies and classified in the seventeenth ne- glected tropical diseases (NTD), occurs in trop- ical and subtropical areas of some 98 coun- tries of the world (1). More than 350 million people are living in areas at risk of leish- maniasis and an estimated 2 million new cas- es of the disease occur annually. Distribution and incidence of leishmaniasis is not the same in endemic areas. Seven countries; Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria have about 90% of cutaneous leishman- iasis (CL) cases (2). Two clinical types of cutaneous leishman- iasis including anthroponotic cutaneous leish- maniasis (ACL) and zoonotic cutaneous leish- maniasis (ZCL) with causative agents of Leish- mania tropica and L. major respectively, have been reported from several parts of Iran (3, 4). Cutaneous leishmaniasis is the main vector- borne disease in Iran with an annual average of more than 22,000 cases that about 80% of Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ file:///G:/JAD/NO.%2039/1330-1361%20%20%20Pourmohammadi/behrad89@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 234 http://jad.tums.ac.ir Published Online: Sep 30, 2022 them are zoonotic form. The endemic foci of this type of the disease are in rural areas of 18 out of 31 provinces (5, 6). Incidence of CL in Damghan District had been reported around 111 per 100,000 in 2009 (7). There are more than 800 phlebotomine sand fly species, but 166 species are proven or sus- pected vectors of human leishmaniasis in the world. Furthermore, about 50 species of them have been found to be naturally infected with Leishmania species and have been introduced as the vectors of leishmaniasis (8-10). Gerbil rodents (Muridae: Gerbillinae) are the main res- ervoir hosts of ZCL in the most areas of Iran (5, 11, 12) including Damghan District (13, 14). Iranian sand flies includes 48 species in two genera of Phlebotomus and Sergentomy- ia. Each genus has 6 subgenera in the Country (15). The subgenus Sergentomyia have no role in the transmission of leishmaniasis to human. They are vectors of lizard leishmania- sis. Phlebotomus papatasi is the main vector of ZCL, while Ph. salehi is also found to be infected with L. major in some foci of the dis- ease. Phlebotomus salehi is mainly reported in the southern part of the country. Phleboto- mus ansari, Ph. salehi and Ph. caucasicus group are known to have a role in zoonotic cycle of CL between rodents (5, 16). Phleboto- mus alexandri have been reported in the most studies on sand flies in Iran and it is found to be infected with L. infantum (17). Damghan District in in Semnan Province, northern Iran. It is situated 342km east of Tehran on the high-road to Mashad, at an ele- vation of 1,250m above sea level. At the 2016 census, the County's population was 94,190 in 30,296 households. This County is an im- portant focus of ZCL, with annually more than 200 new cases of the disease in the last years (18). An outbreak of the disease, with about 1500 cases, has occurred in the rural areas of Damghan District in 1999 (18). Rodents such as Rhombomys opimus, Nesokia indica and Meriones libycus have been reported as the main reservoir hosts (14, 19) and Ph. papatasi has been introduced as the main vector of the disease in rural areas of the district (20). At the beginning of the disease outbreak, leishmaniasis was only limited to the rural areas, but in recent years, ZCL agents were detected in the patients who had previously traveled to the endemic rural areas of Damghan (7, 18). This finding reinforces the hypothesis that the disease is spreading to the city and that there is a need to consider the necessary measures to pre- vent and control the disease. For vector control strategies, it was necessary to detect the leish- mania parasites in the phlebotomine sand flies as the vectors of ZCL in Damghan focus. There- fore, this study was designed and conducted to identify the vectors (sand fly species) and their infection with Leishmania parasites in the city. Materials and Methods Study area This descriptive cross-sectional study was conducted during 20 March 2016 to 20 Janu- ary 2018 in different parts of Damghan City, in Semnan Province. Damghan is located in 1170m above sea level and 350km east of the capital, Tehran, in northern part of Iran (13). The city is situated between Shahroud and Semnan Cities and is bounded on the moun- tainous areas in the north (Alborz Mountains) with 9.8 ℃ and the plain areas in the south (Kaveer Desert) with 23.5 ℃ average temper- atures. The annual rainfall and average tem- perature of the city are about 120mm and 16.3 ℃ respectively (21). The city has generally a warm weather in summer and cold in winter. Sand fly collection Sand flies were collected from indoors (bed- room, guestroom, toilet and stable) and outdoors (e.g. rodent burrows and wall cracks) biweek- ly by sticky traps in different parts of the city. In each sampling, 300 sticky paper traps (30 indoors and 30 outdoors) were fixed in the sun- set and collected in the next morning before sunrise. Phlebotomine sand flies were collect- http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/Iran https://en.wikipedia.org/wiki/Tehran https://en.wikipedia.org/wiki/Mashad J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 235 http://jad.tums.ac.ir Published Online: Sep 30, 2022 ed and kept in ethanol (70%). The head and last abdominal segments of female sand flies were removed and mounted on a microscope slide in a drop of Puri’s medium, and taxo- nomically identified according to valid taxo- nomic criteria (22). Each sample identified at species level, according to the standard identi- fication keys (23, 24). The remaining portion of the female sand flies′ bodies were subjected to DNA extraction and molecular detection of Leishmania infection. Molecular identification Totally 179 samples of the female sand flies were selected for molecular detection of leishmania infection. Each female sample was separately checked for Leishmania infection by nested-PCR assay using specific primers (25). DNA Extraction Each sample were homogenized in a mix- ture of 200μl lysis buffer (50μl Tris-HCl [pH 7.6], 1% Tween 20 and 1μl EDTA), 12μl of proteinase K solution (19μl of the enzyme/ ml), in a 1.5ml sterile microcentrifuge tube. The homogenate incubated at 37 °C overnight, and then 300μl phenol: chloroform: isoamyl alcohol mixture (25:24:1, by vol.) were add- ed. After shaking vigorously, the mixture was centrifuged (10,000 RPM for 10min). The ex- tracted DNA in the supernatant was precipi- tated with 400μl of cold and pure ethanol, re- suspended in 50μl of double-distilled water, and stored at -20 °C until using for detection of Leishmania parasites′ kDNA. Amplification of Kinetoplast Minicircle DNA Nested PCR assay was carried out in two rounds using the CSB1XR (ATT TTT CGC GAT TTT CGC AGA ACG) and CSB2XF (CGA GTA GCA GAA ACT CCC GTT CA) primers for the first round and LiR (TCG CAG AAC GCC CCT) and 13Z (ACT GGG GGT TGG TGT AAA ATAG) for the second round. First, a total reaction mixture (25μl) was prepared which contained 5μl of template DNA, 12μl Master mix (containing deoxynu- cleoside triphosphate (Sinaclon, Tehran, Iran), Taq polymerase, MgCl2, Tris-HCl (pH 7.6)), 1μl of CSB1XR and 1μl of CSB2XF, and 6μl of Doubled Distilled Water (DDW). PCR re- action protocol was set in a thermocycler (Ep- pendorf AG; Humbug, Germany). The tem- perature program was set at 94 °C for 5min for the first extension, followed by 30 cycle (which was repeated at 94 °C for 30s, 55 °C for 1min, and 72 °C for 1.5min), and then a fi- nal extension at 72 ° C for 10min. For the sec- ond round, 1μl of the first-round products’ di- lution (1:9, by vol.) was used as the templates. The reaction for the second round was the same as the first round with an exception in the vol- ume of the reaction mixture changed to 30μl (as the DDW volume was changed from 6μl to 11μl for the second round), and the use of 13Z and LiR primers. Finally, 5μl of the final products were run on 1.5% (V/V) agarose gel marked with ethidium bromide and visualized by ultraviolet trans-illumination. The size of bands was estimated by comparison with the size of the reference strains (12, 25). Reference strains of L. major (MHOM/IR/ 54/LV 39), L. infantum (MCAN/IR/96/Lon 46) and L. tropica (MHOM/IR/89/ARD 2) were used as standards from Department of Medi- cal Parasitology, Shiraz University of Medical Sciences. Also, double-distilled water was in- cluded in each run as a negative control. A band of 560bp, 680bp, and 750bp indicated the presence of L. major, L. infantum and L. trop- ica respectively. Results Totally, 1862 sandflies (822 females and 1040 males) including 8 species (six Phleboto- mus spp. and two Sergentomyia spp.) were trapped and identified in a period of 22 months. They were belonging to; Ph. papatasi (48.8%), Ph. mongolensis (2%), Ph. caucasicus (7.7%), Ph. sergenti (1.2%), Ph. alexandri (0.7%), Ph. andrejevi (8.3%), Se. murgabiensis sintoni (29.3 %) and Se. sumbarica (2%) (Table 1). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 236 http://jad.tums.ac.ir Published Online: Sep 30, 2022 The sand flies′ sex ratio (ratio of male to female) was 126.5/100. Totally, 580 (31.1%) and 1282 (68.9%) specimens were collected from the indoor and the outdoor places respec- tively. 43.2% of the sandflies collected from the outdoors and 61% from the indoors were belonging to P. papatasi. 52.3 percent of the abdominal status of sand flies were empty, and the others were blood fed (10.3%), gravid (16%), and semi gravid (21.4 %) (Table 1). Four (2.2%) of the 179 molecularly exam- ined sand flies were shown to be infected with L. major parasites with band size about 560bp (figure 1). Of which, one was from indoor, 2 from rodent barrows and another one was from outdoor. 75% (3/4) of infected sandflies were collected in August and September, but 25% (1/4) were trapped in June. Table 1. Details of sand flies collected in Damghan City, 2016–2018 Outdoor (%) Indoor (%) Abdominal status No. (%) Female No. (%) Male No. (%) Total No. (%) Species Gravid Semi-gravid Empty Blood fed 554 (61) 354(39) 96 (26) 75 (20.4) 147 (40) 50 (13.6) 368 (40.5) 540 (59.5) 908 (48.8) Phlebotomus papatasi 35 (94.6) 2(5.4) 1 (12.5) 2 (25) 3 (37.5) 2 (25) 8 (21.6) 29 (78.4) 37 (2) Phlebotomus mongolensis 122 (85.3) 21(14.7) 3 (11.1) 7 (26) 11 (40.7) 6 (22.2) 27 (18.9) 116 (81.1) 143 (7.7) Phlebotomus caucasicus 12 (100) - - - 2 (100) - 2 (16.7) 10 (83.3) 12 (0.7) Phlebotomus alexandri 15 (68.2) 7(31.8) - - 4 (100) - 4 (18.2) 18 (81.8) 22 (1.2) Phlebotomus sergenti 142 (91) 14(9) 5 (16.7) 8 (26.6) 12 (40) 5 (16.7) 30 (19.2) 126 (80.8) 156 (8.3) Phlebotomus andrejevi 364 (66.7) 182(33.3) 26 (7.2) 82 (22.7) 235 (65.1) 18 (5) 361 (66.1) 185 (33.9) 546 (29.3) Sergentomyia murgabiensis sintoni 38 (100) - 1 (4.5) 2 (9) 16 (72.8) 3 (13.7) 22 (57.9) 16 (42.1) 38 (2) Sergentomyia sumbarica 1282 (68.9) 580 (31.1) 132 (16) 176 (21.4) 430 (52.3) 84 (10.3) 822 (44.1) 1040 (55.9) 1862 Total Fig. 1. Gel electrophoresis of Nested-PCR products of Leishmania major kDNA in Phlebotomus papatasi caught from urban areas of Damghan, 2016–2018. The wells correspond to L. major in Ph. papatasi (1-4), Reference strains of L. major (5), Negative control (DW) (6), and molecular weight marker (7) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 237 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 2. Map of the distribution of Leishmania major infected sand flies in Damghan City, Semnan Province, Iran Discussion The identification of eight sandflies spe- cies in this study showed that these insects have a relatively high diversity in the city. So far, 44 species of both genera Phlebotomus and Sergentomyia of sand flies have been identified and reported in Iran (5, 20). Only four of the Iranian sand fly species were shown to be infected with leishmania para- sites. Promastigotes of leishmania parasites have been found and reported in 13 various species of these significant biologic vectors from endemic foci of cutaneous and visceral leishmaniasis (5). In the present study Ph. papatasi was dom- inant species and had the highest abundance in indoor and outdoor places and in rodent bur- rows. This species of sand flies, in most other studies, has also shown the highest frequency and they have been collected and reported from various parts of the country with an eight to 1756m altitude range (5). The study area, Damghan City, with an altitude of 1170m, ar- id and semi- arid climate is a suitable place for this species to survive. Besides climatic sit- uations, some other factors such as the con- struction of new buildings and accumulation http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 238 http://jad.tums.ac.ir Published Online: Sep 30, 2022 of their wastes in different parts of the city, the presence of reservoir rodents in those places may affect the spread of the disease. Phlebotomus papatasi is spread in the arid and semi-arid zones of Mediterranean, Eu- rope, North Africa, Middle East, and the Indi- an subcontinent (11, 26-28) and it has been reported as a dominant species in other cuta- neous leishmaniasis foci in Iran, notably in rural areas of the country and has been in- troduced as proven vector of ZCL (11, 29-33). Among the molecularly examined sand- flies, only the species of Ph. papatasi were shown to be infected with L. major parasites (4 out of 147; 2.7%). Similar infection rates have been reported by other researchers from various parts of the country (5, 20, 32, 34). Other species of the examined sand flies were not observed to be infected with L. major par- asites. Furthermore, natural infection of Ph. papatasi and Nesokia indica with L. major has previously been reported from rural areas of Damghan District (13, 20). Based on the detection of L. major in Ph. papatasi collect- ed from the studied area and the presence of ZCL human cases in this region, it may con- firm the ZCL cycle in Damghan County. Another species of the identified sand flies in this study, Ph. sergenti, were not shown to be infected with Leishmania parasites. Phleboto- mus sergenti is the main vector of ACL in Iran and many other countries (35-39). Based on the presence of Ph. sergenti as the potential vector of ACL in Damghan City, further epi- demiological study is recommended on hu- man and animal reservoirs such as dogs. Identification of Ph. alexandri was anoth- er important finding of this survey. Natural infection of Ph. alexandri with L. infantum as the causative agent of visceral leishmaniasis (VL) has already been reported from Iran (17, 40), and it has been introduced as vector of the disease in some other parts of the world (41-45). Moreover, the authors have recently reported the natural infection of Nesokia indi- ca with L. infantum in the studied area (11). Therefore, beside of animal-sand fly-animal cycle of zoonotic visceral leishmaniais, ani- mal-sand fly-human cycle of the disease can be expected in the studied region. Phlebotomus caucasicus accounted for 6.2% of the surveyed sandflies. None of the species was found to be infected with L. major in this study. In a study, 3.3–20% of examined Ph. caucasicus collected from rodent barrows have been reported to be infected with L. major pro- mastigotes in central part of Iran (4, 20, 40). Most of the studied female sand flies (52.3 %) with digestive tract were checked for PCR. Of those, the largest number was belonging to Ph. papatasi and the largest number of infect- ed sand flies (75%) was observed in the same specimens. In these sand flies, the parasites have passed blood digestion stages behind, and more importantly, they have been able to pass the peritrophic membrane (46). In the current study, 75% of infected sand flies were trapped from rodent's burrows and outside places. Therefore, it is recommended that local people wear adequate dress and ap- ply insect repellent to avoid bites outsides. In homes also the use of screens and bed nets impregnated with insecticide to prevent dis- ease is recommended. Conclusion This study confirms the report of Ph. pa- patasi infection with L. major from Damghan City by other researchers (20). This finding could justify the reason for ZCL of people dwelling in the city with any history of travel- ling to the ZCL foci. Moreover, the existence of the other species of sand flies, including Ph. sergenti and Ph. alexandri, the vectors of L. tropica and L. infantum respectively, raises the possibility of future outbreaks of anthro- ponotic cutaneous leishmaniasis and visceral leishmaniasis in the study area. Therefore, further studies accompanied by designing and performing the vector control programs in the city are seriously recommended. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 233–242 S Mohammadi-Azni et al.: Molecular Detection of … 239 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Acknowledgements This study was financially supported by Semnan University of Medical Sciences (Pro- ject No.731). We would like to extend our appreciation to the research deputy of Semnan University of medical sciences. Ethical considerations Ethical approval for this study was obtained from the Ethics Committee at Semnan Univer- sity of Medical Sciences, Iran (IR.SEMUMS. REC.1397.136). 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