J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 315 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Original Article First Report of Natural Infection of Phlebotomus mongolensis to Leishmania major and Leishmania turanica in the Endemic Foci of Zoonotic Cutaneous Leishmaniasis in Iran Azad Absavaran1,2, Mehdi Mohebali3, Vahideh Moin-Vaziri4, Alireza Zahraei-Ramazani1, Amir Ahmad Akhavan1, Sayena Rafizadeh5, Amirhossin Rassi6, Alireza Barmaki1, *Yavar Rassi1 1Department of Medical Entomology and Vector control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical entomology and Vector control, Zanjan University of Medical Sciences, Zanjan, Iran 3Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5Ministry of Health and Medical Education, Tehran, Iran 6Faulty of Medicine, Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Prof Yavar Rassi , E-mail: rassiy@tums.ac.ir (Received 06 July 2021; accepted 03 Sep 2022) Abstract Background: The primary aim of this study is to determine infection to Leishmania parasites in the wild population of Phlebotomus caucasicus and Phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in Iran. Methods: Sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap pa- per. In order to detect and identify of Leishmania parasites in females Ph. caucasicus and Ph. mongolensis, the Nested– PCR amplification of ITS2-rDNA region was performed to generate amplicon with 245bp for Leishmania major, 206bp for L. gerbilli and 141bp for L. turanica. Results: In the current study we found DNA of different gerbil parasites such as L. major and L. turanica, and mixed infection of L. major/L. turanica in Ph. caucasicus and Ph. mongolensis. It should be noted that, in Iran, natural infec- tion with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Conclusion: Both species of Ph. caucasicus and Ph. mongolensis not only may participate in the ZCL transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans. Keywords: Leishmaniasis; Phlebotomus caucasicus; Phlebotomus mongolensis; Leishmania major; Leishmania turanica Introduction Leishmaniasis is a group of vector-borne diseases caused by a protozoan parasite be- longing more than 20 Leishmania species. The disease spreads to tropics, subtropics, and the Mediterranean basin, as well as to 98 tropical countries in Asia (Middle East), Europe (South- ern Europe and the Mediterranean), Africa (Tropics, North, West, and East Africa), and the United States (Mexico, Central and South America). More than 1 billion people are at risk for leishmaniasis in endemic areas. The prevalence of this disease is 12 million cases worldwide and it is estimated that 700 000 to one million new cases occur annually (1, 2). 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/ mailto:rassiy@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 316 http://jad.tums.ac.ir Published Online: Dec 31, 2022 There are three main forms of leishmaniases, including cutaneous leishmaniasis (CL), vis- ceral leishmaniasis (VL), also known as Kala azar, and mucocutaneous leishmaniasis (MCL) (2). Cutaneous leishmaniasis is the most com- mon form of the disease and in 2021 over 85 % of new CL cases occurred in 10 countries: Afghanistan, Algeria, Brazil, Colombia, Syria, Libya, Tunisia, Pakistan, Iraq, and Iran (2, 3). There are two epidemiological types of cuta- neous leishmaniasis in Iran: anthroponotic cu- taneous leishmaniasis (ACL) or urban/ dry form and zoonotic cutaneous leishmaniasis (ZCL) or rural/wet form. Zoonotic cutaneous leishman- iasis is a major public health problem in Iran which is endemic in many rural regions in 19 out of 31 provinces and about 85% of con- firmed leishmaniasis cases in the country are of ZCL type. The causative agent of ZCL in Iran is Leishmania major and the main animal reservoirs of disease are rodents of the sub- family Gerbillinae (4). Of more than 1000 species of identified phlebotomine sand flies, 31 species of Phleboto- mus (Old World) and 47 species of Lutzomyia (New World) are proven vectors of human leishmaniasis (5–7). According to recent stud- ies, to date 48 confirmed phlebotomine sand flies have been reported from Iran, including 30 species of the genus Phlebotomus and 18 species of the genus Sergentomyia (8–12). Phlebotomus (Phlebotomus) papatasi is the proven and main vector of L. major to human in endemic foci of ZCL in Iran. Phlebotomus caucasicus group belongs to the subgenus Paraphlebotomus, which has been considered as species group including Ph. caucasicus, Ph. mongolensis and Ph. andrejevi, playing a main role in maintenance of enzootic cycle of L. ma- jor among rodent reservoir hosts (8). Phleboto- mus caucasicus and Ph. mongolensis not only participate in the transmission cycle of ZCL among reservoir hosts but also play an im- portant role as secondary vectors in the trans- mission of leishmaniasis to humans (8). The females of these species have similar taxonom- ic characteristics and are isomorphic but based on recent study, morphometric analysis and mor- phological characters have been used for dis- crimination of these closely related species (13). Natural promastigote infection was iso- lated from Ph. caucasicus collected from ger- bil and jird burrows in the focus of Esfahan Province in Iran and typed by isoenzymes as- says as L. major zymodeme MON-26 (14). The traditional or classical methods such as sand fly dissection and culture of parasite have been used for Leishmania detection, but these tech- niques are time consuming and requires many sand fly specimens and also are less sensitive than molecular techniques and are not able to differentiate Leishmania parasite species (15). The present study has used a Nested-PCR meth- od, which able to differentiate Leishmania par- asite species and is a specific alternative meth- od to classical techniques (16). In recent years, molecular techniques are frequently used in epidemiological studies spe- cifically on phlebotomine sand flies as vectors of ZCL in endemic foci of Iran for detection and identification of Leishmania infection in phlebotomine sand flies (17–27). The objec- tive of present study was to use molecular methods for the first time to detect and identi- fy of Leishmania infection within wild caught Ph. caucasicus and Ph. mongolensis in some important zoonotic cutaneous leishmaniasis foci in Iran. Materials and Methods Sand flies’ collection and species identification Sand flies were collected from the differ- ent allopatric locations in the provinces of Es- fahan and Fars (central and southern Iran, re- spectively) and sympatric locations in Goles- tan Province (northeastern Iran). From June through October 2016, sand flies from the ac- tive rodent burrow colonies were collected us- ing sticky trap papers (castor oil coated white papers, 21×30cm) from 16 collecting sites. Col- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 317 http://jad.tums.ac.ir Published Online: Dec 31, 2022 lected sand flies were stored in 96% ethanol and kept in -20 °C for morphological and mo- lecular assays. At first, for removing castor oil on specimen’s body surface, collected speci- mens were washed twice in 1% detergent and sterile distilled water and then was dissected in a drop of sterile normal saline by sterilized forceps. The head and the last two abdominal segments were cut off and slide mounted in Puris' medium for species identification and identified after 24–72 hours using valid iden- tification keys (13, 28–30). The remaining body (abdomen, wings, and legs) were preserved in 1.5ml sterile micro-tubes containing 96% eth- anol for DNA extraction and detection of Leish- mania parasite. Molecular detection and identification of Leishmania species GeneAll® ExgeneTM Tissue Kit (GeneAll Biotechnology Company, South Korea) was used to extract genomic DNA. To detect and to identify Leishmania parasites we used the Nested-PCR assay developed by Akhavan et al. (16). Nested-PCR method has been used to amplify the Leishmania spp. regions of ITS2, primers as follows: Leish out F (5′-AAA CTC CTC TCTGGT 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′). PCR products were sepa- rated by 1.5% (w/v) agarose gel electrophore- sis in TBE buffer (0.09mM Tris, 0.09mM bo- ric acid and 20mM EDTA, pH 8.3), visual- ized under ultraviolet light after staining with Safe Stein (0.5μg/ml) and photographed. Ref- erence strains of L. major (MRHO/IR/75/ER), L. gerbilli (MRHO/CN/60/GERBILLI) and L. turanica (MRHO/SU/1983/MARZ-051) were used as positive controls. Also, double dis- tilled water was included in each run as a neg- ative control (16). In order to sequencing, the PCR products of the second-round (nested) PCR for all posi- tive samples were purified using the Gel Puri- fication Kit (ExpinTM PCR SV, GeneAll Bio- technology Company, South Korea). Both forward and reverse strands of amplified DNA were sequenced with the PCR primers. Nucleotide homologies of the sequenced products were evaluated with Leishmania spp. sequences available in GenBank and then checked by using Basic Local Alignment Search Tool (BLAST) analysis software (http://www.ncbi.nlm.nih.gov/BLAST) Results A total of 176 female sand flies were se- lected in this study (64 specimens of Ph. cau- casicus and 112 specimens of Ph. mongolen- sis), and extraction of genomic DNA was con- ducted to identify Leishmania parasites in these phlebotomine sand flies. In 17 female sand fly specimens including 6 specimens of Ph. cau- casicus and 11 specimens of Ph. mongolensis, Leishmania parasites were detected. Out of these 17 specimens of Leishmania-infected sand flies, seven, eight, and two specimens were in- fected to L. major, L. turanica, and mixed in- fection of both L. major and L. turanica re- spectively. The positive specimens produced species-specific band/s corresponding to L. ma- jor (245 and 233bp), and L. turanica (141bp) (Fig. 1). It is important to note that L. major and L. turanica parasites were detected in Es- fahan and Golestan provinces in both Ph. cau- casicus and Ph. mongolensis, but in Fars Prov- ince (Sadegh abad) only L. major parasite from Ph. mongolensis was detected in one case (Table 1). In this study, the infection rate for Leishmania parasites were estimated to be 9.3 % for Ph. caucasicus and 9.8% for Ph. mon- golensis. Details of Leishmania parasites de- tected in six specimens of Ph. caucasicus and 11 specimens of Ph. mongolensis in different collection sites with their abdominal status mentioned in tables 1 and 2. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 318 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Table 1. Leishmania parasite positive PCR detected in specimens of Phlebotomus caucasicus and Ph. mongolensis based on collection sites in Esfahan, Golestan and Fars provinces, Iran, 2016 Positive samples No of Exam- ined specimens Collection sites Species (Leishmania positive samples) Mix of L. ma- jor + L. turanica L. turanica L. major Total - 2 2 4 32 Habib Abad (Esfahan) Ph. caucasicus (6 specimens) - - - - 14 Ali Abad (Esfahan) - - - - 12 Nik Abad (Esfahan) - 1 - 1 1 Ezhiyeh (Esfahan) - - 1 1 5 Agh Tagheh (Golestan) - 1 - 1 8 Sian (Esfahan) Ph. mongolensis (11 specimens) - 1 - 1 3 Abbas Abad (Esfahan) - - - - 2 Nik Abad (Esfahan) - - - - 4 Heydar Abad (Esfahan) - - - - 3 Ezhiyeh (Esfahan) - - - - 16 Raja Abad (Fars) - - 1 1 7 Sadegh Abad (Fars) - - - - 3 Kouh Sabz (Fars) - - - - 2 Band Amir (Fars) - - - - 8 Ghareh Gol (Golestan) - 1 - 1 12 Ouch Quee (Golestan) 1 - 3 4 26 Agh Tagheh (Golestan) 1 2 - 3 18 Narlidagh (Golestan) 2 8 7 17 176 Total Fig. 1. Agarose (1.5%) gel electrophoresis of Nested-PCR products for Leishmania parasite infection in Phlebotomus caucasicus and Ph. mongolensis in Esfahan, Golestan and Fars provinces. Lanes M, 50 bp Ladder (ExcelBandTM, SMOBIO Technology); Lane 1, L. major (245 bp, detected in Ph. caucasicus); Lane 2, L. turanica (detected in Ph. cau- casicus); Lanes 3-4-8-9-11, L. turanica (detected in Ph. mongolensis); Lane 5, L. major (245bp, detected in Ph. mongo- lensis); Lane 6, L. major (233 bp, detected in Ph. mongolensis); Lane 7, mix infection with L. major (233 bp) and L. turanica (detected in Ph. mongolensis); Lane 10, mix infection with L. major (245 bp) and L. turanica (detected in Ph. mongolensis); Lane 12, L. major (positive control); Lane 13, L. turanica (positive control); Lane 14, negative control (distilled water) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 319 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Table 2. Leishmania parasite positive PCR detected in Phlebotomus caucasicus and Ph. mongolensis specimens based on abdominal status in Esfahan, Golestan and Fars provinces, Iran, 2016 Abdominal status of specimens Leishmania positive samples Specimens Species G SG FF UF L. major + L. turanica L. turanica L. major Total - 1 1 4 - 3 3 6 64 Ph. caucasicus 2 - - 9 2 5 4 11 112 Ph. mongolensis 2 1 1 13 2 8 7 17 176 Total UF= Unfed, FF= Fresh fed, SG= Semi gravid, G= Gravid Discussion Identification of phlebotomine sand flies as vectors of leishmaniasis is very important and crucial for leishmaniasis control programs. In this study, molecular detection, and identi- fication of Leishmania parasites based on Nest- ed-PCR method allowed us to detect more Leishmania infections than previously in Ira- nian sand flies. Numerous natural sand fly pro- mastigote infections in ZCL foci in Iran have been reported based on parasitological meth- ods and direct examinations (31–35), identifi- cation of Leishmania parasite using isoenzyme electrophoresis (14, 36), and molecular meth- ods based on polymerase chain reactions (17– 27). In the present study two species of Leish- mania parasites including L. major and L. tu- ranica as well as L. major + L. turanica mixed infection in Ph. caucasicus and Ph. mongolen- sis were detected using Nested-PCR of ITS2- rDNA region. These parasites are in concordance with identified gerbils’ parasites and Ph. caucasicus and Ph. mongolensis belongs to Kazakhstan, Uzbekistan, Turkmenistan and China (37, 38) and Iran (23, 39). Phlebotomus caucasicus is an Asiatic species, first recorded from the Trans- caucasia region and is distributed from the ge- ographical area of Iran to China. It is a com- mon species in sandy deserts and hills, and usu- ally lives in rodent and bird nests, bites rarely humans (40). Various transmissions of gerbil parasites have occurred in Iran's northern neighbors in Central Asia, including L. major, L. turanica, and L. gerbilli, within or near the nest of the great gerbil of Rhombomys opimus (41). Ro- dent colonies provide habitat for many spe- cies of sand flies, increasing the risk of the Leishmania parasite being spread to mammals (42). Phlebotomus caucasicus was first intro- duced by Adler and Theodor in 1957 and was identified among Central Asian rodents as a suspected vector of L. major and L. gerbilli parasites (43). It is also considered an L. tu- ranica vector in Turkmenistan (37, 44), and L. donovani vector in Central Asia and Ka- zakhstan (45). In this research, three samples of L. major parasite (Esfahan and Golestan provinces) and three samples of L. turanica parasite (Esfahan province) were identified from six Ph. cauca- sicus specimens. Typing of parasites isolated from this sand fly species by isoenzyme meth- od led to the precise diagnosis of parasite as L. major Mon-26 and proved to be the same type of human parasite, also Ph. papatasi as vector and Rh. opimus as reservoir (14). Phlebotomus caucasicus is also confirmed to be 12.5 and 7.5% natural leptomonad infec- tion in the Nikabad and Borkhar regions of Esfahan Province respectively (14). In 2008, Parvizi and Ready identified two species of L. major and L. gerbilli from Ph. caucasicus in http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 320 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Esfahan Province and L. gerbilli in Golestan Province based on Nested-PCR method by se- quencing of ITS1-5.8S rDNA region (17). Us- ing the RAPD-PCR method, L. major parasites were reported from four Ph. caucasicus group specimens (4.2%) caught from rodent burrows in Shahroud County (Semnan Province) in Iran (39). Once again, using the Nested-PCR meth- od of kDNA genome and using the RFLP meth- od of ITS1-rDNA region, the L. major para- site was reported from Ph. caucasicus group of sand flies collected in Damghan city (Sem- nan province) in Iran (46). According to these reports, along with the 20% anthropohilic index for Ph. caucasicus (14, 47), strong evidence shows that Ph. cau- casicus is a natural vector of L. major, L. tu- ranica and L. gerbilli parasites among reser- voir rodents as well as a secondary or sus- pected vector of L. major for humans in ZCL foci in Iran (8). In the current study, four samples of L. major (Golestan and Fars provinces), five sam- ples of L. turanica (Esfahan and Golestan prov- inces) and two mixed infection samples of both L. major and L. turanica (Golestan Province) were found from 11 collected Ph. mongolen- sis specimens. It should be noted that, in Iran, natural infection with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Phlebotomus mongolensis is al- so an Asiatic species and is a dominant spe- cies in the sandy deserts and hills and usually lives in rodents burrow (41). It is also known to be L. turanica and L. gerbilli vector in Turk- menistan (37, 44), L. turanica in China (38), L. donovani and L. major in Central Asia and Kazakhstan (45). Based on the findings of this study, it is possible that both Ph. caucasicus and Ph. mongolensis are potential vectors of the gerbil Leishmania parasites. However, the detection of genomic DNA of Leishmania in sand flies does not confirm that they have been vector and PCR-based methods can not differentiate Leishmania promastigotes in both infectious and non-infectious forms. The pref- erence of sand flies to human blood, and the growth and development of parasites in the external cycle and experimental bite transmis- sion have been the most important criteria for disease parasite transmission (48–50). Considering that another criterion for the definition of a sand fly as a vector is the pres- ence of the infective form of metacyclic pro- mastigotes in the thoracic areas of midgut, fore- gut and mouth sections of sand flies, the posi- tion of the sand flies’ abdomen is also very critical in determining the vector. In this study, among the 17 Ph. caucasicus and Ph. mongo- lensis sand flies’ specimens with Leishmania infection, 13 specimens with empty (unfed) abdominal status, indicating that the parasite of Leishmania was successfully developed in midgut areas. The transmission dynamics of Leishmania parasites depend not only on the Leishmania species diversity but also on the parasite's intra-species strain diversity. In this research, according to the results of molecular identification of Leishmania para- sites, it can be concluded that Ph. caucasicus and Ph. mongolensis were considered as po- tential vectors of L. major and L. turanica par- asites and allowing them to circulate of Leish- mania parasites among rodents and probably humans. Acknowledgements This study was financially supported by the Deputy of Research, Tehran University of Medical Sciences, Grant No. 27252. Ethical considerations This experiment was carried out under the guidance of the Ethics Committee of the Teh- ran University of Medical Sciences (IR.TUMS. REC.1394.144). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 315–324 A Absavaran et al.: First Report of … 321 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Conflict of interest statement Authors declare that there is no conflict of interest. References 1. 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