J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 8 http://jad.tums.ac.ir Published Online: March 31, 2020 Original Article Molecular Identification of Leishmania Species in Phlebotomus alexandri (Diptera: Psychodidae) in Western Iran Abdollah Naghian1,2; Mohammad Ali Oshaghi1; Vahideh Moein-Vaziri3; Yavar Rassi1; Mohammad Mehdi Sedaghat1; Ehsan Mostafavi4; Arshad Veysi5; Hassan Soleimani6; Hossein Dehghan7; Alireza Zahraei-Ramazani1; Hossein Mirhendi8; Mohammad Hassan Amini1; *Mohammad Reza Yaghoobi-Ershadi1; *Amir Ahmad Akhavan1 1 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Medical Entomology and Vector Control, School of Public Health, Urmyia University of Medical Sciences, Urmyia, Iran 3 Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4 Department of Epidemiology, Pasteur Institute of Iran, Tehran, Iran 5 Zoonoses Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran 6 Yazd Health Research Station, National Institute of Health Research, Yazd, Iran 7 Department of Public Health, School of Public Health, Jiroft University of Medical Sciences, Jiroft, Iran 8 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Esfahan, Iran (Received 06 Aug 2018; accepted 05 Dec 2018) Abstract Background: Visceral and cutaneous leishmaniasis are common in some areas of Iran and consider as health problems. Phlebotomus alexandri has been incriminated as a suspected vector for the both form of leishmaniasis. Methods: This study was carried out in 4 western provinces of Iran. Sand flies were collected using sticky traps and light traps from indoor and outdoor resting places. Nested PCR was employed to detect Leishmania parasites among collected sand flies. Results: Seven hundred and twenty two P. alexandri females were collected and pooled in 179 batches. Results of nest- ed PCR showed, out of 9 samples from East Azerbaijan Province, only one sample was infected by Leishmania infan- tum. Of 34 individual and pooled samples from Kermanshah Province, only one pooled sample was infected with Leishmania major and among 30 individual and pooled samples in Fars Province, five specimens were infected by L. major, L. infantum, Leishmania donovani and Leishmania tropica. Furthermore, out of 108 individual and pooled sam- ples from Khuzestan Province, 10 samples showed infection with L. major and L. infantum. Conclusion: The results of this study showed that P. alexandri is more active in hot zones than in moderate zones and this species may be considered as a permissive species. Keywords: Leishmaniasis; Phlebotomus alexandri; Leishmania; Iran Introduction Leishmaniasis is a parasitic infectious dis- ease that is transmitted by the bite of infected phlebotominae sandflies, and it is grouped in the seventeen neglected tropical diseases (NTD) (1) which has been recorded from 98 countries (2). There are three main forms of disease in the world including visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutane- ous leishmaniasis (MCL) (3). So far two types of the disease including CL and VL, has been reported in Iran (4, 5). Cutaneous leishmaniasis is the most common form of the disease in the *Corresponding authors: Prof Mohammad Reza Yaghoobi-Ershadi, Email: yaghoobia@gmail.com, Dr Amir Ahmad Akhavan, Email: aaakhavan@tums.ac.ir http://jad.tums.ac.ir/ mailto:yaghoobia@gmail.com mailto:aaakhavan@tums.ac.ir J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 9 http://jad.tums.ac.ir Published Online: March 31, 2020 world as well as in Iran (5, 6). Although the new VL foci have increased remarkably, zoon- otic visceral leishmaniasis (ZVL) incidence has decreased recently in the country (Ministry of Health and Medical Education (MOHME)), 2015, unpublished data). There are about 1000 sand fly species and subspecies reported in the world (7), of these only about 93 species transmit 20 Leishmania species to humans (2). Till now, 29 species and 2 subspecies of Phlebotomus and 19 species of Sergentomyia sand flies were reported in Iran (8). Six sand fly species including Phlebotomus salehi, Phlebotomus mongolensis, Phlebotomus al- exandri, Phlebotomus andrejevi, Phlebotomus caucasicus and Phlebotomus ansarii known as suspected vectors and Phlebotomus papatasi known as the main proven vector of zoonotic cutaneous leishmaniasis (ZCL) due to Leish- mania major in the country (4). Moreover, an- throponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica occurs in 14 foci of 7 provinces in Iran. Four species of the sub- genus Larrussious and P. alexandri of the sub- genus Paraphlebotomus were incriminated as probable vectors of ZVL due to Leishmania infantum in seven new and old foci of the country (4, 5). Phlebotomus alexandri report- ed as a proven vector for ZVL (2) and an- throponotic visceral leishmaniasis (AVL) in china (9). Furthermore, it is incriminated as the probable vector of AVL and ZVL in Iraq, Oman, Mongolia, Turkey and China (2, 10). Leishmania infection rate for P. alexandri re- ported as 1.74% in Khuzestan endemic re- gions (11). It was also found infected by L. infantum (12) and L. major (13) using molec- ular methods in Fars Province in South West and Sarakhs in North Eastern of Iran respec- tively. Furthermore, P. alexandri was found in- fected in Turkemanistan (14) and it is suspect- ed to transmission of Leishmania killicki in Tu- nisia (15, 16). Considering the studies have been conducted so far in Iran and other countries, it seems that more studies are needed to clarify the role of P. alexandri in different Leishmania parasite transmission cycles. The current study was designed and conducted in four western endemic and non-endemic provinces of Iran. Materials and Methods Study area This study was carried out in 4 western and south western provinces of Iran including East Azerbaijan, Fars, Kermanshah and Khuzestan from 2011 to 2012. In Eastern Azerbaijan Prov- ince (36˚, 45’ to 39˚, 26’ N and 45˚ 5’ to 48˚ 21’ E) in Northwest of Iran, maximum and mini- mum temperature were +45 ˚C in Julfa and -25 ˚C in Bostanabad respectively and the rainfall varied between 196 to 563mm. In Kermanshah Province (33˚, 41’ to 35˚, 17’ N and 45˚ 24’ to 48˚ 6’ E) in the West of Iran, maximum and minimum temperature were +50.4 ˚C in Soomar and -17.4 ˚C in Sonqur respectively and the rainfall varied between 273 to 573mm. In Fars Province (27˚, 3’ to 31˚, 40’ N and 50˚ 36’ to 55˚ 35’ E) in South West of Iran, maximum and minimum of temperature were +49.8 ˚C in La- mard and -14.6 ˚C in Safashahr respectively and the rainfall varied between 83 to 1007mm. In Khuzestan Province (29˚, 53’ to 33˚, 0’ N and 47˚ 40’ to 50˚ 33’E ) in south west of Iran, maxi- mum and minimum of recorded temperature in this province were +52.6 ˚C in Shushtar and - 5.6 ˚C in Dehdaz respectively and the rainfall varied between 73 to 702mm (17). Sand fly collection and identification Sand flies were collected using light traps and sticky traps during active seasons of sand flies in 127 places and 599 places in 2011, re- spectively (Fig. 1). Collected sand flies were conserved in 96% alcohol, kept in 4 ˚C refriger- ator. The head and the last two segments of sand flies were detached on the slide in sterile condition and mounted in Puris medium. The rest of the bodies including abdomen and thorax were transferred into a sterile 1.5ml micro tube and were kept in -20 ˚C until use. The speci- mens were identified using valid morphological keys (18, 19). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 10 http://jad.tums.ac.ir Published Online: March 31, 2020 Molecular experiments Genomic DNA was extracted and purified using a GeneAll kit (Exgene™ Tissue SV mini) according to the protocol of the kit. PCR conditions Polymerase Chain Reaction amplification has done in an Applied Biosystems thermocy- cler. The first step of Nested-PCR contained 0.6μM of each forward (AAACTCCTCTCT GGTG-CTTGC; Leish out F) and reverse (AAA CAAAGGTTGTCGGGGG; Leish out R) exter- nal primers (20), 12.5μl Taq DNA polymerase, 2X Master Mix RED (Amplicon, Denmark) and sterile distilled water to a final volume of 25 μl. First denaturation step at 95 °C for 5min was continued by 30 cycles of denaturation at 94 °C for 30s, annealing at 60 °C for 45s and ex- tension at 72 °C for 1min, with a final extension step of 72 °C for 5min. The second step of Nested-PCR was performed in a final volume of 20μl containing 1μl of a 1:10 dilution in distilled water of the first-round PCR product as template, 0.3μM of each forward (AATT CAA CTTCGCGTT GGCC; Leish in F) and reverse (CCTCTC TTTTTTCTC-TGTGC; Leish in R) (20), 10μl of Taq DNA polymerase and 2X Master mix RED. The second round PCR was cycled under the following conditions: 95 °C for 2min, 25 cycles of 94 °C for 15s, 62 °C for 30s, 72 °C for 45s followed by 72 °C for 5min. PCR products were electrophoresed on 1.5% (w/v) agarose gel in TBE buffer (0.09 mM Tris, 0.09mM boric acid and 20mM EDTA, pH 8.3), visualized with safe stain (0.5μg/ml) and photographed. Reference strains of L. infantum, L. major and L. tropica were used as positive controls and distilled water was used as negative control ac- cordingly. The PCR product of the negative con- trol of the first step of PCR was used as the neg- ative control in the second step and the PCR product of the positive control of the first step PCR was used as positive control in the second step as well. To avoid cross-contamination, nec- essary precautions were taken. The products of nested PCR were sent to corresponding com- pany for nucleotide sequencing. Results Sand flies were collected using 207 light traps and 7633 sticky traps during active seasons of sand flies in the studied areas. Totally, 722 fe- male P. alexandri were collected in 4 studied provinces including 9 from East Azerbaijan, 114 from Fars, 90 from Kermanshah, and 509 spec- imens from Khuzestan. Collected sand flies were pooled in 179 batches according to five param- eters including: locality (counties of provinces), places of catch (indoor or outdoor), type of trap (light trap or sticky trap), topography (moun- tains, foothills or plains) and physiological sta- tus (unfed, blood-fed, semi-gravid and gravid) of the sand flies. The locations where P. alex- andri sand flies were caught and their Leish- mania infection are shown in (Fig. 1). In Kermanshah Province, of 34 pooled sam- ples, only one was infected by L. major (Fig. 2). This sample was contained 13 unfed female P. alexandri sand flies, which collected from 4 vil- lages (Kalashi Lulem, Kalashi Bakhan, Mezran and Melah Rash) of Javanrud County (Table 1). In East Azerbaijan, collected P. alexandri sand flies were pooled in 9 samples. Among these samples only one was infected by L. in- fantum (Fig. 1, Table 1). The infected sample was contained 1 unfed female sand fly that was col- lected in a mountainous cave near the village of Ghyzil gaya in Ahar County. In Fars Province, collected P. alexandri sand flies were pooled in 31 batches. Of these 5 sam- ples were infected by Leishmania parasites (Fig. 1, Table 1). Two samples were infected by L. infantum. One of the infected sample by L. in- fantum was contained 3 blood-fed P. alexandri female which were collected in 2 villages (Shor- ab-Chamgole, and PirehSorkh) of Mamasani County. One of the infected sample was infected by L. donovani contained an unfed P. alexandri female that was collected in a cave located in Nur Abad City. The other one showed a mixed http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 11 http://jad.tums.ac.ir Published Online: March 31, 2020 infection of L. infantum and L. tropica, contained an unfed P. alexandri female which was col- lected in Pireh Sorkh Village of Mamasani. Two samples were found to be infected by L. major, one of them was collected in Khumeh Zar Village of Mamasani County that was a pooled of 14 unfed female sand flies and another was a mixed infection of L. major and L. tropica contained an unfed female that was collected in Shir Espari Village of Mamasani County. In Khuzestan Province, all collected P. al- exandri female sand flies were pooled in 108 samples, of these 10 samples were infected by Leishmania parasites and all were unfed. Re- sults showed, in Seyyed Taher Village that lo- cated in plain area in north of Ahvaz County, three samples were infected by L. major, and three samples were infected by L. infantum (Fig. 1, Table 1). Furthermore, another sample was infected with L. infantum in Sar Dasht (Kohe Gandom) village in mountainous area in north of Dezful County (Fig. 1). Apart from these, a sample contained one P. alexandri sand fly had infection of L. infantum was collected in Dehe- now Bagher Village located in plain area in south of Dezful County, (Fig. 1, Table 1). More- over, a sample contained a sand fly that was infected with L. major collected in Magtu Vil lage in the south of Ahvaz County, (Fig. 1, Table 1). The geographical and epidemiological param- eters of pooled samples are shown in Table 1. Fig. 1. The map of Leishmania infection of Phleboto- mus alexandri in the studied areas, Iran, 2011 and 2012 Table 1. The geographical and epidemiological parameters of positive pooled samples of Phlebotomus alexandri in the studied regions Geographical status Province Place Pooled samples code Leishmania species No. sand flies in sample Plain Khuzestan Indoor 108 L. infantum 1 Indoor 98.3 L. infantum 23 Outdoor 119 L. infantum 2 Indoor 97.3 L. major 16 Indoor 89.2 L. major 18 Indoor 89.1 L. major 4 Outdoor 152 L. infantum 1 Outdoor 116 L. major 1 Foothills Fars Outdoor 24.2 L. major 14 Indoor 3 L. infantum 3 Outdoor 19 L. major and L. tropica 1 Indoor 2m L. infantum and L. tropica 1 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 12 http://jad.tums.ac.ir Published Online: March 31, 2020 Mountain Khuzestan Indoor 122 L. infantum 1 Fars Outdoor 28 L. donovani 1 Kermanshah Outdoor 41 L. major 13 East Azerbaijan Outdoor 65.1 L. infantum 1 Fig. 2. The 1.5% agarose gel electrophoresis of nested PCR products in Khuzestan and Kermanshah Provinces sam- ples. Lanes 1 and 17 are 50bp ladder; lane 2- negative control; lane 3- Leishmania infantum positive control; lane 4- Leishmania major positive control; lane 5- Leishmania tropica positive control; lane 6–15- samples of Khuzestan Province; lane 16 sample from Kermanshah Province Fig. 3. The gel electrophorus image of nested PCR results in Fars and Eastern Azerbaijan provinces’ samples. Lene 1 and 12 are 50bp ladder; Lane 2 is negative control; Lane 3, 4 and 5 are positive control of Leishmania infantum, Leish- mania major and Leishmania tropica, respectively; Lane 6, 7, 9 and 11 samples from Fars, Lane 8 a sample from East Azerbaijan Table 1. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 13 http://jad.tums.ac.ir Published Online: March 31, 2020 Discussion The result of this study showed that, P. al- exandri harbored different species of Leishma- nia parasites in the endemic and non-endemic areas. Sand flies infection by L. major from Ja- vanrud County villages in Kermanshah Prov- ince showed, despite there is no CL cases in these areas, it is postulated that, there is an enzootic foci in this region which can pose a risk to extend ZCL to people in the future. This is the first report of P. alexandri sand fly infection due to L. major in Javnrud County in Kermanshah Province. This region is located near Qasr-e Shirin and Sare Pole Zahab Coun- ties, where the previous studies reported ZCL cases among endogenous people (21). In East Azerbaijan, the study regions are located near Meshkin Shahr City in the Ardabil Province where has been known as the first main en- demic foci of ZVL in Iran (4). The infection of P. alexandri due to L. infantum has been report- ed in Fars Province as well (12). Recently, Phlebotomus tobbi was found infected with L. infantum in East Azerbaijan (22). In northwest of Iran, P. kandelakii (23) and P. perfiliewei (24) were reported infected by L. infantum as well. Here, P. alexandri has been introduced as the fourth suspected ZVL vector in the north west of Iran. Since the infected P. alexandri with L. infantum was collected in a fox nest in the border of Karamlu and Khalifan Villages of Ahar County, it implies that foxes can play a role in enzootic cycle of parasite in this re- gion. The results of our studies in Fars Prov- ince showed that, the infection of P. alexandri with Leishmania parasites is higher compar- ing to other provinces. This province known as ACL and ZVL foci in the country (4). Ac- cording to recent report of the MOHME, the occurrence of CL in this province is very high. Leishmania major infection in P. alexandri is reported for the first time in Mamasani Coun- ty in Fars Province, although L. infantum in- fection in P. alexandri has been reported in this area previously (12). Since the occurring mixed infection in P. alexandri, this species might play role as a permissive vector which supports mul- tiple Leishmania parasite inside the digestive tract. Additionally, it is the first report of P. al- exandri infection with L. donovani in Nurabad City as well. In the current study P. alexandri found infected with L. infantum in Chamgol and Pireh Sorkh Villages of Mamasani County for the first time in Fars Province. In Khuzestan Province, P. alexanderi was found infected with 2 Leishmania parasites including, L. major and L. infantum. On the other hand, in East Azer- baijan, P. alexandri was found infected with L. infantum in a fox nest as well. In contrast to the results of previous studies and also current study in other regions, P. alexandri mostly col- lected in the plain areas in Khuzestan Province. According to the MOHME internal reports in 2015, Fars and Khuzestan Provinces had the first and second incidences of CL in Iran re- spectively, but Kermanshah Province had the ninth incidence at the same time. Also these reports state that Fars and East Azerbaijan Provinces had the first and second incidence of ZVL in Iran respectively, but Khuzestan had just 1 case in the year 2015. Microscopic Leishmania infection of P. alexandri in Shush and Ahvaz Counties was reported 4 decades ago, but was not detected by molecular tech- niques and did not show any successful inoc- ulation in sensitive animals (25). In agreement with previous work, our study introduced P. alexandri as a probable vector of ZCL and ZVL in Iran. In the current study the majority of infected P. alexandri sand flies was unfed and it reflects that, they are capable to transmit the parasite to the new host through next blood feeding. Furthermore, the finding highlights that P. alexandri mostly occurred in hot regions (Khuzestan) rather than cold regions (other stud- ied provinces). This is the first report of L. in- fantum and L. major infections in P. alexandri in Khuzestan Province, and is the first report of infection to L. tropica by P. alexandri using mo- lecular technique in the world. In Khuzestan Province, the occurrence of CL is common ac- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 8–16 A Naghian et al.: Molecular Identification of … 14 http://jad.tums.ac.ir Published Online: March 31, 2020 cording to the MOHME reports, but only 1 case was reported in this province for ZVL in 2015 (26). On the other hand, according to WHO and some other studies, in eastern provinces of Iraq ZVL is endemic (27-29). The 90% of ZVL cases in Iraq occur among less than 5 years old chil- dren. The number of reported cases were 3900 in Iraq in 1992 and increased to 1050 cases in 2012 (28). Conclusion The results of this study showed, P. alex- andri could harbor infection of 4 Leishmania species (L. major, L. tropica, L. donovani and L. infantum) so that, it supports the hypothesis of permissiveness of this species. Furthermore, the reporting of natural infection of P. alexan- deri in some presumably free areas of ZCL, e.g. in Kermanshah, revealed the cycling of par- asite among its host and sand flies vector at least in enzootic cycle. Acknowledgements This study was financially supported by School of Public Health, Tehran University of Medical Sciences. Our sincere thanks go to Mrs Kazeruni, Mr Ghazizadeh, Mr Kamali, Mr Alizadeh for their kind collaboration and as- sistance in the sand flies collection. 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