J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 124 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article PCR Positivity of Gerbils and Their Ectoparasites for Leishmania Spp. in a Hy- perendemic Focus of Zoonotic Cutaneous Leishmaniasis in Central Iran Sahar Azarmi1, *Alireza Zahraei-Ramazani1, Mehdi Mohebali2, Yavar Rassi1, Amir Ahmad Akhavan1, Amrollah Azarm1, Omid Dehghan1, Samira Elikaee2, Rahimeh Abdoli1, Mohsen Mahmoudi2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Corresponding Author: Dr Alireza Zahraei-Ramazani, E-mail: azahraei@tums.ac.ir (Received 02 Sep 2020; accepted 02 May 2022) Abstract Background: Various arthropods, including Rhipicephalus sanguineus and Ctenocephalides felis felis have been sug- gested as secondary vectors of Leishmania spp. many years ago. This study was conducted to determine zoonotic cuta- neous leishmaniasis (ZCL) PCR positivity of reservoir hosts and their ectoparasites for Leishmania spp. in Segzi plain in Esfahan Province from October 2016 to October 2017. Methods: Microscopic examination and nested PCR were used to detect and identify Leishmania spp. isolated from rodents' ears and ectoparasites, and then, the results were confirmed by two methods, PCR-restriction fragment length polymorphism and sequencing. Results: Totally, 93 rodents (92 Rhombomys opimus and one Nesokia indica) and nine different species of ectoparasites (n=527) including fleas, mites, and ticks were collected during different seasons in the study area. Fourteen R. opimus were positive for Leishmania spp. by microscopic examination while one N. indica and 77 R. opimus were positive by nested PCR. The infection rate of rodents with Leishmania major and Leishmania turanica was 39.79% (n=37) and 15.05% (n=14), respectively. Mixed natural infections with L. major and L. turanica were seen in rodents. Moreover, 72.22% of fleas (39/54), 75.0% of mites (5/8), and 100% of tick nymph (1/1) were PCR positive for Leishmania parasites. Conclusions: The highest rate of infection with L. major and L. turanica in R. opimus populations was observed in summer and spring, respectively. It is suggested that the role of L. turanica and the probable role of ectoparasites in the epidemiology of disease should be investigated. A Xenodiagnostic test is recommended for future study. Keywords: Ectoparasite; Leishmania gerbilli; Leishmania major; Leishmania turanica; Rhombomys opimus Introduction Zoonotic Cutaneous Leishmaniasis (ZCL) caused by Leishmania major Yakimoff and Schokhor, 1914 is a public health problem in several countries affecting a large number of people (1). The disease is endemic in in 18 out of 31 provinces of Iran (2). The great gerbils (Rhombomys opimus Lichtenstein, 1823) is the main reservoir hosts of ZCL in Iran and this rodent acts as a reservoir host of ZCL in Es- fahan Province, central Iran (1). The main and biological vector of L. major is Phlebotomus pa- patasi Scopoli, 1786 in Iran (3). However, sec- ondary forms of Leishmania transmission by oth- er arthropods have been reported in the litera- ture, but their role is still unclear in the epide- miology of leishmaniasis (4–7). Some arthro- pods, such as the brown dog tick (Rhipicepha- lus sanguineus Latreille, 1806), have been in- troduced as probable mechanical vectors for some Leishmania parasites (4–8). Ticks and fleas may play a role in the transmission of Leishmania infantum Nicolle, 1908 between 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:azahraei@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 125 http://jad.tums.ac.ir Published Online: June 30, 2022 dogs (8). Two recent studies have suggested that the Leishmania parasite could be trans- mitted by tick and flea bites (6, 7). Ticks that have high reproductive rates and long lifespan, are widely distributed and abun- dant, and can maintain high population densi- ties. They are potential vectors of several ver- tebrate pathogens (9). According to reports of human parasitism by R. sanguineus (10) and transmission of Leishmania parasites by the brown dog tick (5, 6), ticks may have a role in the survival of Leishmania parasites between dogs and sporadic transmission of this para- site from dogs to humans (6, 11). Like ticks, fleas, which are widely distrib- uted due to their high reproductive rate, can attain a high population density. If a source of blood is available, fleas can live for 200 days. Their blood feeding habit and high longevity may turn fleas into a potential vector of leish- maniasis in endemic areas over time (12). A recent study found that the Ctenocephalides felis felis Bouche, 1835 fleas collected from dogs showed the presence of promastigotes in smears stained by Giemsa. Moreover, it was also shown that fleas could transmit Leishma- nia chagasi Cunha and amp, Chagas, 1937 from the infected dogs to hamsters in laboratory con- ditions. This investigation was confirmed by pol- ymerase chain reaction (PCR) and the indirect fluorescent antibody test (IFAT) assays (13). This study was conducted to determine the PCR positivity of ZCL reservoir hosts and their ectoparasites for Leishmania spp. in a hyperen- demic area of Esfahan Province, Iran. Materials and Methods Study area This study was done in Segzi plain (32.694866° N, 52.120171° E), 35km east of the city of Esfahan, Esfahan Province over a period of 12 months from October 2016 to October 2017. Segzi is located at an average altitude of 1545 meters above sea level and has hot summers and cold winters. Most of the veg- etation in the desert area of Segzi is Salsola rigida and Haloxylon ammodendron (black saxaul). Collection and identification of rodents and their ectoparasites Rodents were captured using 30 Sherman live traps (18×18×30cm) at intervals of 45 days, 2 times in each season from different residen- tial, agricultural, and desert areas in Segzi. The traps were placed in front of the holes of active colonies before sunset and sunrise. In case of trapping before sunset, the traps were collect- ed the next morning. Also, the traps placed be- fore sunrise were removed until noon of the same day. The captured rodents were trans- ferred to the laboratory of the Esfahan Health Research Station and anesthetized (14). Ecto- parasites were collected from each rodent by brushing the hairs of the hosts on a water sur- face and stored in 96% ethyl alcohol separate- ly. The ectoparasites were mounted on micro- scopic slides and then identified morphologi- cally (15–18). The rodents were identified based on their morphological and morphometrical char- acteristics using identification keys (19). Microscopic examination While the rodents were anesthetized, two different microscopic slides (thin smear) were prepared from each ear lobe and cutaneous le- sions for the examination of the presence of Leishmania amastigotes. After Giemsa staining, the slides were evaluated using a light micro- scope (×1000). Before and after smear prepa- ration, the ears of the rodents were sterilized with 70% alcohol (1). The rodents were trans- ferred to the Esfahan Health Research Station animal house and bred for other educational and research purposes. Nested PCR assay The prepared smears from ear lobes and cutaneous lesions in the rodents' ears were used for DNA extraction and investigation of Leish- mania infection in rodents. The genomic DNA http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 126 http://jad.tums.ac.ir Published Online: June 30, 2022 was extracted and purified according to the GeneAll® kit instruction. The extracted DNA samples were kept in 40μl of elution buffer at -20 °C. The partial sequence of the internal tran- scribed spacer (ITS2) of the ribosomal RNA gene (rDNA) was used for detection and iden- tification of different species of Leishmania par- asites. The sequences of the primers were as follows: external forward primer (5'-AAA CTC CTC TCT GGT GCT TGC-3'), external reverse primer (5'-AAA CAA AGG TTG TCG GGG G-3'), internal forward primer (5'- AAT TCA ACT TCG CGT TGG CC-3') and internal re- verse primer (5'-CCT CTC TTT TTT CTC TGT GC-3') (20). Three microliters of template DNA, 1.5μl of each external primers, 12.5μl of Ampliqon (Taq DNA Polymerase 2x Master Mix Red- MgCl2 1.5mM/2mM), and 6.5μl of sterile dis- tilled water were used in the initial PCR. Ther- mal cycle steps of the initial PCR were as fol- lows: at 95 °C for 5 minutes (initial denatur- ation), 30 cycles at 94 °C for 30 seconds (de- naturation), 60 °C for 45 seconds (annealing), and 72 °C for 1 minute (extension) with a final extension step at 72 °C for 5min. The second-round (nested) PCR included 3μl of the diluted product of the first-round PCR (1:10 dilution in distilled water), 1.5μl of each internal primer, 10μl of Ampliqon, and 6 μl of sterile distilled water. The thermal cycle steps of the second-round PCR were as fol- lows: 95 °C for 2 minutes (initial denatura- tion), 30 cycles at 94 °C for 15 seconds (dena- turation), 62 °C for 30 seconds (annealing) and 72 °C for 45 seconds (extension) with a final extension step at 72 °C for 5min. The PCR products were analyzed by 1.5% agarose gel electrophoresis. Bands created in the gel were visualized using UV ray and gel stain and photographed. The standard strain L. major (MRHO/IR/75/ER) was used as posi- tive control and distilled water was used as a negative control. The predicted size of ITS2 products in the second-round PCR was 247bp for L. major (GenBank accession numbers: FJ753394), 206bp for Leishmania gerbilli Wang, Qu and Guan, 1964 (GenBank accession num- bers: AJ300486), and 141bp for Leishmania tu- ranica Strelkova etc, 1990 (GenBank accession numbers: AJ272382). Also, the ectoparasites collected from PCR positive rodents were tested for detection of Leishmania parasites using nested PCR assay as described. PCR-RFLP and sequencing The results of the second-round PCR prod- ucts were confirmed by two methods; sequenc- ing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using restriction digestion with MnlI (Jena Bi- oscience, Germany). PCR-RFLP was done by mixing 1μl of the MnlI enzyme and 1.5μl of G buffer with 12.5μl of the positive nested PCR product in a final volume of 15μl (21). In this technique, 120, 73, 43 and 11bp band lengths were expected for L. major (GenBank accession number: FJ753394), 158, 37 and 11bp band lengths for L. gerbilli (GenBank accession num- ber: AJ300486), 131 and 10bp band lengths for L. turanica (GenBank accession number: AJ272382). Moreover, 30μl of 10 positive nested PCR products were sent to Macrogen, South Korea for sequencing after purification using the Bi- oneer kit. The fragments were sequenced us- ing internal forward and reverse primers. Nu- cleotide sequences were edited and aligned using the Chromas Pro v2.1.3 and were com- pared with the sequences in the GenBank us- ing the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotech- nology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST). Then, the phylogenetic tree was constructed by the Mega 7 software and Maximum Likelihood Tree method with a bootstrap value of 1000 repli- cates. All the obtained sequences in this study were recorded in the GenBank. http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/BLAST J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 127 http://jad.tums.ac.ir Published Online: June 30, 2022 Statistical analysis Fisher's exact test was used to evaluate the difference in the infected rodents between dif- ferent seasons by SPSS 22.00 software and the p-values less than 0.05 were considered signif- icant. To estimate evolutionary relationships be- tween two nucleotide sequences, the correla- tion coefficient was calculated using Mega 7 software and the pairwise distance matrices method using the P distance model. So, the cor- relation coefficient between two distance ma- trices was used to estimate the similarity of two sequences and infer the exact genetic re- lationship between species of the Leishmania. Results Totally, 92 R. opimus (52 females and 40 male) and one Nesoki indica Gray and Hard- wicke, 1832 (one male) were captured. Nine- teen rodents were trapped in the fall, 11 in the winter, 28 in the spring, and 35 in the summer and 9 different species of ectoparasites (527) were collected from them. The identified ec- toparasites included one tick species (one nymph of R. sanguineus), one sucking louse species (Polyplax spp. Enderlein, 1904), five flea species (399 Xenopsylla nuttalli Ioff, 1930, 46 Echidnophaga oschanini Wagner, 1930, 5 Nosopsyllus ziarus Klein, 1963, 5 Cop- topsylla mesghalii Farhang-Azad, 1966 and one Nosopsyllus turkmenicus turkmenicus Vlasov and Ioff, 1937) and two mite species (28 Hirstio- nyssus sp. Fonseca, 1948 and 41 Dermanyssus sanguineus Hirst, 1914 (synonym: Liponyssoides sanguineus)) (Fig. 1). The amastigote form of Leishmania para- sites was seen in 14 (15.2%) out of 92 collected R. opimus using microscopic examination. While 77 of R. opimus and one N. indica (83.87%) were positive using nested PCR. Table 1 shows the infection of rodents with different species of Leishmania in different seasons using nest- ed PCR. All positive smears were positive by nested PCR. The percentage of rodents infect- ed with L. major and L. turanica was 39.79% (37) and 15.05% (14), respectively. Mixed nat- ural infections were seen in 15.05% of rodents (14) with L. major and L. turanica, 7.53% of the rodents (7) with L. major and L. gerbilli, and 6.45% of the rodents (6) with the three Leishmania species. Nesoki indica was infect- ed with L. major. Fisher's exact test showed a significant difference in the leishmanial infec- tion rate between different seasons (P= 0.004). The rodents Leishmania spp. infection rate was the highest (94.29%) in the summer and the lowest (45.45%) in the winter. Mixed leish- manial infection of L. major and L. turanica was observed in all the seasons (Table 1). Moreover, 39 out of 54 fleas (72.22%), five out of eight mites (75%), and one tick nymph (100%) were found Leishmania PCR positive using nested PCR. Leishmania DNA was de- tected from 67.5% (27/40) of X. nuttalli, 100% (3/3) of N. ziarus, 100% (3/3) of C. mesghalii, 75% (6/8) of E. oschanini, 80% (4/5) of D. sanguineus, 66.66% (2/3) of Hirstionyssus sp., and 100% (1/1) of the nymphs of R. sanguineus. Of five PCR positive specimens of X. nuttalli fleas with gravid abdomen status, one (female) was L. major PCR positive for, two (males) were positive for both of L. major and L. turanica, and two (females) for both of L. major and L. gerbilli. Moreover, one of two L. major PCR positive Hirstionyssus sp. mites was gravid and the rest of the PCR positive ectoparasites were blood-fed. DNA of the three species of Leish- mania parasites and L. turanica was found in two E. oschanini (male) and one N. ziarus (male) fleas, respectively (Table 2). Out of ten positive ITS2 nested PCR prod- ucts, nine were sequenced well. All these se- quences were compared with the sequences of ITS2 in the GenBank. The GenBank accession numbers for the ITS2 region of the Leishma- nia parasites included MK372246 (L. turani- ca; host: R. opimus), MK372247 (L. infantum; host: R. opimus), MK372248 (L. major; host: Hirstionyssus sp.), MK372249 (L. major; host: X. nuttalli), MK372250 (L. major; host: R. opi- mus), MK372251 (L. major; host: N. ziarus), http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 128 http://jad.tums.ac.ir Published Online: June 30, 2022 MK372252 (L. major; host: R. sanguineus), MK372253 (L. major; host: C. mesghalii) and MK372254 (L. major; host: D. sanguineus). One of the sequenced samples was similar to L. in- fantum with accession number MG831328.1 (Query cover= 98%, E value= 1e-72 and Ident= 96%). Leishmania tropica Wright, 1903 ITS2 se- quence was obtained from GenBank and used as an out group for construction of phylogenetic tree. All the L. major (n=7) sequences obtained in this study clustered with L. major from GenBank (accession number: AJ786164) and formed a separate clade in the tree. Also, L. trunica obtained in this study was sister taxon with L. turanica from GenBank (accession num- ber: HF545838) and both associated with L. gerbilli (accession number: HF545839) and formed separate clade (Fig. 2). The genetic dis- tance coefficient showed no difference between the sequences of L. major in this study and the sequence recorded in the GenBank. The evo- lutionary correlation coefficient between the L. tropica and the sequence of L. major and L. turanica was 0.516 and 0.550, respectively. In addition, the difference of evolutionary corre- lations between the L. turanica sequence rec- orded in the GenBank and the sequence of L. turanica in this study was 0.008 (Table 3). Table 1. Abundance and rate of seasonal Leishmania parasite infection in rodents detected by nested-PCR assay of ITS2-rDNA gene, Segzi area, Esfahan Province, October 2016–October 2017 Season A ll c a p tu r e d r o d e n ts Infected rodents with Leishmania spp. Total infected rodents (%) L. major (%) L. turanica (%) L. major + L. turanica (%) L. major + L. gerbilli (%) L. major + L. turan- ica + L. gerbilli (%) Fall 19 11(57.9) 1(5.26) 1(5.26) 3(15.79) 0(0) 16(84.21) Winter 11 1(9.09) 0(0) 3(27.28) 0(0) 1(9.09) 5(45.45) Spring 28 9(32.14) 7(25) 3(10.72) 2(7.14) 3(10.72) 24(85.71) Summer 35 16(45.73) 6(17.14) 7(20) 2(5.71) 2(5.71) 33(94.29) Total 93 37(39.79) 14(15.05) 14(15.05) 7(7.53) 6(6.45) 78(83.87) Fig. 1. Different species of collected Ectoparasites collected from rodents in Segzi, Esfahan province, 2016-2017; a: Coptopsylla mesghalii, b: Echidnophaga oschanini, Nosopsyllus turkmenicus turkmenicus (c), Nosopsyllus ziarus (d), Xenopsylla nuttalli (e), Rhipicephalus sanguineus (f), Polyplax spp. (g), Hirstionyssus sp. (h) and Dermanyssus san- guineus (i) (Original photos) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 129 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 2. Abdominal status and Leishmania parasite infection in rodents’ ectoparasites detected by nested-PCR assay of ITS2-rDNA gene, Segzi area, Esfahan Province, October 2016–October 2017 Species of ectoparasites N o . o f e x a m in e d s a m - p le s PCR-positivity of the ectoparasites with Leishmania spp. N o . o f p o si ti v e sa m p le s B lo o d -F e d B lo o d -d ig e st e d L. major L. turani- ca L. major + L. turani- ca L. major + L. gerbilli L. major +L. turanica + L. gerbilli Xenopsylla nuttalli 40 27 22 5 23 0 2 2 0 Echidnophaga oschanini 8 6 6 0 4 0 0 0 2 Nosopsyllus ziarus 3 3 3 0 2 1 0 0 0 Coptopsylla mesghalii 3 3 3 0 3 0 0 0 0 Dermanyssus sanguineus 5 4 4 0 4 0 0 0 0 Hirstionyssus sp. 3 2 1 1 2 0 0 0 0 Rhipicephalus sanguineus 1 1 1 0 1 0 0 0 0 Total 63 46 40 6 39 1 2 2 2 Fig. 2. Phylogenetic tree of the partial ITS2 gene sequences of the isolated Leishmania parasites from ectoparasites (MK372248-49 and MK372251-54) and smear of rodents' ear lobes (MK372246-50) and the ITS2 sequences obtained from GenBank. Bootstrap values that are reported as percentages at the nodes were obtained from 1000 bootstrap repli- cates. Scale bar corresponds to 0.1 change per nucleotide http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 130 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 3. Comparison of the ITS2 sequences of the isolated Leishmania parasites from ectoparasites and smears of ear lobes of rodents, Segzi area, Esfahan Province, October 2016–October 2017 Leishmania species with GenBank accession numbers 1 2 3 4 5 6 7 8 9 10 11 12 M K 3 7 2 2 4 6 H F 5 4 5 8 3 8 H F 5 4 5 8 3 9 M K 3 7 2 2 4 8 M K 3 7 2 2 4 9 M K 3 7 2 2 5 0 M K 3 7 2 2 5 1 M K 3 7 2 2 5 2 M K 3 7 2 2 5 3 M K 3 7 2 2 5 4 A J 7 8 6 1 6 4 K R 7 0 6 3 7 4 1 L. turanica MK372246 0.000 2 L. turanica HF545838 0.008 3 L. gerbilli HF545839 0.042 0.042 4 L. major MK372248 0.203 0.211 0.203 5 L. major MK372249 0.203 0.211 0.203 0.000 6 L. major MK372250 0.203 0.211 0.203 0.000 0.000 7 L. major MK372251 0.203 0.211 0.203 0.000 0.000 0.000 8 L. major MK372252 0.203 0.211 0.203 0.000 0.000 0.000 0.000 9 L. major MK372253 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 10 L. major MK372254 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 0.000 11 L. major AJ786164 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 12 L. tropica KR706374 0.550 0.559 0.542 0.516 0.516 0.516 0.516 0.516 0.516 0.516 0.516 0.000 Note. These sequences were compared with the available ITS2 sequences in GenBank (AJ786164.1, HF545839.1, HF545838.1 and KR706374.1). The correlation coefficient was calculated using Mega 7 soft- ware and pairwise distance matrices method using P distance model. Discussion The current study showed that R. opimus was found in large numbers in Segzi. The dis- tribution of R. opimus has a strong correlation with the climatic conditions and topography of the area, such as the seasonal rainfall and aver- age annual temperature (22). In recent years, because of the population growth, making fac- tories and residential houses near the colonies of the reservoir hosts, and expansion of the city, leishmanial infections in rodent populations and humans have increased (23). Also, with the emergence of drought in the Segzi plain, rodents have invaded the outskirts of the city. So, con- tact of rodents with humans has increased and subsequently, the leishmanial infection rates have increased. In a study on ZCL by Akha- van et al. (20) in three rural districts (Borkhar, Segzi and Badrood) of Esfahan Province, 21 out of 95 R. opimus were positive by micro- scopic examination and 48 of them by nested PCR. The results of our and other recent stud- ies (20, 21, 24) indicate that L. major, L. gerbilli, and L. turanica, and mixed natural infections exist in R. opimus populations (Table 1). In this http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 131 http://jad.tums.ac.ir Published Online: June 30, 2022 study, the highest infection rates with L. ma- jor and L. turanica in R. opimus populations were observed in summer and spring respec- tively, and the highest percentage of L. major and L. turanica coinfections was seen in win- ter. The least infection rate of L. major was in the winter (Table 1). Mixed natural infections with L. major and L. turanica in R. opimus pop- ulations are typical in central Asia (25). Leish- mania turanica raises the persistence of L. major infection in R. opimus (26). Therefore, mixed natural infections with L. major and L. turanica help to preserve leishmaniasis in rodent popu- lations. According to the results of this study, young R. opimus gerbils matured after the win- ter which more than 40% of them were infect- ed (Table 1) at the emerging of P. papatasi and probable vectors in late May. Due to the suitable environmental and climatic conditions and the consequent abundance of food resources, gerbils reproduce twice or more per year (19, 27). In the Segzi region, which receives the most rainfall in late winter and early spring, a signif- icant increase in population rates was observed in spring. From early spring to summer, the in- creasing trend in the numbers of R. opimus de- clined (27) and the rate of bites per rodent in- creased. So, it is expected that the chances of parasite ingestion by P. papatasi and ectopar- asites from infected rodents are then greater. In our study, phylogenetic analysis results showed no significant difference between the L. major sequences and L. major isolated from rodents and ectoparasites were located in the same clade or monophyletic group. It means that L. major parasite extracted from rodents were not ge- netically different from those extracted from ec- toparasites and were similar (Fig. 2 and Table 3). Zoonotic cutaneous leishmaniosis is increas- ing Esfahan Province, central Iran (28), while the leishmanial infection rates of the main vec- tor are low in the endemic areas (29). Therefore, it could be suggested that other vectors could have a role in disease transmission. In Aran and Bidgol city in Esfahan Province, Doroodgar et al. (29) reported that 17.8% of R. opimus, 71.4% of human isolates and 1.9% of P. pa- patasi infected with L. major. The possibility of the transmission of Leishmania parasites by ectoparasites has long been discussed (6, 11, 30) and has been already proved in laboratory conditions (5, 13, 31). The present study showed that ectoparasites of the rodents can easily in- gest Leishmania parasites during blood-feed- ing. However, just only this evidence cannot prove that these ectoparasites are vectors of ZCL. In our study, PCR analysis of ectoparasites specimens to detect the DNA of Leishmania spp. showed 73.02% (46/63 ectoparasites) positivi- ty and the DNA of L. major was detected in 7 of ectoparasite species, including R. sanguineus, X. nuttalli, E. oschanini, N. ziarus, C. mesghalii, Hirstionyssus sp. and D. sanguineus by nested PCR (Table 2). The high positivity rate in the ectoparasites is related to their life habits and the long duration of blood feeding. Around 52.6 % (41/78) of the rodents infected with Leish- mania spp. had been infested with the ecto- parasites. Two studies by McKenzie and Fer- reira et al. showed transmission of Leishmania parasites by R. sanguineus and C. felis in labor- atory conditions (6, 7). In the mid-1980s, McKenzie (6) demonstrat- ed that the collected R. sanguineus from natu- rally infected dogs can inject Leishmania par- asites into the healthy dogs during blood-feed- ing and infect them. Ferreira et al. (7) demon- strated that C. felis fleas collected from infect- ed dogs could transmit Leishmania spp. non- infected laboratory hamsters and it was observed that 18.1% of the hamsters were positive by both methods of PCR and enzyme-linked immuno- sorbent assay (ELISA). Coutinho et al. (5), found that six R. sanguineus ticks (15.4%) were pos- itive for L. chagasi using the PCR technique. They showed that R. sanguineus could transfer L. chagasi from infected dogs to hamsters in la- boratory conditions. In another study, the pro- mastigotes of L. chagasi were observed in 4 stained smears out of 207 (1.9%) C. felis fe- lis specimens collected from dogs, whereas Leishmania spp. infection was reported in 43 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 132 http://jad.tums.ac.ir Published Online: June 30, 2022 out of 144 (29.9%) fleas by PCR (13). In a study in northwest of Iran by Azarm, in which three species of fleas; C. canis Cur- tis, 1826, C. felis and Pulex irritans Linnaeus, 1758 were collected from dogs, 75% of C. canis and 66.7% of C. felis collected from in- fected dogs were PCR-positive for L. infantum. But Leishmania DNA was not detected from P. irritans (32). In fact, Coutinho et al. (5), McKen- zie (6), Ferreira et al. (7), and Coutinho and Li- nardi (13) demonstrated that Leishmania para- sites can be viable in the blood-sucking ar- thropods (such as tick and fleas) in laboratory conditions and infected their vertebrate hosts, but, they did not prove that these arthropods could act as vectors of the Leishmania para- site in nature. In the current study, five specimens of X. nuttalli fleas and one Hirstionyssus sp. mite, which were gravid, were found Leishmania DNA PCR positive (Table 2). In Xenopsylla spp. fleas, blood digestion lasts 2–9 days de- pending on the temperature, relative humidity, and the host species. In fleas, the duration of digestion is shorter at low relative humidity than high relative humidity (33). In a study by Colombo, L. infantum was detected in 23% of the fleas and 50% of the ticks collected from the infected dogs by reverse transcription PCR (RT-PCR), real time PCR, and ELISA. In ad- dition, RNA analysis of the tick specimens col- lected from infected dogs after seven to ten days showed that the parasites were alive. Moreover, the alive parasites were isolated from adult ticks that had molted recently (34). In a study con- ducted in Brazil, the results of immunohisto- chemistry (IHC) and real time PCR showed Leishmania spp. promastigotes in the intestine, ovaries, and salivary glands of the R. sanguineus ticks collected from infected dogs (35). Prob- ably Leishmania spp. may remain in these ec- toparasites such as X. nuttalli and Hirstionyssus sp., at least until full digestion of blood meal. However, the detection of Leishmania DNA is not sufficient evidence of parasite survival in X. nuttalli and Hirstionyssus sp., and this re- quired more careful study. In another study in Turkey, to investigate the presence of L. major, the pools of R. san- guineus ticks on Meriones unguiculatus Milne- Edwards, 1867 were examined by RT-PCR. The results showed that none of the pools was infected with L. major (36). However, in this study the main reservoir has not been tested for the potency of ticks in the transmission of L. major. A study by Rakhshanpour et al. (37), in Iran showed that approximately 67% of the R. sanguineus ticks collected from dogs were infected with L. infantum using the semi-nest- ed PCR. However, none of the parasitological (Giemsa staining and cultivation of parasite) and molecular (nested PCR) tests results were positive when the transmission of L. infantum by stained R. sanguineus between dogs was stud- ied in laboratory conditions. In fact, blood-feeding arthropods are suscep- tible to infection with different types of patho- genic microorganisms, but it does not mean that they can transmit all of them (38). Although there is no strong evidence indicating that ec- toparasites act as a vector of the Leishmania parasites; recent studies suggest that this theo- ry is important. Evolutionarily, it is unclear how long it would take for the Leishmania parasites to adapt to other blood-feeding arthropods. This is a fact that the Leishmania parasites have a long evolutionary history with the main ecto- parasites of Leishmanial spp. reservoirs, such as L. infantum with R. sanguineus and C. felis (dog ectoparasites) (5, 7) and L. major with X. nuttalli (ectoparasite of R. opimus) (39, 40) and they have been in contact during this time. Therefore, it is possible that the ectoparasites will retain Leishmania parasites over time and act between reservoirs as potential vectors. Conclusion In conclusion, this study found that L. major, L. gerbilli, and L. turanica exist in R. opimus populations in Segzi area. The highest infec- tion rates of R. opimus populations with L. ma- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 124–135 S Azarmi et al.: PCR Positivity of … 133 http://jad.tums.ac.ir Published Online: June 30, 2022 jor and L. turanica were observed in the sum- mer and spring respectively, and the highest percentage of L. major and L. turanica coin- fection was seen in the winter. According to the results of this study, it is possible that L. major remains in the ectoparasites such as X. nuttalli and Hirstionyssus sp., at least until the blood fed is digested. Therefore, the presence of L. major in the ectoparasites indicates the proba- ble importance of rodents’ ectoparasites in ZCL dispersion. Thus, to study the probable role of ectoparasites in the transmission of L. major in rural areas between reservoirs and humans, it should be investigated whether the parasites remain viable inside the ectoparasites and per- formed Xenodiagnosis test on the main reser- voirs for experimental infections. Acknowledgements All authors sincerely thank the head and staff of Esfahan Health Research Station for their assistance in the project. Ethical considerations Animal experiments were admired by the Ethics Committee of the Tehran University of Medical Sciences, Tehran, Iran (the ethical code: IR.TUMS.SPH.REC.1396.2804). Conflict of interest statement Authors declare that there is no conflict of interest. References 1. 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