Esfahan is known as one of the important foci for ZCL in Iran. Segzi has a population of about 6500. According to the reports of Esfahan Health Centers, cutaneous leishmaniasis is reported on average 10 cases per 1000 people annually. R. opimus is the main reservoir host for ZCL in the northeast parts of the central plateau of Iran, and N. indica has been identified as the secondary reservoir in Esfahan . The current study showed that R. opimus rodents were found in large numbers in Segzi. The distribution of R. opimus has a strong correlation with the climatic conditions and topography of the area, such as the seasonal rainfall and average annual temperature . In recent years, because of the population growth, making factories and residential houses near the colonies of the reservoir hosts, and expansion of the city, leishmanial infections in rodent populations and humans have increased. Also, with the emergence of drought in the Segzi plain, rodents have invaded the outskirts of the city. So, contact of rodents with humans has increased and subsequently, the leishmanial infection rates have increased. In a study on ZCL by Akhavan et al in three rural districts (Borkhar, Segzi and Badrood) of Esfahan Province, 21 out of 95 R. opimus were positive by microscopic examination and 48 of them by nested PCR . The results of our and other recent studies [20, 21] indicate that L. major, L. gerbilli, and L. turanica, and mixed natural infections exist in R. opimus populations (Table 1). In this study, the highest infection rates with L. major and L. turanica in R. opimus populations were observed in the summer and spring respectively, and the highest percentage of L. major and L. turanica coinfections was seen in the winter. 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 populations are typical in central Asia . L. turanica raises the persistence of L. major infection in R. opimus . Therefore, mixed natural infections with L. major and L. turanica help to preserve leishmaniasis in rodent populations. According to the results of this study, it can be said that young R. opimus gerbils mature after the winter, with more than 40% of them (Table 1) being infected at the beginning of the active season of Ph. papatasi and probable vectors. Because of rodents breeding in early spring, rodent populations increase and the transmission of ZCL begins in the Segzi area when the sand flies emerge in late May. From early spring to summer, the increasing trend in the numbers of R. opimus declines  and the proportion of infected animals rises. So, the highest leishmanial infection rate of R. opimus is in late summer and the chances of parasite ingestion by Ph. papatasi and ectoparasites that bite infected rodents are then greater. In our study, phylogenetic analysis results showed no significant difference between the L. major sequences and they were monophyletic (Figure 1 and Table 3).
Zoonotic cutaneous leishmaniosis is increasing Esfahan Province, central Iran , while the leishmanial infection rates of the main vector are low in the endemic areas . Therefore, it could be suggested that other vectors could have a role in disease transmission. In the Aran o Bidgol city in Esfahan province, Doroodgar et al reported that 17.8% of R. opimus, 71.4% of human isolates and 1.9% of P. papatasi infected with L. major . The possibility of the transmission of Leishmania parasites by ectoparasites has long been discussed [6, 11, 28] and has been already proved in laboratory conditions [5, 13, 29]. The ectopasites collected from infected rodents were selected for genomic DNA detection of Leishmania parasites using nested PCR. The present study showed that ectoparasites of the rodents can easily ingest Leishmania parasites during blood-feeding. However, just only this evidence cannot prove that these ectoparasites are vctors of ZCL.
In our study, PCR analysis of ectoparasites specimens to detect the DNA of Leishmania spp. showed 73.02% (46/63 ectoparasites) positivity 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 Leishmania spp. had been infested with the ectoparasites. Two studies by K. K. McKenzie and Ferreira et al. showed transmission of Leishmania parasites by R. sanguineus and C. felis in laboratory conditions [6, 7].
In the mid-1980s, McKenzie, KK demonstrated that the collected R. sanguineus from naturally infected dogs can inject Leishmania parasites into the healthy dogs during blood-feeding and infect them . Ferreira, MGPA et al demonstrated that the collected C. felis fleas from infected dogs can transmit Leishmania spp. to the uninfected hamsters and it was observed that 18.1% of the hamsters were positive by both methods of PCR and enzyme-linked immunosorbent assay (ELISA), 45% of the hamsters were positive only by PCR method, and 9% of the hamsters were only by ELISA . Coutinho et al, found that 6 R. sanguineus ticks (15.4%) were positive for L. chagasi using the PCR technique. They showed that R. sanguineus could transfer L. chagasi from infected dogs to hamsters in laboratory conditions . In another study, the promastigotes of L. chagasi were observed in 4 stained smears out of 207 (1.9%) C. felis felis specimens collected from dogs, whereas Leishmania spp. infection was reported in 43 out of 144 (29.9%) fleas by PCR . In fact, K. K. McKenzie, Ferreira, MGPA et al, Coutinho et al, and Coutinho and Linardi [5, 6, 7, 13]demonstrated that Leishmania parasites can be viable in the blood-sucking arthropods (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 parasite in nature.
In the current study, five specimens of X. nuttalli fleas and one Hirstionyssus sp. mite, which their blood fed was digested, were found PCR-positive for Leishmania DNA (Table 2). In Xenopsylla spp. fleas, blood digestion lasts 2-9 days depending 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 . In a study by Colombo et al., L. infantum was detected in 23% of the fleas and 50% of the ticks collected from the infected dogs by RT-PCR, real time PCR, and ELISA. In addition, RNA analysis of the tick specimens collected from infected dogs after 10 to 7 days showed that the parasites were alive. Moreover, the alive parasites were isolated from adult ticks that had molted recently . In a study conducted in Brazil, the results of immunohistochemistry (IHC) and real-time PCR (RT-PCR) showed Leishmania spp. promastigotes in the intestine, ovaries, and salivary glands of the R. sanguineus ticks collected from infected dogs . Probably Leishmania spp. may remain in these ectoparasites such as X. nuttalli and Hirstionyssus sp., at least until the blood fed is digested. However, the detection of Leishmania DNA is not sufficient evidence of parasite survival in X. nuttalli and Hirstionyssus sp., and this required more careful study.
In another study in Turkey, to investigate the presence of L. major, the pools of R. sanguineus ticks on Meriones unguiculatus were examined by RT-PCR. The results showed that none of the pools was infected with L. major . The study conducted in Turkey could yield more precise results if it used the main reservoirs related to study potency of ticks in the transmission of parasites. A study by Rakhshampour et al, in Iran showed that approximately 67% of the R. sanguineus ticks collected from dogs were infected with L. infantum using the semi-nested 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 studied in laboratory conditions .
In fact, blood-feeding arthropods are susceptible to infection with different types of pathogenic microorganisms, but it does not mean that they can transmit all of them . Although there is no strong evidence indicating that ectoparasites act as a vector of the Leishmania parasites, recent studies suggest that this theory 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 ectoparasites 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) [36, 37] 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.