Different morphologies of Leishmania major amastigotes with no molecular diversity in a neglected endemic area of zoonotic cutaneous leishmaniasis in Iran

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Suspected patients [n = 165] were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012- 2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b [Cyt b] markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Only L. major was identified in patients containing regular amastigotes' shapes [oval or round] with a size of 2-4 microm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes [spindle, pear, or cigarette] were observed separately in non-classical wet lesions with more than 4 microm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation [chi [2] P > 0.05], including only one common haplotype [GenBank access no. EF413075]. Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits

Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran

One of the well-known foci of zoonotic cutaneous leishmaniasis [ZCL] in Iran is Turkemen Sahara, which is located in north eastern Iran. ZCL is a disease of mammals, and humans can become infected as accidental hosts. Many researchers have argued that Rhombomys opimus is the only main reservoir host of ZCL in this region of the Golestan province. No other rodents or mammals are thought to host or have been reported to host Leishmania parasites in this region. This research was designed and developed to isolate, detect and firmly identify Leishmania parasites in mammals and rodents other than R. opimus. Wild mammals were caught from gerbil burrows. Leishmania parasites were detected to assess the infection of reservoir hosts in 2010. Each genomic DNA sample was screened for Leishmania infection via nested PCR and sequencing using the internal transcribed spacer ribosomal DNA [ITS-rDNA] identification protocol for parasites. The greatest number of infections [8/19] were found in Menones libycus. One in three infections was found in Hemiechinus auritus, and this is the first report of infection in this species. Only Leishmania major was definitively identified and unambiguously typed in M. libycus and H. auritus. The infection rates in these two wild mammals were not significantly different, and no other gerbil parasites were detected in M. libycus or H. auritus at our study site. Recent findings of Leishmania turanica in R. opimus and failures to detect L. turanica in M. libycus may be attributable to unidentified Leishmania infections in two M. libycus due to unreadable sequences. These cases may represent mixed infections by L. major and L. turanica. The assumptions that gerbil parasites can be co-infectors provide a starting point for the identification of the causative and potential parasites responsible for the frequent infections that are mainly mediated via sandfly vectors

Distribution, fauna and seasonal variation of sandflies, simultaneous detection of nuclear internal transcribed spacer ribosomal DNA gene of leishmania major in rhombomys opimus and phlebotomus papatasi, in natanz district in central part of Iran

Zoonotic cutaneous leishmaniasis [ZCL] due to Leishmania major is increasing in many parts of Iran. This disease originally is a disease found in gerbils. Leishmania parasites are transmitted by sandflies that live and breed in gerbil burrows. Nested PCR amplified Leishmania ITS1-5.8S rRNA gene in both main reservoir host "Rhombomys opimus" and in the "Phlebotomus papatasi" main vector of ZCL, in Iran. Population differentiation and seasonal variation of sandflies were analyzed at a microgeographical level in order to identify any isolation by distance, habitat or seasons. Populations of sandflies were sampled from the edges of villages in Natanz, Isfahan province, Iran, using the Centers for Disease Control traps and sticky papers. Individual sandflies were identified based on external and internal morphological characters. Nested PCR protocols were used to amplify Leishmania ITS1-5.8S rRNA gene, which were shown to be species-specific via DNA sequence. A total of 4500 sandflies were collected and identified. P. papatasi, Phlebotomus sergenti and Phlebotomus jacusieli from genus Phlebotomus and Sergentomyia sintoni and Sergentomyia clydei from genus Sergentomyia were identified in this region. P. papatasi was the most abundant sandfly in the collections. Ten out of 549 female P. papatasi and four out of 19 R. opimus were found to be infected with L. major. Seasonal activity of sandflies starts in June and ends in November. Abundance of P. papatasi was in September. Finding and molecular typing of L. major in P. papatasi and R. opimus confirmed the main vector and reservoir in this region

Molecular investigation of the population differentiation of phlebotomus papatasi, important vector of leishmania major, in different habitats and regions of Iran

Phlebotomus papatasi is an important vector of L. major in Iran. P. papatasi was collected from peridomestic animal shelters, inside and around the houses and also the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in several provinces in Iran. Mitochondrial Cytochrome b [Cyt b] of sandflies, which is a maternally-inherited gene marker, was used to see if there is any "isolation by distance" over a large geographical scale among Iranian provinces. The analyses were based on the last 717 bp of the Cyt b gene followed by 20 bp of intergenic spacer and the transfer tRNA ser [TCN] gene, i.e. the 737 bp fragment [without primers] amplified with the primers CB1-SE and CB-R06. Cyt b Long fragment sequences were obtained from 149 out of 177 specimens of P. papatasi and 49 haplotypes were identified. Based on the Cyt b Long fragment examined, P. papatasi showed only recent divergence in Iran, because the genetic distances between haplotypes were small. However, some evidence for isolation by distance was found. First, all the haplotypes from Iran did not belong to a single network, whereas most from Golestan province [Iran] did belong to a single network. Second, there were some abundant haplotypes that were found only in one province