Dataset Information

Genetic diversity of Leishmania major strains isolated from Phlebotomus papatasi and Phlebotomus alexandri in western Iran based on minicircle kDNA.

ABSTRACT: This study aimed to screen the natural infection rate of Leishmania major in Phlebotomus papatasi and Phlebotomus alexandri in two counties (Mehran and Dehloran) of Ilam province as cutaneous leishmaniasis endemic areas in the west of Iran. Furthermore, the genetic diversity of parasite species that are isolated from vectors, was investigated. Sandflies were collected by sticky traps from May 2018 to October 2018. Afterward, specimens were prepared for species identification by morphological features. DNA was extracted from female sandflies, and minicircle kDNA was used to identify Leishmania isolates through nested-PCR, followed by genetic diversity between Leishmania isolates was investigated by sequence analysis of the amplified minicircle kDNA. Natural infection of the L. major was shown in all positive specimens using nested-PCR. Analysis of data from 14 isolates displayed a high level of genetic diversity in L. major. In the phylogenetic trees, all of the L. major isolates occurred in six clusters. Clusters I, II, III, and VI contained isolated strains from P. papatasi. While clusters IV and V contained isolated strains from P. alexandri. Genetic diversity of L. major isolated from vectors was investigated in western Iran for the first time. According to the results of this study, probably "various clones of L. major populations are distributed in the study area.

SUBMITTER: Khoshnood S

PROVIDER: S-EPMC8368947 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Genetic diversity of Leishmania major strains isolated from Phlebotomus papatasi and Phlebotomus alexandri in western Iran based on minicircle kDNA.

Khoshnood Saleh S Tavalla Mehdi M Mohaghegh Mohammad Ali MA Khamesipour Faham F Hejazi Seyed Hossein SH

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20210310 3

This study aimed to screen the natural infection rate of Leishmania major in Phlebotomus papatasi and Phlebotomus alexandri in two counties (Mehran and Dehloran) of Ilam province as cutaneous leishmaniasis endemic areas in the west of Iran. Furthermore, the genetic diversity of parasite species that are isolated from vectors, was investigated. Sandflies were collected by sticky traps from May 2018 to October 2018. Afterward, specimens were prepared for species identification ...[more]

PMID: 34475667

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Project description:Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.

Project description:BackgroundLeishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.MethodsDNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.ResultsThe qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.ConclusionsA new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Project description:Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

Dataset Information

Genetic diversity of Leishmania major strains isolated from Phlebotomus papatasi and Phlebotomus alexandri in western Iran based on minicircle kDNA.

Publications

Genetic diversity of <i>Leishmania major</i> strains isolated from <i>Phlebotomus papatasi</i> and <i>Phlebotomus alexandri</i> in western Iran based on minicircle kDNA.

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