BackgroundThe aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale.Methodology and Principal FindingsLeishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate?Conclusions and SignificanceWe propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

BackgroundThe recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported.Methods/Principal FindingsA common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am.ConclusionsDespite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential ri...

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Developmental regulation of mRNA levels in trypanosomatid protozoa is determined post-transcriptionally and often involves sequences located in the 3-untranslated regions (3-UTR) of the mRNAs. We have previously identified a developmentally regulated gene family in Leishmania encoding the amastin surface proteins and showed that stage-specific accumulation of the amastin mRNA is mediated by sequences within the 3-UTR. Here we identified a 450-nt region within the amastin 3-UTR that can confer amastigote-specific gene expression by a novel mechanism that increases mRNA translation without an increase in mRNA stability. Remarkably, this 450-nt 3-UTR element is highly conserved among a large number of Leishmania mRNAs in several Leishmania species. Here we show that several of these mRNAs are differentially expressed in the intracellular amastigote stage of the parasite and that the 450-nt conserved element in their 3-UTRs is responsible for stage-specific gene regulation. We propose that the 450-nt conserved element, which is unlike any other regulatory element identified thus far, is part of a common mechanism of stage-regulated gene expression in Leishmania that regulates mRNA translation in response to intracellular stresses.Parasites of the genus Leishmania cause cutaneous, mucocutaneous, and visceral infections affecting ϳ400,000 people each year of the 397 million that are at risk worldwide (1). During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sand fly vector as an extracellular promastigote and the acidic phagolysosomes of macrophages as an intracellular amastigote. Differentiation of the parasite into the amastigote form is a prerequisite for its intracellular survival. Several environmental factors including acidic pH, elevated temperature, and the harmful phagolysosomal milieu trigger cytodifferentiation accompanied by the differential expression of a variety of genes (2-6). Such stage-specific gene expression is crucial for adaptation because Leishmania differentiates from an extracellular to an intracellular parasite. Gene regulation in Leishmania and related trypanosomatids shares unique features that include polycistronic transcription of large RNA units by an ␣-amanitin-sensitive RNA polymerase II, probably in the absence of promoter elements, and pre-mRNA processing into monocistronic mRNAs through a post-transcriptional control mediated by trans-splicing and polyadenylation (7-9). trans-Splicing and polyadenylation are mechanistically coupled in trypanosomatids and recognize regulatory signals that consist of polypyrimidine-rich sequences (10,11).Numerous examples in Leishmania species support the notion that developmental regulation of mRNA levels is determined post-transcriptionally by sequences located in the 3Ј-untranslated regions (3Ј-UTR) 1 that usually control mRNA stability (12-17). More recently, a novel mechanism of stagespecific regulation affecting pre-mRNA processing has been reported in Leishmania mexicana (18). The role of 3Ј-UTRs and/or int...

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Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 M (0.12 g/ml), 55 g/ml, 95 g/ml, and 0.12 g/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferaseexpressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.Leishmaniasis is a significant cause of morbidity and mortality in several countries of the world (19). A vertebrate host is infected with flagellated extracellular promastigote forms via the bite of a sand fly. Promastigotes are rapidly transformed into nonflagellated amastigotes, which divide actively within the mononuclear phagocytes of the vertebrate host.The basic treatment for leishmaniasis consists of the administration of sodium stibogluconate (Pentostam), meglumine (Glucantime), or pentamidine. Treatment failure, especially in kala-azar, mucosal leishmaniasis, and diffuse cutaneous leishmaniasis, is becoming a common problem in many areas where the diseases are endemic. There are now strong indications that treatment failure may be partly due to the drug resistance of the parasite (15,18,21,25). In addition, numerous cases of relapse or unresponsiveness have been reported during the treatment of patients coinfected with human immunodeficiency virus and Leishmania spp. (1). Although rapid assays for drug screening of Leishmania promastigotes have been devised (5), promastigotes are usually less sensitive to various drugs than amastigotes (11,29). Although animal models are well established for drug testing, they are not suitable for largescale primary drug screens. The development of new drugs has been impeded by the lack of a simple, reliable, and rapid evaluation system allowing the simultaneous determination of drug activity at the mammalian stage under both axenic and intracellular conditions. The development of a system for the in vitro cultivation of amastigotes under axenic conditions enabled the development of models for in vitro drug sc...