The 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. Leishmania 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? We 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 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. A 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. Despite 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 risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories. Recent projections on global warming indicate a constant rise of temperatures in the Mediterranean subregion in the near-mid future. While this phenomenon already caused geographical expansion of several arthropod-borne diseases, it is likely to affect also temporal parameters of seasonally transmitted diseases such as leishmaniasis, a protozoan infection spread by the bite of phlebotomine sand flies. Phenology of sand flies consists in the periods of emergence of adults and their disappearance from collections following an activity period during warm months, which can be characterized by peaks of abundance. Current phenological observations can be important for continuing investigations on sand fly dynamics that may impact on leishmaniasis transmission in the future. With this aim, partners from eight Mediterranean countries identified sites with documented Leishmania infantum transmission by six different vector species and performed multiannual trappings. From the high number of 56,000 specimens collected throughout an area spanning from Portugal at west to Georgia at east, the current seasonal dynamics of Mediterranean vectors was obtained. Both, period of adults emergence and type of density trend were found to be significantly correlated with latitude or mean annual temperature of trapping sites. At the southernmost latitudes, vector activity started as early as begin of April and ended by late November, showing that the no-risk period of potential exposure to L. infantum lasted only 4 months.

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. Dengue viruses (DENV) are generally maintained in a cycle which requires horizontal transmission via their arthropod vector, Ae. aegypti, to the vertebrate host. One important consequence of this process is the interference of the virus with the immune systems of both the mosquito and its host. While infection of humans causes disease, the presence of DENV in mosquitoes gives rise to life-long and persistent infection with active viral replication in the salivary glands. In the present study, we have evaluated the mosquito's immune response following DENV infection by analyzing the gene expression profile of infected and uninfected salivary glands. The results show that DENV infection activates signaling pathways and induces the expression of gene products that are involved in the innate immune response to DENV infection, and in particular a putative antibacterial cecropin-like peptide. The immature and mature forms of this peptide were found to be active against a variety of pathogens including DENV and Chikungunya viruses, as well as the Leishmania parasite. This study is the first to establish a comparative analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We demonstrate that certain DENV-induced peptides possess broad-spectrum anti-pathogenic activity and may have therapeutic potential in the treatment of human infectious disease.

Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microassay, previously described as a means of quantifying Leishmania amazonensis in vitro at the amastigote stage (D. Sereno and J. L. Lemesre, Parisitol. Res., in press), we have compared the activities of seven drugs, including those currently used to treat leishmaniasis, against axenically grown amastigote and promastigote forms of three Leishmania species (L. amazonensis, L. mexicana, and L. infantum, responsible for diffuse cutaneous, cutaneous, and visceral leishmaniasis, respectively). The ability of axenically cultured amastigote organisms to be used in an investigation of antileishmanial agents was first evaluated. We have confirmed the toxicities of sodium stibogluconate (Pentostam), pentamidine, and amphotericin B to active and dividing populations of axenically cultured amastigotes. The toxicity of potassium antimonyl tartrate trihydrate, which is generally higher than that of Pentostam, seemed to indicate that pentavalent antimony can be metabolized in vivo to compounds, possibly trivalent in nature, which are more active against the amastigote organisms. When the drug susceptibilities of parasites at both stages were compared, great variations were found for all the drugs studied. These major differences, which show the specific chemosusceptibility of the parasite at the mammalian stage, demonstrate the potential of using cultured amastigotes instead of promastigotes in a drug-screening procedure for early detection. This in vitro model may help in the isolation of active compounds, particularly those with low-grade activities, against the mammalian stage of the parasite.

This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing “Nagana” or animal African trypanosomosis [AAT]), Trypanosoma evansi (“Surra”) and Trypanosoma equiperdum (“Dourine”), and Trypanosoma cruzi, a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called “atypical human infections by animal trypanosomes” [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on “One Health,” by advancing and preserving animal, human and environmental health. Graphical Abstract