Vector soup: high‐throughput identification of Neotropical phlebotomine sand flies using metabarcoding

Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological‐based methods for sand fly species identifications are time‐consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1‐ha forest plot in French Guiana. Besides providing reliable molecular data for species‐level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high‐throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco‐epidemiological studies.     

Leishmania major: species specific delayed hypersensitivity reaction induced by exogenous secreted antigen in the guinea pig

The cellular response to Leishmania major (L. major) is usually evaluated in vivo by the delayed-type-hypersensitivity (DTH) test using leishmanin. Leishmanin can give false-positive reactions in areas where there is a background of leishmaniasis. In a previous study, it was shown that a 56 kDa antigen purified from promastigote and culture supernatant of L. major induce strong DTH reactions in sensitized guinea pigs. In this study, the species-specificity of this antigen was further investigated. Three groups of guinea pigs were sensitized with L. major, L. tropica, and L. infantum and both flanks of sensitized animal were injected intradermally with purified 56 kDa antigen or soluble leishmania antigen (SLA). The extent of indurations were measured after 24, 48, and 72 h. In animals which were sensitized with three species of leishmania, only those immunized with L. major showed skin reactions to purified antigen by an increase in skin thickness. Since complex antigen mixtures such as SLA and leishmanin show cross-reactivity and can be non-specific, the result obtained here suggest that 56 kDa antigen may be a useful diagnostic tool for species specific diagnosis in field studies of leishmaniasis.     

Meeting report: fourth international congress of the Society for Melanoma Research

The 4th international melanoma congress of the Society for Melanoma Research (SMR), organized by Marianne Berwick (University of New Mexico), Paul Chapman (Memorial Sloan-Kettering Cancer Center), Rene Gonzalez (University of Colorado) and Ze'ev Ronai (Burnham Institute), was held at the Marriott Hotel in downtown New York on November 2007. The congress was attended by a record high number of attendees (over 500 delegates) who joined to discuss recent advances in melanoma biology and therapy. About 40% of the participants arrived from 39 countries, a testament to the high impact of this annual gathering on the international melanoma community. Over 120 of the participants were students or postdoctoral fellows, representing a most impressive fraction of young scientists engaged in melanoma research. The meeting consisted of more than 50 plenary and minisymposia presentations, stimulating the exchange of unpublished data and novel ideas, and helping to forge new collaborations that are anticipated to facilitate significant advances in basic, translational and clinical melanoma research. Another major focus of this meeting was over 160 posters, which were heavily attended and provided an effective forum for extensive informal discussions. This report will highlight the major scientific themes and advances of this most successful meeting, and provide a useful perspective on the current state of melanoma research, as well as where the field should be heading.     

High-resolution crystallography and drug design

Ultra-high-resolution X-ray crystallography of macromolecules (i.e. resolution better than 0.8 Angstroms) is a rising field that promises to provide new insight into the structure-function relationships of biomacromolecules. The picture emerging from macromolecular structures at this resolution is far more complex than previously understood, requiring for its study improved tools for structure refinement, analysis and annotation. Some of these problems were highlighted during the recent High Resolution Drug Design Meeting (Bischenberg-Strasbourg, France, 13-16 May 2004). We will review here some of the results and discussions that took place during that meeting and elaborate on the trends and challenges ahead in this emerging new field of research.     

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.     

Dressed for success: the surface coats of insect-borne protozoan parasites

Three major human diseases, malaria, sleeping sickness and leishmaniasis, are caused by protozoan parasites that are transmitted by blood-sucking insects. These insects are not mere 'flying syringes' that mechanically transfer parasites from one mammal to the next. Instead, they provide a specific environment--albeit not a particularly hospitable one--in which the parasites differentiate, proliferate and migrate to the correct tissues to ensure transmission to the next mammalian host. Recent studies on the role of parasite surface molecules in insect vectors have delivered some surprises and could provide insights on ways to interrupt transmission.     

Specific recognition of Leishmania major poly-beta-galactosyl epitopes by galectin-9: possible implication of galectin-9 in interaction between L. major and host cells

Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-beta-galactosyl epitope (Galbeta1-3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host beta-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-beta-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-beta-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galbeta1-3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host.     

Lessons for the stem cell discourse from the gene therapy experience

The discovery of human embryonic stem cells has pointed to the potential use of these cells in developing new approaches to therapy for many major human diseases. While there is general agreement that such applications are possible and will require a great deal of additional basic and preclinical research, some discussions of the therapeutic applications of human embryonic stem cells have been characterized by the kinds of exaggerations and elevated expectations that characterized the field of human gene therapy a decade ago. In the case of gene therapy, public perception of and confidence in the field were damaged by the hype. Most unfortunate of all, the hopes of patients and their advocates were disappointed. The eventual success of a gene therapy approach, albeit one still plagued by serious adverse events, has come through scientific advance and careful clinical application. The probable eventual use of human embryonic stem cells for therapy of human disease will also require thorough basic and clinical research, but that goal is endangered by the current level of inaccurate representations and undeliverable promises.     

Natural product based leads to fight against leishmaniasis

The growing incidence of parasitic resistance against generic pentavalent antimonials, specifically for visceral disease in Indian subcontinent, is a serious issue in Leishmania control. Notwithstanding the two treatment alternatives, that is amphotericin B and miltefosine are being effectively used but their high cost and therapeutic complications limit their use in endemic areas. In the absence of a vaccine candidate, identification, and characterization of novel drugs and targets is a major requirement of leishmanial research. This review describes current drug regimens, putative drug targets, numerous natural products that have shown promising antileishmanial activity alongwith some key issues and strategies for future research to control leishmaniasis worldwide.     

Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.     

Leishmania major soluble exo-antigens (LmSEAgs) protect neonatal BALB/C mice from a subsequent challenge with L. major and stimulate cytokine production by Leishmania-naïve human peripheral blood mononuclear cells

The vaccine potential and immunogenicity of soluble Leishmania major exo-antigens (LmSEAgs), a potentially novel source for vaccine candidates for leishmaniasis, were evaluated in neonatal BALB/c mice and with human peripheral blood mononuclear cells. Vaccinated neonatal BALB/c mice resisted infection with L. major, and lymphoid cells from the mice proliferated when restimulation with LmSEAgs and produced interferon-gamma and some interleukin-4. In addition, LmSEAgs stimulated human peripheral blood mononuclear cells to produce large amounts of interferon-gamma and some interleukin-5. This finding suggests that LmSEAgs may be a vaccine candidate for leishmaniasis in humans.     

Progress in vaccine research and possible effector mechanisms in visceral leishmaniasis

Visceral leishmaniasis represents a serious public health concern in endemic regions and is rapidly emerging as an opportunistic infection in HIV patients. The disease is difficult to diagnose and prevent, and available treatment is associated with toxicity and drug resistance. Even though significant headway has been made in the development of vaccines against cutaneous leishmaniasis, visceral leishmaniasis has received limited attention. The fact that a large proportion of the people living in endemic areas have self-resolving subclinical infection and individuals once recovered are immune to reinfection provides a rationale for designing immunoprophylactic strategies against visceral leishmaniasis. The primary aim of this paper is to review advances in vaccination strategies against visceral leishmaniasis, suggesting possible effector mechanism leading to resistance. It also covers the role of immunostimulators and gives an account of the adjuvants used against visceral leishmaniasis. Vaccine strategies in different established experimental models have also been dealt with which can provide potential leads for their application in humans. In light of the available observations made during the course of studies performed on experimental models of visceral leishmaniasis there is increasing evidence that a successful approach towards a vaccine involves the requirement of Th1 subset of CD4+ cells along with Th2, CD8+, and B cells. In this review we present the possible mechanism of interaction of these cells and their effector molecules in providing resistance against visceral leishmaniasis for the future design of effective vaccine against this disease.     

The Brazilian legal framework on the scientific use of animals

Brazil has an exceptionally dynamic research sector in Latin America in health, biotechnology, and pharmacology, backed by defined government policies on science and technology and a health research agenda focusing on important neglected diseases: malaria, leishmaniasis, Chagas disease, turberculosis, leprosy, and dengue. The Brazilian health research policy promotes partnerships and networks among scientists in academic institutions in both wealthy industrialized and disease-endemic countries, and in these efforts the government's guidelines for animal use in biomedical research are considered fundamental to guarantee both animal welfare and the quality of research. Given international discussions of animal experimentation regulations and guidelines, in this article we describe current Brazilian legislation governing the use of animals in scientific investigations. We conclude that, despite advances in the implementation of the 3Rs (reduction, refinement, replacement), the new regulatory framework does not sufficiently incorporate ethical considerations, lacking explicit reference to the 3Rs as well as measures for their full application. The more humane use of animals in research will depend on the approach adopted by Brazil's National Council for the Control of Animal Experimentation to promote the 3Rs and to improve internal regulations as well as data collection and analysis in research institutions. In Brazil as elsewhere, one of the greatest challenges to policymakers is to harmonize the myriad and intertwined legal provisions without hindering biomedical research.     

Molecular identification of Leishmania spp. isolates causes cutaneous leishmaniasis (CL) in Sanliurfa Province,Turkey, where CL is highly endemic

Cutaneous leishmaniasis (CL) is an important public health problem in Turkey. CL has been most frequently seen in Sanliurfa. There is an expectation of increase in the population of leishmaniasis cases with the influence of Syrian refugees arriving in Turkey. In this study we aimed to diagnosis of CL and identifying of parasite from Leishmania isolates by using ITS 1 PCR RFLP. Samples were collected from 135 CL patients in Sanliurfa. After the specimens were inoculated in medium NNN, the ones which were cultures positive were cultivated in RPMI 1640 followed by PCR-RFLP. Genomic DNA was extracted phenol-chloroform procedure. Samples were examined by using ITS 1 PCR followed by RFLP analysis. Our results indicated that two species, L. tropica (132 samples) and L. major (3 samples), are responsible for cutaneous leishmaniasis in Sanl?urfa. Our study is the first scientific study in which it is reported molecular analyses of cutaneous leishmaniasis cases caused by L. major in Sanliurfa in Southestern Anatolia Region. Because CL cases caused by L.major are detected in our study, it is considered that genotyping is important for diagnosis of Leishmania and following change of epidemiology.     

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

Hybrid formation within the genus Leishmania?

Leishmanial organisms isolated from a desert rodent (Psammomys obesus) and a feral dog (Canis familiaris) in the Eastern Province of Saudi Arabia were isoenzymically distinct from Leishmania major and L. arabica, organisms usually associated with human and wild animal cutaneous leishmaniasis in this area. Further examination of isoenzyme banding patterns of cloned populations of these organisms, together with karyotyping using orthogonal field alternation gel electrophoresis and the use of highly specific kinetoplast DNA probes, has produced evidence suggesting that these organisms isolated from the dog and the rodent are hybrids of L. major and L. arabica.     

A gp63 based vaccine candidate against Visceral Leishmaniasis

Visceral leishmaniasis is a macrophage associated disorder which leads to a profound decrease in the natural immunotherapeutic potential of the infected subjects to combat the disease. The major surface glycoprotein gp63 has been found to be a significant vaccine candidate against visceral leishmaniasis. The current study addresses the levels of similarity and identity in the gp63 obtained from different species of Leishmania viz donovoni, chagasi and infantum linked to the cause of visceral leishmaniasis. The results from BLAST, Phylogram and Cladogram studies indicate significant identity, similarity and conservation of important residues in the protein which lead us to conclude that a common gp63 based vaccine can be used as a therapeutical tool against visceral leishmaniasis caused by different species strains of leishmania.     

DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major

Epidemiological field studies on leishmaniasis have been hampered by the laborious, and often inefficient, methods used to assess the rates of infection of sandfly vectors (Diptera; Phlebotominae) by species of the causative disease organisms, protozoal parasites of the genus Leishmania (Kinetoplastida; Trypanosomatidae). We report the rapid and accurate identification of both sandfly vector (Phlebotomus (Phlebotomus) papatasi (Scopoli] and infecting Leishmania major Yakimov & Schokov by DNA hybridizations to squash-blotted sandflies. Large numbers of whole (infected) sandflies can be quickly squashed on to nylon hybridization filters and (following standard procedures) the filter-bound DNA can be hybridized sequentially to cloned, multicopy genomic sequences that are specific for species of Leishmania (kinetoplast DNA) or for the sandfly (ribosomal (r) DNA). Our sandfly probe consists of a 3.2 kb fragment of the intergenic 'non-transcribed' spacer of rDNA of P. papatasi that we have detected only in this species: it is present in all six geographically isolated populations tested (from Tunisia through to India) but cannot be detected in the morphologically similar P. (Phlebotomus) duboscqi Neveu-Lemaire, the vector of Leishmania major south of the Sahara; it also cannot be detected in Phlebotomus species of the subgenera Larroussius and Paraphlebotomus that together with P. papatasi are the dominant man-biting sandflies in north African foci of zoonotic cutaneous leishmaniasis, where (as in many arid regions of western Asia) P. papatasi is believed to be the sole vector of L. major.     

First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR‐Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase‐Thymidylate Synthase (DHFR‐TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod‐2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off‐target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR‐Cas9‐mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.