Leishmania donovani complex: genotyping with the ribosomal internal transcribed spacer and the mini-exon.

Intergenic region typing by restriction analysis of the ribosomal internal transcribed spacer (ITS) and mini-exon provide diagnostic markers for some Leishmania. Here, we evaluate restriction analysis of these targets for genotyping and phylogenetic analysis within the Leishmania donovani complex (agents of visceral leishmaniasis). Each method was useful for genotyping of both L. donovani complex strains and Old World Leishmania species. The targets produced less robust groups than gp63 intergenic regions, but support the need for re-evaluation of the taxonomy of the L. donovani complex

Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

Background Many cases of cutaneous leishmaniasis (CL) have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. Methods Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA) sequence. Excreted factor (EF) serotyping and multilocus enzyme electrophoresis (MLEE) were also applied. Results This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT) done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains. Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria. Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains came. Conclusions The Palestinian strains that were assigned to different genetic groups differed in their MLEE profiles and their EF types. A new zymodeme, zymodeme MON-307 was discovered that seems to be unique to the northern part of the Palestinian West Bank. What seemed to be a straight forward classical situation of L. tropica causing anthroponotic CL in the Jenin District might be a more complex situation, owing to the presence of two separate sub-types of L. tropica that, possibly, indicates two separate transmission cycles involving two separate types of phlebotomine sand fly vector

Comparative Microsatellite Typing of New World Leishmania infantum Reveals Low Heterogeneity among Populations and Its Recent Old World Origin

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Protozoal parasites of the genus Leishmania are transmitted by sand fly bites to humans and animals. Three major forms of disease are caused by these parasites: cutaneous leishmaniasis, responsible for disfiguring skin wounds; mucocutaneous leishmaniasis, causing non-healing ulceration around the mouth and nose; and the potentially fatal visceral leishmaniasis, involving internal organs such as the spleen and liver. More than 2 million new human infections are caused annually by leishmaniasis globally, it is endemic in more than 88 countries and prevalent also as an imported disease in non-endemic regions due to travel and tourism. Most species of Leishmania that infect humans are zoonotic and transmitted from animal reservoir hosts. As various leishmanial parasites cause disease with similar symptoms, but require different therapeutic regimens and have dissimilar prognoses, reliable, sensitive and rapid diagnostic assays are needed. This study focuses on the five main species that cause leishmaniasis in the Old World. It presents a new assay for rapid detection, species identification and quantification of leishmanial parasites in clinical samples, reservoir hosts and sand flies. This technique could be especially valuable in regions where several leishmanial species exist, in non-endemic regions where infected patients require a rapid diagnosis, and for epidemiological host and vector studies leading to prevention programs

Multifaceted Population Structure and Reproductive Strategy in Leishmania donovani Complex in One Sudanese Village

Leishmania species of the subgenus Leishmania and especially L. donovani are responsible for a large proportion of visceral leishmaniasis cases. The debate on the mode of reproduction and population structure of Leishmania parasites remains opened. It has been suggested that Leishmania parasites could alternate different modes of reproduction, more particularly clonality and frequent recombinations either between related individuals (endogamy) or between unrelated individuals (outcrossing) within strongly isolated subpopulations. To determine whether this assumption is generalized to other species, a population genetics analysis within Leishmania donovani complex strains was conducted within a single village. The results suggest that a mixed-mating reproduction system exists, an important heterogeneity of subsamples and the coexistence of several genetic entities in Sudanese L. donovani. Indeed, results showed significant genetic differentiation between the three taxa (L. donovani, L. infantum and L. archibaldi) and between the human or canine strains of such taxa, suggesting that there may be different imbricated transmission cycles involving either dogs or humans. Results also are in agreement with an almost strict specificity of L. donovani stricto sensu to human hosts. This empirical study demonstrates the complexity of population structure in the genus Leishmania and the need to pursue such kind of analyses at the smallest possible spatio-temporal and ecological scales

The history of leishmaniasis

In this review article the history of leishmaniasis is discussed regarding the origin of the genus Leishmania in the Mesozoic era and its subsequent geographical distribution, initial evidence of the disease in ancient times, first accounts of the infection in the Middle Ages, and the discovery of Leishmania parasites as causative agents of leishmaniasis in modern times. With respect to the origin and dispersal of Leishmania parasites, the three currently debated hypotheses (Palaearctic, Neotropical and supercontinental origin, respectively) are presented. Ancient documents and paleoparasitological data indicate that leishmaniasis was already widespread in antiquity. Identification of Leishmania parasites as etiological agents and sand flies as the transmission vectors of leishmaniasis started at the beginning of the 20th century and the discovery of new Leishmania and sand fly species continued well into the 21st century. Lately, the Syrian civil war and refugee crises have shown that leishmaniasis epidemics can happen any time in conflict areas and neighbouring regions where the disease was previously endemic

Genetic Polymorphisms and Drug Susceptibility in Four Isolates of Leishmania tropica Obtained from Canadian Soldiers Returning from Afghanistan

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease transmitted by the bite of sandflies, resulting in sores on the skin. No vaccines are available, and treatment relies on chemotherapy. CL has been frequently diagnosed in military personnel deployed to Afghanistan and returning from duty. The parasites isolated from Canadian soldiers were characterized by pulsed field gels and by sequencing conserved genes and were identified as Leishmania tropica. In contrast to other Leishmania species, high allelic polymorphisms were observed at several genetic loci for the L. tropica isolates that were characterized. In vitro susceptibility testing in macrophages showed that all isolates, despite their genetic heterogeneity, were sensitive to most antileishmanial drugs (antimonials, miltefosine, amphotericin B, paromomycin) but were insensitive to fluconazole. This study suggests a number of therapeutic regimens for treating cutaneous leishmaniasis caused by L. tropica among patients and soldiers returning from Afghanistan. Canadian soldiers from this study were successfully treated with miltefosine

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens

Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used