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Sample records for unicellular parasite leishmania

  1. Motility, Force Generation, and Energy Consumption of Unicellular Parasites.

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    Hochstetter, Axel; Pfohl, Thomas

    2016-07-01

    Motility is a key factor for pathogenicity of unicellular parasites, enabling them to infiltrate and evade host cells, and perform several of their life-cycle events. State-of-the-art methods of motility analysis rely on a combination of optical tweezers with high-resolution microscopy and microfluidics. With this technology, propulsion forces, energies, and power generation can be determined so as to shed light on the motion mechanisms, chemotactic behavior, and specific survival strategies of unicellular parasites. With these new tools in hand, we can elucidate the mechanisms of motility and force generation of unicellular parasites, and identify ways to manipulate and eventually inhibit them. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites.

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    Kaczanowski, Szymon; Sajid, Mohammed; Reece, Sarah E

    2011-03-25

    Apoptosis-like programmed cell death (PCD) has recently been described in multiple taxa of unicellular protists, including the protozoan parasites Plasmodium, Trypanosoma and Leishmania. Apoptosis-like PCD in protozoan parasites shares a number of morphological features with programmed cell death in multicellular organisms. However, both the evolutionary explanations and mechanisms involved in parasite PCD are poorly understood. Explaining why unicellular organisms appear to undergo 'suicide' is a challenge for evolutionary biology and uncovering death executors and pathways is a challenge for molecular and cell biology. Bioinformatics has the potential to integrate these approaches by revealing homologies in the PCD machinery of diverse taxa and evaluating their evolutionary trajectories. As the molecular mechanisms of apoptosis in model organisms are well characterised, and recent data suggest similar mechanisms operate in protozoan parasites, key questions can now be addressed. These questions include: which elements of apoptosis machinery appear to be shared between protozoan parasites and multicellular taxa and, have these mechanisms arisen through convergent or divergent evolution? We use bioinformatics to address these questions and our analyses suggest that apoptosis mechanisms in protozoan parasites and other taxa have diverged during their evolution, that some apoptosis factors are shared across taxa whilst others have been replaced by proteins with similar biochemical activities.

  3. Detection of Leishmania RNA virus in Leishmania parasites.

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    Haroun Zangger

    Full Text Available Patients suffering from cutaneous leishmaniasis (CL caused by New World Leishmania (Viannia species are at high risk of developing mucosal (ML or disseminated cutaneous leishmaniasis (DCL. After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2 stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

  4. Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

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    Ginouvès, Marine; Simon, Stéphane; Bourreau, Eliane; Lacoste, Vincent; Ronet, Catherine; Couppié, Pierre; Nacher, Mathieu; Demar, Magalie; Prévot, Ghislaine

    2016-01-01

    In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country. © The American Society of Tropical Medicine and Hygiene.

  5. Leishmania development in sand flies: parasite-vector interactions overview.

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    Dostálová, Anna; Volf, Petr

    2012-12-03

    Leishmaniases are vector-borne parasitic diseases with 0.9 - 1.4 million new human cases each year worldwide. In the vectorial part of the life-cycle, Leishmania development is confined to the digestive tract. During the first few days after blood feeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. This binding is strictly stage-dependent as it is a property of nectomonad and leptomonad forms only. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan (LPG), this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to the stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Transformation to metacyclic stages highly infective for the vertebrate host is the other prerequisite for effective transmission. Here, we review the current state of knowledge of molecular interactions occurring in all these distinct phases of parasite colonization of the sand fly gut, highlighting recent discoveries in the field.

  6. Description of Leishmania (Leishmania forattinii sp. n., a new parasite infecting opossums and rodents in Brazil

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    Elizaide L. A. Yoshida

    1993-09-01

    Full Text Available A new parasite species of Leishmania is described, L. (Leishmania forattinii sp. n., which was isolated from a pooled triturate of liver and spleen of a opossum (Didelphis marsupialis aurita and from skin samples from a rodent (Proechmys iheringi denigratus, captured in primary forest on the Atlantic Cost of Brazil. Our results on the basis of biological and molecular criteria indicate that this taxonomically distinct parasite ias a new species of the L. mexicana complex, but closely related to L. (L. aristidesi Laison & shaw, 1979, as revelated by phenetic and phylogenetic numerical analyses of the enzyme data. L. forattinii was clearly distinguishable from other Leishmania species of the genus usisng enzyme electrophoresis, monoclonal antibodies, molecular karyotypes, analysis of restriction enzyme digestion patterns of kinetoplast DNA (kDNA, as well as the use of kDNA hybridization procedures.

  7. Preparation of live attenuated leishmania parasites by using laser technology

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    Hussain, Nabiha; Alkhouri, Hassan; Haddad, Shaden

    2018-05-01

    Leishmaniasis is a parasitic disease of humans, affecting the skin, mucosal and/or internal organs, caused by flagellate protozoa Leishmania of the Trypanosomatidae family. Leishmania would be one for which a vaccine could be developed with relative ease. Many studies mount an effective response that resolves the infection and confers solid immunity to reinfection and suggesting that infection may be a prerequisite for immunological memory. Genetically altered live attenuated parasites with controlled infectivity could achieve such immunological memory. Recent concepts include use of genetically modified live-attenuated Leishmania parasites, and proteomics approach for the search of a cross-protective leishmanial vaccine that would ideally protect against both cutaneous and visceral forms of the disease. No licensed vaccine is available till date against any form of leishmaniasis. The present study evaluated role of laser technology in development of a safe live Leishmania vaccine, a vaccine is a biological preparation that improves immunity to a particular disease, and is often made from weakened or killed forms of LPs. The parasite culture was expanded in RPMI 1640 medium with 10% fetal calf serum (FCS) and grown until stationary phase for experiments. 80 samples of leishmania promastigotes (Culture media of LPs) were exposed to Nd:YAG laser (wavelength 1064 nm, single spot or double) with different outputs powers (7w, 100 Hz, 99.03w/cm2, 0.99 J/cm2 and 8 w, 100 Hz, 113.18w/cm2 1.13J/cm2)) for suitable exposer times. The effect of semiconductor laser (wavelength 810 nm, 7w, 2000 Hz, 99.03w/cm2, 0.05 J/cm2) or (7 w, 500 Hz, 99.03 w/cm2, 0.2J/cm2) single spot or double with long exposure times. The viability of Leishmania parasites was measured using XTT method; viable parasites were decreased with long exposure times. XTT test referred both these wavelengths were effective in killing percentage of Leishmania promastigotes, the remaining were devoid flagellum that

  8. Fitness of Leishmania donovani parasites resistant to drug combinations.

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    Raquel García-Hernández

    2015-04-01

    Full Text Available Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line. In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.

  9. Deception and Manipulation: The Arms of Leishmania, a Successful Parasite

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    Cecílio, Pedro; Pérez-Cabezas, Begoña; Santarém, Nuno; Maciel, Joana; Rodrigues, Vasco; Cordeiro da Silva, Anabela

    2014-01-01

    Leishmania spp. are intracellular parasitic protozoa responsible for a group of neglected tropical diseases, endemic in 98 countries around the world, called leishmaniasis. These parasites have a complex digenetic life cycle requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The clinical manifestations associated with leishmaniasis depend on complex interactions between the parasite and the host immune system. Consequently, leishmaniasis can be manifested as a self-healing cutaneous affliction or a visceral pathology, being the last one fatal in 85–90% of untreated cases. As a result of a long host–parasite co-evolutionary process, Leishmania spp. developed different immunomodulatory strategies that are essential for the establishment of infection. Only through deception and manipulation of the immune system, Leishmania spp. can complete its life cycle and survive. The understanding of the mechanisms associated with immune evasion and disease progression is essential for the development of novel therapies and vaccine approaches. Here, we revise how the parasite manipulates cell death and immune responses to survive and thrive in the shadow of the immune system. PMID:25368612

  10. Deception and Manipulation: the arms of Leishmania, a successful parasite

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    Pedro eCecílio

    2014-10-01

    Full Text Available Leishmania spp. are intracellular parasitic protozoa responsible for a group of neglected tropical diseases, endemic in 98 countries around the world, called leishmaniasis. These parasites have a complex digenetic life cycle requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The clinical manifestations associated with leishmaniasis depend on complex interactions between the parasite and the host immune system. Consequently, leishmaniasis can be manifested as a self-healing cutaneous affliction or a visceral pathology, being the last one fatal in 85-90% of untreated cases. As a result of a long host-parasite co-evolutionary process, Leishmania spp. developed different immunomodulatory strategies that are essential for the establishment of infection. Only through deception and manipulation of the immune system, Leishmania spp. can complete its life cycle and survive. The understanding of the mechanisms associated with immune evasion and disease progression is essential for the development of novel therapies and vaccine approaches. Here, we revise how the parasite manipulates cell death and immune responses to survive and thrive in the shadow of the immune system.

  11. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection.

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    Semini, Geo; Paape, Daniel; Paterou, Athina; Schroeder, Juliane; Barrios-Llerena, Martin; Aebischer, Toni

    2017-08-01

    Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse-chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low-density lipoproteins indicated that retention of this source of cholesterol is increased in parasite-containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann-Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites' membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA-encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol-dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. Leishmania parasite detection and quantification using PCR-ELISA

    Czech Academy of Sciences Publication Activity Database

    Kobets, Tetyana; Badalová, Jana; Grekov, Igor; Havelková, Helena; Lipoldová, Marie

    2010-01-01

    Roč. 5, č. 6 (2010), s. 1074-1080 ISSN 1754-2189 R&D Projects: GA ČR GA310/08/1697; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : polymerase chain reaction * Leishmania major infection * parasite quantification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.362, year: 2010

  13. Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages

    International Nuclear Information System (INIS)

    Handman, E.; Schnur, L.F.; Spithill, T.W.; Mitchell, G.F.

    1986-01-01

    Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. The authors have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study they have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here they show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, they show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro

  14. Neutrophils reduce the parasite burden in Leishmania (Leishmania amazonensis-infected macrophages.

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    Erico Vinícius de Souza Carmo

    2010-11-01

    Full Text Available Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L major, whereas less information is available for L. (L amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L. amazonensis (C3H/HePas. In contrast, the susceptible strain (BALB/c displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L. amazonensis-infected macrophages in vitro.Mouse peritoneal macrophages infected with L. (L. amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1 intracellular parasites were efficiently destroyed in the co-cultures; 2 the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas or susceptible (BALB/c to L. (L. amazonensis; 3 parasite destruction did not require contact between infected macrophages and neutrophils; 4 tumor necrosis factor alpha (TNF-α, neutrophil elastase and platelet activating factor (PAF were involved with the leishmanicidal activity, and 5 destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L. amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.

  15. Serological and molecular survey of Leishmania parasites in apparently healthy dogs in the West Bank, Palestine

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    Hamarsheh Omar

    2012-08-01

    Full Text Available Abstract Background Canine visceral leishmaniasis (CVL is caused by Leishmania infantum in all Mediterranean countries. The Leishmania parasite is transmitted by the bite of a corresponding sand fly vector and primarily maintained in nature by wild and domestic reservoirs, including dogs, foxes and jackals. Infected dogs are the primary reservoir host in endemic regions and are the most significant risk disposing humans to infection. The present study aimed at assessing the prevalence of infection with Leishmania and identification of Leishmania infantum in domestic dogs in the West Bank, Palestine. Methods The infection rate among domestic dogs collected from seven districts in the Palestinian West Bank was investigated by examination of parasites in culture from the buffy coat using serological and molecular methods; based on ELISA, internal transcribed spacer 1 (ITS1 and cysteine protease (CPB PCR. Results Out of 215 dogs examined for Leishmania, 36 (16.7% were positive in at least one method. Twenty three animals (11.5% were positive for Leishmania DNA, whereas, ELISA and culture revealed 16 (7.5%, and 4 (1.5% respectively. CPB-PCR on one of three culture-positive isolates revealed Leishmania infantum as the causative agent for Leishmania infection in dogs. Conclusions Our study showed that canine leishmania infection is prevalent with varying degrees in all the seven studied districts in Palestine despite the absence of human VL cases in 4 of these districts. The causative agent was confirmed to be Leishmania infantum.

  16. Anti-Leishmania activity of new ruthenium(II) complexes: Effect on parasite-host interaction.

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    Costa, Mônica S; Gonçalves, Yasmim G; Nunes, Débora C O; Napolitano, Danielle R; Maia, Pedro I S; Rodrigues, Renata S; Rodrigues, Veridiana M; Von Poelhsitz, Gustavo; Yoneyama, Kelly A G

    2017-10-01

    Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. The many complications presented by the current treatment - including high toxicity, high cost and parasite resistance - make the development of new therapeutic agents indispensable. The present study aims to evaluate the anti-Leishmania potential of new ruthenium(II) complexes, cis‑[Ru II (η 2 -O 2 CR)(dppm) 2 ]PF 6 , with dppm=bis(diphenylphosphino)methane and R=4-butylbenzoate (bbato) 1, 4-(methylthio)benzoate (mtbato) 2 and 3-hydroxy-4-methoxybenzoate (hmxbato) 3, in promastigote cytotoxicity and their effect on parasite-host interaction. The cytotoxicity of complexes was analyzed by MTT assay against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Leishmania) infantum promastigotes and the murine macrophage (RAW 264.7). The effect of complexes on parasite-host interaction was evaluated by in vitro infectivity assay performed in the presence of two different concentrations of each complex: the promastigote IC 50 value and the concentration nontoxic to 90% of RAW 264.7 macrophages. Complexes 1-3 exhibited potent cytotoxic activity against all Leishmania species assayed. The IC 50 values ranged from 7.52-12.59μM (complex 1); 0.70-3.28μM (complex 2) and 0.52-1.75μM (complex 3). All complexes significantly inhibited the infectivity index at both tested concentrations. The infectivity inhibitions ranged from 37 to 85%. Interestingly, the infectivity inhibitions due to complex action did not differ significantly at either of the tested concentrations, except for the complex 1 against Leishmania (Leishmania) infantum. The infectivity inhibitions resulted from reductions in both percentage of infected macrophages and number of parasites per macrophage. Taken together the results suggest remarkable leishmanicidal activity in vitro by these new ruthenium(II) complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form.

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    Yao, Chaoqun; Wilson, Mary E

    2016-04-12

    The Leishmania spp. protozoa, the causative agents of the "neglected" tropical disease leishmaniasis, are transmitted to mammals by sand fly vectors. Within the sand fly, parasites transform from amastigotes to procyclic promastigotes, followed by development of virulent (metacyclic) promastigote forms. The latter are infectious to mammalian hosts. Biochemical components localized in the parasite plasma membrane such as proteins and sterols play a pivotal role in Leishmania pathogenesis. Leishmania spp. lack the enzymes for cholesterol synthesis, and the dynamics of sterol acquisition and biosynthesis in parasite developmental stages are not understood. We hypothesized that dynamic changes in sterol composition during metacyclogenesis contribute to the virulence of metacyclic promastigotes. Sterols were extracted from logarithmic phase or metacyclic promastigotes grown in liquid culture with or without cholesterol, and analyzed qualitatively and quantitatively by gas chromatograph-mass spectrometry (GC-MS). TriTrypDB was searched for identification of genes involved in Leishmania sterol biosynthetic pathways. In total nine sterols were identified. There were dynamic changes in sterols during promastigote metacyclogenesis. Cholesterol in the culture medium affected sterol composition in different parasite stages. There were qualitative and relative quantitative differences between the sterol content of virulent versus avirulent parasite strains. A tentative sterol biosynthetic pathway in Leishmania spp. promastigotes was identified. Significant differences in sterol composition were observed between promastigote stages, and between parasites exposed to different extracellular cholesterol in the environment. These data lay the foundation for further investigating the role of sterols in the pathogenesis of Leishmania spp. infections.

  18. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

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    Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

    2018-01-01

    Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the

  19. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

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    Helena Messlinger

    2018-01-01

    Full Text Available Activated natural killer (NK cells release interferon (IFN-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani. When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells

  20. Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection.

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    Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

    2016-08-19

    Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival.

  1. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

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    Mohammad Akhoundi

    2016-03-01

    Full Text Available 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.

  2. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

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    Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

    2016-01-01

    Background 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. Methodology and Principal Findings 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? Conclusions and Significance 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

  3. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

    Science.gov (United States)

    Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

    2016-03-01

    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.

  4. Detection of Leishmania parasites in the blood of patients with isolated cutaneous leishmaniasis.

    Science.gov (United States)

    Nakkash-Chmaisse, Hania; Makki, Raja; Nahhas, Georges; Knio, Khouzama; Nuwayri-Salti, Nuha

    2011-07-01

    The consequences of the spread of Leishmania parasites to the blood from lesions in patients with cutaneous leishmaniasis are numerous. To assess the magnitude of this invasion we conducted the present study on patients referred to the American University of Beirut Medical Center for cutaneous leishmaniasis. Patients referred for the management of cutaneous leishmaniasis were included in the study. Skin and blood cultures for Leishmania were taken from these patients. One hundred sixty-two patients were proven to have cutaneous leishmaniasis by pathology; 52% were males and 44% females (gender information was missing for 4%). Patient age ranged from 5 months to 70 years. None of the patients had received treatment for Leishmania. We obtained parasite isolates from 85 patients (52.5%), proven by cultures from skin and blood/blood components. Interestingly, the parasite was isolated in the blood and blood components of 50 patients (30.9%). Isoenzyme analysis confirmed the fact that the organisms in blood and skin were the same; from the 28 isolates that were positive in both skin and blood, eight isolates were Leishmania major and two were Leishmania tropica. The remaining isolates, whether positive in both blood and skin or in either of these tissues, skin or blood and its products, were Leishmania infantum sensu lato. In the current study, the detection rate of parasites in the blood of patients with cutaneous leishmaniasis was high. This illustrates the invasive characteristic of the parasite that has escaped the skin. Testing should be considered in areas other than Lebanon, especially around the Mediterranean basin. Whether these findings support the administration of systemic treatment for cutaneous leishmaniasis or not needs to be confirmed in larger prospective studies. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  5. The Leishmania nicotinamidase is essential for NAD(+) production and parasite proliferation

    OpenAIRE

    Gazanion, Elodie; Garcia, Deborah; Silvestre, R.; Gérard, C.; Guichou, J. F.; Labesse, G.; Seveno, Martial; Cordeiro-Da-Silva, A.; Ouaissi, A.; Sereno, Denis; Vergnes, Baptiste

    2011-01-01

    NAD(+) is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD(+) metabolism revealed that Leishmania protozoan parasites are NAD(+) auxotrophs. Consequently, these parasites require assimilating NAD(+) precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD(+) by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that ca...

  6. Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis

    NARCIS (Netherlands)

    Mugasa, Claire M.; Laurent, Thierry; Schoone, Gerard J.; Basiye, Frank L.; Saad, Alfarazdeg A.; El Safi, Sayda; Kager, Piet A.; Schallig, Henk Dfh

    2010-01-01

    ABSTRACT: BACKGROUND: Molecular methods to detect Leishmania parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to

  7. The polymerase chain reaction can reveal the occurrence of naturally mixed infections with Leishmania parasites

    DEFF Research Database (Denmark)

    Ibrahim, M E; Smyth, A J; Ali, M H

    1994-01-01

    On isolation and characterization of Leishmania parasites from Sudanese patients with visceral leishmaniasis (VL), four cases of mixed infections were found. Three of those cases were from the Eastern Sudan focus of VL. In one case the patient was found to be concomitantly infected with Leishmania...

  8. Subversion of Immunity by Leishmania amazonensis Parasites: Possible Role of Phosphatidylserine as a Main Regulator

    Directory of Open Access Journals (Sweden)

    Joao Luiz Mendes Wanderley

    2012-01-01

    Full Text Available Leishmania amazonensis parasites cause progressive disease in most inbred mouse strains and are associated with the development of diffuse cutaneous leishmaniasis in humans. The poor activation of an effective cellular response is correlated with the ability of these parasites to infect mononuclear phagocytic cells without triggering their activation or actively suppressing innate responses of these cells. Here we discuss the possible role of phosphatidylserine exposure by these parasites as a main regulator of the mechanism underlying subversion of the immune system at different steps during the infection.

  9. Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

    Science.gov (United States)

    Badirzadeh, Alireza; Taheri, Tahereh; Taslimi, Yasaman; Abdossamadi, Zahra; Heidari-Kharaji, Maryam; Gholami, Elham; Sedaghat, Baharehsadat; Niyyati, Maryam; Rafati, Sima

    2017-07-01

    Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite

  10. In vitro activity of the beta-carboline alkaloids harmane, harmine, and harmaline toward parasites of the species Leishmania infantum.

    Science.gov (United States)

    Di Giorgio, C; Delmas, F; Ollivier, E; Elias, R; Balansard, G; Timon-David, P

    2004-01-01

    Harmane, harmine, and harmaline were investigated for their in vitro antileishmanial activity toward parasites of the species Leishmania infantum. Harmane and Harmine displayed a moderate antiproliferative activity toward human monocytes and exerted a weak antileishmanial activity toward both the promastigote and the amastigote forms of the parasite. Their mechanism of action on the promastigote form of the parasite involved interactions with DNA metabolism leading to an accumulation of parasites in the S-G(2)M phases of the cell-cycle. Harmaline, at the contrary, was deprived from toxicity toward human cells and Leishmania promastigotes, however it exerted a strong antileishmanial activity toward the intracellular amastigote form of the parasite. This property was shown to partly result from the capacity of the molecule to prevent parasite internalization within macrophages by inhibiting Leishmania PKC activity.

  11. A microculture technique for isolating live Leishmania parasites from peripheral blood of visceral leishmaniasis patients.

    Science.gov (United States)

    Hide, M; Singh, R; Kumar, B; Bañuls, A L; Sundar, S

    2007-06-01

    Current procedures for diagnosing Leishmania parasites from patients involve invasive and dangerous tissue aspiration. We have developed a non-invasive and highly sensitive microculture method that can isolate parasites from the buffy coat of the patient's peripheral blood. The parasites were cultured in 96-well culture plates. Nineteen parasitologically proven visceral leishmaniasis (VL) patients were included in the study. Using this technique, we were able to isolate parasites from 16 (84%) samples. However, all 19 (100%) samples were positive on culture of splenic aspirates. We conclude that this technique is useful for the isolation and cryoconservation of parasites from patients' blood. This simple method could be tried as a first-instance alternative before other more sensitive procedures such as splenic aspirate; however, negative results should be confirmed by tests with higher sensitivity.

  12. Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction

    OpenAIRE

    Dostálová, Anna

    2012-01-01

    of PhD. thesis named "Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction", Anna Dostálová, 2011 This thesis focuses on the development of protozoan parasites of the genus Leishmania in their insect vectors, sand flies. It sums up results of three projects I was involved in during my PhD studies. Main emphasis was put on permissive sand fly species that support development of various species of Leishmania. Using a novel method of...

  13. Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection

    Czech Academy of Sciences Publication Activity Database

    Kurey, Irina; Kobets, Tetyana; Havelková, Helena; Slapničková, Martina; Quan, L.; Trtková, Kateřina; Grekov, Igor; Svobodová, M.; Stassen, A. P. M.; Hutson, A.; Demant, P.; Lipoldová, Marie

    2009-01-01

    Roč. 61, č. 9 (2009), s. 619-633 ISSN 0093-7711 R&D Projects: GA ČR GA310/06/1745; GA MŠk(CZ) LC06009 Grant - others:EC(XE) 05-1000004-7761 Institutional research plan: CEZ:AV0Z50520514 Keywords : Leishmania major * Parasite elimination * QTL mapping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.988, year: 2009

  14. Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites.

    Science.gov (United States)

    Dodge, Matthew A; Waller, Ross F; Chow, Larry M C; Zaman, Muhammad M; Cotton, Leanne M; McConville, Malcolm J; Wirth, Dyann F

    2004-03-01

    Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

  15. In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites.

    Directory of Open Access Journals (Sweden)

    Rubén E Varela-M

    Full Text Available BACKGROUND: The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. CONCLUSIONS/SIGNIFICANCE: Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania

  16. Mapping the antigenicity of the parasites in Leishmania donovani infection by proteome serology.

    Directory of Open Access Journals (Sweden)

    Michael Forgber

    Full Text Available BACKGROUND: Leishmaniasis defines a cluster of protozoal diseases with diverse clinical manifestations. The visceral form caused by Leishmania donovani is the most severe. So far, no vaccines exist for visceral leishmaniasis despite indications of naturally developing immunity, and sensitive immunodiagnostics are still at early stages of development. METHODOLOGY/PRINCIPLE FINDINGS: Establishing a proteome-serological methodology, we mapped the antigenicity of the parasites and the specificities of the immune responses in human leishmaniasis. Using 2-dimensional Western blot analyses with sera and parasites isolated from patients in India, we detected immune responses with widely divergent specificities for up to 330 different leishmanial antigens. 68 antigens were assigned to proteins in silver- and fluorochrome-stained gels. The antigenicity of these proteins did not correlate with the expression levels of the proteins. Although some antigens are shared among different parasite isolates, there are extensive differences and no immunodominant antigens, but indications of antigenic drift in the parasites. Six antigens were identified by mass spectrometry. CONCLUSIONS/SIGNIFICANCE: Proteomics-based dissection of the serospecificities of leishmaniasis patients provides a comprehensive inventory of the complexity and interindividual heterogeneity of the host-responses to and variations in the antigenicity of the Leishmania parasites. This information can be instrumental in the development of vaccines and new immune monitoring and diagnostic devices.

  17. Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites.

    Science.gov (United States)

    Fotouhi-Ardakani, Reza; Dabiri, Shahriar; Ajdari, Soheila; Alimohammadian, Mohammad Hossein; AlaeeNovin, Elnaz; Taleshi, Neda; Parvizi, Parviz

    2016-12-01

    The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 - KU680871). Four genes had a remarkable number of informative sites (P=0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P=0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D=-2.2, PLeishmania parasites. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut

    Directory of Open Access Journals (Sweden)

    Vanessa Diniz Atayde

    2015-11-01

    Full Text Available Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

  19. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

    Science.gov (United States)

    Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

    2011-10-01

    NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. © 2011 Blackwell Publishing Ltd.

  20. Leishmania infantum EndoG is an endo/exo-nuclease essential for parasite survival.

    Directory of Open Access Journals (Sweden)

    Eva Rico

    Full Text Available EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.

  1. Programmed cell death in Leishmania: biochemical evidence and role in parasite infectivity

    Directory of Open Access Journals (Sweden)

    Sreenivas eGannavaram

    2012-07-01

    Full Text Available Demonstration of features of a programmed cell death (PCD pathway in protozoan parasites initiated a great deal of interest and debate in the field of molecular parasitology. Several of the markers typical of mammalian apoptosis have been shown in Leishmania which suggested the existence of an apoptosis like death in these organisms. However studies to elucidate the down stream events associated with phosphotidyl serine exposure, loss of mitochondrial membrane potential, cytochrome c release and caspase-like activities in cells undergoing such cell death remain an ongoing challenge. Recent advances in genome sequencing, chemical biology should help solve some of these challenges. Leishmania genetic mutants that lack putative regulators/effectors of PCD pathway should not only help demonstrate the mechanisms of PCD but also provide tools to better understand the putative role for this pathway in population control and in the establishment of a successful infection of the host.

  2. Seroprevalence rates of antibodies againstLeishmania infantum and other protozoan and rickettsial parasites in dogs

    Directory of Open Access Journals (Sweden)

    Silvana de Cássia Paulan

    Full Text Available Canine visceral leishmaniasis (CVL is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT to analyze L. infantum,Ehrlichia spp., Babesia canis,Toxoplasma gondii and Neospora caninuminfection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.

  3. Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

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    Prasad K Padmanabhan

    Full Text Available In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/- mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

  4. Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth.

    Science.gov (United States)

    Chua, Ming Jang; Arnold, Megan S J; Xu, Weijun; Lancelot, Julien; Lamotte, Suzanne; Späth, Gerald F; Prina, Eric; Pierce, Raymond J; Fairlie, David P; Skinner-Adams, Tina S; Andrews, Katherine T

    2017-04-01

    Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC 50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC 50  > 20 μM) or S. mansoni schistosomula (IC 50  > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC 50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth

    Directory of Open Access Journals (Sweden)

    Ming Jang Chua

    2017-04-01

    Full Text Available Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM, with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF or human embryonic kidney (HEK 293 cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM or S. mansoni schistosomula (IC50 > 10 μM, however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM. Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days, with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P < 0.05, respectively.

  6. Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

    Czech Academy of Sciences Publication Activity Database

    Šmíd, O.; Matušková, Anna; Harris, S. R.; Kučera, Tomáš; Novotný, M.; Horváthová, L.; Hrdý, I.; Kutejová, E.; Hirt, R. P.; Embley, T. M.; Janata, Jiří; Tachezy, J.

    2008-01-01

    Roč. 4, č. 12 (2008), s. 1-8 ISSN 1553-7366 R&D Projects: GA MŠk LC07032; GA AV ČR IAA501110631 Grant - others:CZ(CZ) B-Bio166/2006 (O.S.). S.H., R.P.H. Institutional research plan: CEZ:AV0Z50200510 Keywords : peptidases * mitochondria * human parasites Subject RIV: EE - Microbiology, Virology Impact factor: 9.125, year: 2008

  7. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    Science.gov (United States)

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

  8. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    Directory of Open Access Journals (Sweden)

    Joana Faria

    2016-01-01

    Full Text Available A growing interest in asparagine (Asn metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

  9. Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience.

    Science.gov (United States)

    Ihalamulla, R L; Rajapaksa, U S; Karunaweera, N D

    2005-09-01

    Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.

  10. Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

    Directory of Open Access Journals (Sweden)

    Ines Lakhal-Naouar

    Full Text Available BACKGROUND: Gene expression analysis in Leishmania donovani (Ld identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS, that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid. However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. CONCLUSION/SIGNIFICANCE: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a

  11. Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

    Science.gov (United States)

    Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

    2017-10-10

    Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

  12. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major

    Directory of Open Access Journals (Sweden)

    Rodrigo Lombraña

    2016-08-01

    Full Text Available Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs. Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

  13. Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability.

    Directory of Open Access Journals (Sweden)

    Wai-Lok Yau

    Full Text Available BACKGROUND: Cyclosporin A (CsA has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function. CONCLUSIONS/SIGNIFICANCE: The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate

  14. Dichotomy in the human CD4+ T-cell response to Leishmania parasites

    DEFF Research Database (Denmark)

    Kemp, M; Kurtzhals, J A; Kharazmi, A

    1994-01-01

    Leishmania parasites cause human diseases ranging from self-healing cutaneous ulcers to fatal systemic infections. In addition, many individuals become infected without developing disease. In mice the two subsets of CD4+ T cells, Th1 and Th2, have different effects on the outcome of experimental...... in humans, and that the balance between subsets of parasite-specific T cells may play an important regulatory role in determining the outcome of the infections....

  15. Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

    Science.gov (United States)

    Moreno-Campos, Rodrigo; Florencio-Martínez, Luis E; Nepomuceno-Mejía, Tomás; Rojas-Sánchez, Saúl; Vélez-Ramírez, Daniel E; Padilla-Mejía, Norma E; Figueroa-Angulo, Elisa; Manning-Cela, Rebeca; Martínez-Calvillo, Santiago

    2016-12-01

    Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

  16. Inorganic phosphate uptake in unicellular eukaryotes.

    Science.gov (United States)

    Dick, Claudia F; Dos-Santos, André L A; Meyer-Fernandes, José R

    2014-07-01

    Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Calcium and magnesium ions modulate the oligomeric state and function of mitochondrial 2-Cys peroxiredoxins in Leishmania parasites.

    Science.gov (United States)

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Castro, Helena; Honorato, Rodrigo V; Oliveira, Paulo S L; Netto, Luis E S; Tomas, Ana M; Murakami, Mario T

    2017-04-28

    Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Calcein+/PI- as an early apoptotic feature in Leishmania.

    Science.gov (United States)

    Basmaciyan, Louise; Azas, Nadine; Casanova, Magali

    2017-01-01

    Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.

  19. Calcein+/PI- as an early apoptotic feature in Leishmania.

    Directory of Open Access Journals (Sweden)

    Louise Basmaciyan

    Full Text Available Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i to better understand the role of apoptosis in unicellular organisms, (ii to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.

  20. Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

    Directory of Open Access Journals (Sweden)

    Mary Dan-Goor

    Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

  1. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  2. Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador: the causative Leishmania parasites and clinico-epidemiological features.

    Science.gov (United States)

    Hashiguchi, Yoshihisa; Gomez, Eduardo A L; Cáceres, Abraham G; Velez, Lenin N; Villegas, Nancy V; Hashiguchi, Kazue; Mimori, Tatsuyuki; Uezato, Hiroshi; Kato, Hirotomo

    2018-01-01

    This study provides comprehensive information on the past and current status of the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador, mainly focusing on the causative Leishmania parasites and clinico-epidemiological features. Available information and data including our unpublished works were analyzed thoroughly. Endemic regions of the Andean-CL (uta) in Peru run from the north Piura/Cajamarca to the south Ayacucho at a wide range of the Pacific watersheds of the Andes through several departments, while in Ecuador those exist at limited and spotted areas in the country's mid-southwestern two provinces, Azuay and Chimborazo. The principal species of the genus Leishmania are completely different at subgenus level, L. (Viannia) peruviana in Peru, and L. (Leishmania) mexicana and L. (L.) major-like (infrequent occurrence) in Ecuador. The Peruvian uta is now prevalent in different age and sex groups, being not clearly defined as found in the past. The precise reasons are not known and should be elucidated further, though probable factors, such as emergence of other Leishmania parasites, non-immune peoples' migration into the areas, etc., were discussed briefly in the text. The Andean-CL cases in Ecuador are more rural than before, probably because of a rapid development of the Leishmania-positive communities and towns, and the change of life-styles of the inhabitants, including newly constructed houses and roads in the endemic areas. Such information is helpful for future management of the disease, not only for Leishmania-endemic areas in the Andes but also for other endemic areas. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Experimental infection and transmission of Leishmania by Lutzomyia cruzi (Diptera: Psychodidae: Aspects of the ecology of parasite-vector interactions.

    Directory of Open Access Journals (Sweden)

    Everton Falcão de Oliveira

    2017-02-01

    Full Text Available Several parameters should be addressed before incriminating a vector for Leishmania transmission. Those may include its ability to become infected by the same Leishmania species found in humans, the degree of attractiveness for reservoirs and humans and capacity to sustain parasite infection under laboratory conditions. This study evaluated the vectorial capacity of Lutzomyia cruzi for Leishmania infantum and gathered information on its ability to harbor L. amazonensis. Laboratory-reared Lu. cruzi were infected experimentally by feeding them on dogs infected naturally with L. infantum and hamsters infected with L. amazonensis. Sand fly attractiveness to dogs and humans was determined using wild caught insects. The expected daily survival of infected Lu. cruzi, the duration of the gonotrophic cycle, and the extrinsic incubation period were also investigated for both parasites. Vector competence was investigated for both Leishmania species. The mean proportion of female sand flies that fed on hosts was 0.40. For L. infantum and L. amazonensis, Lu. cruzi had experimental infection rates of 10.55% and 41.56%, respectively. The extrinsic incubation period was 3 days for both Leishmania species, regardless of the host. Survival expectancy of females infected with L. infantum and L. amazonensis after completing the gonotrophic cycle was 1.32 and 0.43, respectively. There was no association between L. infantum infection and sand fly longevity, but L. amazonensis-infected flies had significantly greater survival probabilities. Furthermore, egg-laying was significantly detrimental to survival. Lu. cruzi was found to be highly attracted to both dogs and humans. After a bloodmeal on experimentally infected hosts, both parasites were able to survive and develop late-stage infections in Lu. cruzi. However, transmission was demonstrated only for L. amazonensis-infected sand flies. In conclusion, Lu. cruzi fulfilled several of the requirements of vectorial

  4. Experimental infection and transmission of Leishmania by Lutzomyia cruzi (Diptera: Psychodidae): Aspects of the ecology of parasite-vector interactions.

    Science.gov (United States)

    Falcão de Oliveira, Everton; Oshiro, Elisa Teruya; Fernandes, Wagner de Souza; Murat, Paula Guerra; Medeiros, Márcio José de; Souza, Alda Izabel; Oliveira, Alessandra Gutierrez de; Galati, Eunice Aparecida Bianchi

    2017-02-01

    Several parameters should be addressed before incriminating a vector for Leishmania transmission. Those may include its ability to become infected by the same Leishmania species found in humans, the degree of attractiveness for reservoirs and humans and capacity to sustain parasite infection under laboratory conditions. This study evaluated the vectorial capacity of Lutzomyia cruzi for Leishmania infantum and gathered information on its ability to harbor L. amazonensis. Laboratory-reared Lu. cruzi were infected experimentally by feeding them on dogs infected naturally with L. infantum and hamsters infected with L. amazonensis. Sand fly attractiveness to dogs and humans was determined using wild caught insects. The expected daily survival of infected Lu. cruzi, the duration of the gonotrophic cycle, and the extrinsic incubation period were also investigated for both parasites. Vector competence was investigated for both Leishmania species. The mean proportion of female sand flies that fed on hosts was 0.40. For L. infantum and L. amazonensis, Lu. cruzi had experimental infection rates of 10.55% and 41.56%, respectively. The extrinsic incubation period was 3 days for both Leishmania species, regardless of the host. Survival expectancy of females infected with L. infantum and L. amazonensis after completing the gonotrophic cycle was 1.32 and 0.43, respectively. There was no association between L. infantum infection and sand fly longevity, but L. amazonensis-infected flies had significantly greater survival probabilities. Furthermore, egg-laying was significantly detrimental to survival. Lu. cruzi was found to be highly attracted to both dogs and humans. After a bloodmeal on experimentally infected hosts, both parasites were able to survive and develop late-stage infections in Lu. cruzi. However, transmission was demonstrated only for L. amazonensis-infected sand flies. In conclusion, Lu. cruzi fulfilled several of the requirements of vectorial capacity for L. infantum

  5. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

    Science.gov (United States)

    Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

    2016-08-09

    Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Nuclear DNA replication and repair in parasites of the genus Leishmania: Exploiting differences to develop innovative therapeutic approaches.

    Science.gov (United States)

    Uzcanga, Graciela; Lara, Eliana; Gutiérrez, Fernanda; Beaty, Doyle; Beske, Timo; Teran, Rommy; Navarro, Juan-Carlos; Pasero, Philippe; Benítez, Washington; Poveda, Ana

    2017-03-01

    Leishmaniasis is a common tropical disease that affects mainly poor people in underdeveloped and developing countries. This largely neglected infection is caused by Leishmania spp, a parasite from the Trypanosomatidae family. This parasitic disease has different clinical manifestations, ranging from localized cutaneous to more harmful visceral forms. The main limitations of the current treatments are their high cost, toxicity, lack of specificity, and long duration. Efforts to improve treatments are necessary to deal with this infectious disease. Many approved drugs to combat diseases as diverse as cancer, bacterial, or viral infections take advantage of specific features of the causing agent or of the disease. Recent evidence indicates that the specific characteristics of the Trypanosomatidae replication and repair machineries could be used as possible targets for the development of new treatments. Here, we review in detail the molecular mechanisms of DNA replication and repair regulation in trypanosomatids of the genus Leishmania and the drugs that could be useful against this disease.

  7. Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

    OpenAIRE

    Sanya Chadha; N. Arjunreddy Mallampudi; Debendra K. Mohapatra; Rentala Madhubala; Ira J. Blader; Greg Matlashewski; Frederick Buckner

    2017-01-01

    ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L.?donovani lysyl-tRNA synthetase (LdLysRS). Two different codin...

  8. Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

    International Nuclear Information System (INIS)

    Wilson, M.E.; Pearson, R.D.

    1986-01-01

    The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of 125 I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH 4 Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH 4 Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%

  9. Immunization with Live Attenuated Leishmania donovani Centrin−/− Parasites Is Efficacious in Asymptomatic Infection

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    Nevien Ismail

    2017-12-01

    Full Text Available Currently, there is no vaccine against visceral leishmaniasis (VL. Toward developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin−/− mutants in naive animal models. In VL endemic areas, asymptomatic carriers outnumber symptomatic cases of VL and are considered to be a reservoir of infection. Vaccination of asymptomatic cases represents a viable strategy to eliminate VL. Immunological correlates of protection thus derived might have limited applicability in conditions where the immunized host has prior exposure to virulent infection. To examine whether LdCen−/− parasites can induce protective immunity in experimental hosts that have low-level parasitemia from a previous exposure mimicking an asymptomatic condition, we infected C57Bl/6 mice with wild-type Leishmania donovani parasites expressing LLO epitope (LdWTLLO 103, i.v.. After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen−/− parasites expressing 2W epitope (LdCen−/−2W 3 × 106 i.v. to characterize the immune responses in the same host. Antigen experienced CD4+ T cells from the asymptomatic (LdWTLLO infected LdCen−/−2W immunized, and other control groups were enriched using LLO- and 2W-specific tetramers, followed by Flow cytometric analysis. Our analysis showed that comparable CD4+ T cell proliferation and CD4+ memory T cell responses (TCM represented by CD62Lhi, CCR7+, and IL-7R+ T cell populations were induced with LdCen−/−2W in both asymptomatic and naive animals that received LdCen−/− immunization. Upon restimulation with peptide, TCM cells differentiated into effector T cells and there was no significant difference in the recall response in animals with asymptomatic infection. Following virulent challenge, comparable reduction in splenic parasite burden was observed in both asymptomatic and naive LdCen−/− immunized animals concomitant with the development of

  10. Molecular Characterization of Leishmania Parasites in Giemsa-Stained Slides from Cases of Human Cutaneous and Visceral Leishmaniasis, Eastern Algeria.

    Science.gov (United States)

    Beldi, Nadia; Mansouri, Roukaya; Bettaieb, Jihene; Yaacoub, Alia; Souguir Omrani, Hejer; Saadi Ben Aoun, Yusr; Saadni, Farida; Guizani, Ikram; Guerbouj, Souheila

    2017-06-01

    In Algeria, visceral leishmaniasis (VL) is due to Leishmania (L.) infantum, while three cutaneous forms (CL) are caused by Leishmania major, Leishmania tropica and Leishmania infantum. In this study, the use of Giemsa-stained slides was evaluated with two PCR techniques, in Eastern Algeria. A total of 136 samples corresponding to 100 CL smears (skin scrapings) and 36 VL slides (bone marrow aspirates) collected from 2008 to 2014 were tested. Upon DNA extraction, two PCRs were used to amplify the ribosomal Internal Transcribed Spacer 1 (ITS1) and mini-exon genes. Amplified products were digested (PCR-RFLP) and profiles analyzed for Leishmania species identification. A statistical analysis was also performed. ITS1-PCR was found significantly more sensitive than mini-exon-PCR (77.95% positives vs. 67.65%; p = 0.001). Comparison of PCR positivity showed statistically significant differences between old and recently prepared slides suggesting a better use of recent slides in PCR analyses. For species identification, PCR-restriction fragment length polymorphism (RFLP) results of ITS1 and mini-exon were concordant. L. infantum was identified from VL cases and L. infantum, L. major, and L. tropica from CL ones. According to geographical origin, L. infantum was found in North-Eastern provinces, while L. major was distributed from the North to the Center-East of Algeria. Interestingly, two L. tropica samples were identified in Annaba, located far North-East Algeria. Distribution of leishmaniasis in Eastern parts of Algeria, besides finding of L. tropica in the far North, is in this study described for the first time using molecular tools, thus confirming the usefulness of slides for PCR identification of Leishmania parasites in retrospective epidemiological investigations.

  11. Monitoring the response of patients with cutaneous leishmaniasis to treatment with pentamidine isethionate by quantitative real-time PCR, and identification of Leishmania parasites not responding to therapy.

    Science.gov (United States)

    Mans, D R A; Kent, A D; Hu, R V; Lai A Fat, E J; Schoone, G J; Adams, E R; Rood, E J; Alba, S; Sabajo, L O A; Lai A Fat, R F; de Vries, H J C; Schallig, H D F H

    2016-08-01

    Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported. To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species. Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed. L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61. L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment. © 2015 The Authors Clinical and Experimental Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists, North American Clinical Dermatologic Society and St Johns Dermatological Society.

  12. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

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    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  13. New Insights Into the Transmissibility of Leishmania infantum From Dogs to Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite Depots at Bite Sites.

    Science.gov (United States)

    Aslan, Hamide; Oliveira, Fabiano; Meneses, Claudio; Castrovinci, Philip; Gomes, Regis; Teixeira, Clarissa; Derenge, Candace A; Orandle, Marlene; Gradoni, Luigi; Oliva, Gaetano; Fischer, Laurent; Valenzuela, Jesus G; Kamhawi, Shaden

    2016-06-01

    Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

  14. Infectivity to Phlebotomus perniciosus of dogs naturally parasitized with Leishmania infantum after different treatments

    Directory of Open Access Journals (Sweden)

    Descalzo Miguel A

    2011-04-01

    Full Text Available Abstract Background In Europe most dogs with clinical leishmaniosis are treated with leishmanicides, typically antimonials combined with allopurinol and good clinical recovery is observed in a high number of these dogs. Through xenodiagnosis, the capacity of a treated animal to infect the vector of the disease under treatment is assessed as a measure of the chemotherapeutic efficacy of the drug used. The objective of the present study was to evaluate through direct xenodiagnosis the infectivity to Phlebotomus perniciosus of dogs naturally parasitized with Leishmania infantum after treatment, and to follow the clinical and parasite course of disease. Thirty two dogs with clinical leishmaniosis were assigned to one of three treatment groups: meglumine antimoniate plus allopurinol (Group A, meglumine antimoniate (Group B or allopurinol (Group C. During the study, the dogs were examined before treatment (Day 0 and bimonthly thereafter until Day 180 (six months post-treatment onset. Results The three groups were scored over time according to the effects of treatment on clinical signs and clinical-pathological variables. Significant differences in clinical scores were observed between Group A and the other two groups, indicating the combined treatment was the most effective. After treatment, bone marrow cultures were positive for the parasite in 30.8% of dogs in some of the check ups (3 or 25% in Group A, 1 or 11.1% in Group B, and 4 or 80% in Group C. Our xenodiagnosis experiments revealed that 15.4% of treated dogs were still able to infect sand flies at some point after treatment (2 dogs or 16.6% in Group A, 2 or 22.2% in Group B and none in Group C. Only 7.7% of the entire study population could infect sand flies as from the second month post-treatment onset. Conclusion The three treatment regimens tested significantly reduced the infectivity of dogs towards sand flies, thus diminishing the epidemiological risks of treated dogs both for human

  15. Leishmania eukaryotic initiation factor (LeIF inhibits parasite growth in murine macrophages.

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    Olga Koutsoni

    Full Text Available The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF, an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment, and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment, and resistance to infection was also observed at both time points tested (19 h and 72 h after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO and reactive oxygen species (ROS, within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α as well as tumor necrosis factor alpha (TNF-α expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably

  16. Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

    Directory of Open Access Journals (Sweden)

    Miriam A Lynn

    Full Text Available Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28 has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28 and the retro-inverso form (RI-BMAP-28, as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

  17. Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

    Science.gov (United States)

    Coleman, Russell E; Hochberg, Lisa P; Swanson, Katherine I; Lee, John S; McAvin, James C; Moulton, John K; Eddington, David O; Groebner, Jennifer L; O'Guinn, Monica L; Putnam, John L

    2009-05-01

    Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

  18. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

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    Rita Marcia Cardoso de Paiva

    2015-12-01

    Full Text Available Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

  19. Biochemical and molecular characterization of Leishmania parasites isolated from an endemic focus in eastern Sudan

    NARCIS (Netherlands)

    Oskam, L.; Pratlong, F.; Zijlstra, E. E.; Kroon, C. C.; Dedet, I. P.; Kager, P. A.; Schönian, G.; Ghalib, H. W.; El-Hassan, A. M.; Meredith, S. E.

    1998-01-01

    Twelve Leishmania isolates from visceral leishmaniasis patients in eastern Sudan were characterized using isoenzyme analysis, Southern blotting and polymerase chain reaction (PCR) 'fingerprinting'. Isoenzyme analysis revealed the presence of 3 zymodemes: MON-18, MON-30 and MON-82, corresponding to

  20. Host modulation by a parasite: how Leishmania infantum modifies the intestinal environment of Lutzomyia longipalpis to favor its development.

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    Vania Cristina Santos

    Full Text Available Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.

  1. Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

    DEFF Research Database (Denmark)

    Cayla, M.; Rachidi, N.; Leclercq, O.

    2014-01-01

    Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even...... though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage...... at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10...

  2. Anti-Parasitic Activities of Allium sativum and Allium cepa against Trypanosoma b. brucei and Leishmania tarentolae.

    Science.gov (United States)

    Krstin, Sonja; Sobeh, Mansour; Braun, Markus Santhosh; Wink, Michael

    2018-04-21

    Background: Garlics and onions have been used for the treatment of diseases caused by parasites and microbes since ancient times. Trypanosomiasis and leishmaniasis are a concern in many areas of the world, especially in poor countries. Methods: Trypanosoma brucei brucei and Leishmania tarentolae were used to investigate the anti-parasitic effects of dichloromethane extracts of Allium sativum (garlic) and Allium cepa (onion) bulbs. As a confirmation of known antimicrobial activities, they were studied against a selection of G-negative, G-positive bacteria and two fungi. Chemical analyses were performed using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Results: Chemical analyses confirmed the abundance of several sulfur secondary metabolites in garlic and one (zwiebelane) in the onion extract. Both extracts killed both types of parasites efficiently and inhibited the Trypanosoma brucei trypanothione reductase irreversibly. In addition, garlic extract decreased the mitochondrial membrane potential in trypanosomes. Garlic killed the fungi C. albicans and C. parapsilosis more effectively than the positive control. The combinations of garlic and onion with common trypanocidal and leishmanicidal drugs resulted in a synergistic or additive effect in 50% of cases. Conclusion: The mechanism for biological activity of garlic and onion appears to be related to the amount and the profile of sulfur-containing compounds. It is most likely that vital substances inside the parasitic cell, like trypanothione reductase, are inhibited through disulfide bond formation between SH groups of vital redox compounds and sulfur-containing secondary metabolites.

  3. Characterization of Leishmania Parasites Isolated From Kala- azar Patients in Kohgiloyeh and Boyerahmad, Using Semi-Nested PCR

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    B Sarkari

    2006-04-01

    Full Text Available ABSTRACT: Introduction & Objective: Visceral leishmaniasis (VL is a disease commonly known as Kala-azar caused by protozoan parasites of the genus Leishmania including L. donovani, L. infantum and L. chagasi. VL is sporadic in many areas of Iran and is endemic in a few provinces such as Fars, Azarbayjan, Bushehr, Ardabil and Qom. VL has been reported from some areas of Kohgiloyeh and Boyerahmad and this study aimed to characterize the causative agent of VL in this region. Materials & Methods: Bone marrow sample was obtained from 6 VL patients from children department in Imam Sajad hospital in Yasuj. DNA was extracted from the obtained samples and was checked by semi-nested PCR to determine the species of the parasite. To do that, a segment of minicircle kinetoplast DNA was amplified, using LINR4 and LIN17 primers. Products of PCR were evaluated by electrophoresis, using 1.5% agarose and stained with ethidium bromide. Results: Parasitologically examination of bone marrow smears demonstrated amastigotes form of the parasite in the samples. For mass cultivation, isolated parasites were cultured in diphasic NNN followed by RPMI 1640 media. All the samples produced a 720 bp band in PCR assay. The isolates were compared with referent strains and it was revealed that all the isolates were L. infantum. Conclusion: Findings of this study demonstrated that the causative agent of VL in Kohgiloyeh and Boyerahmad was L. infantum. Further study is needed to explore other aspects of VL in this region.

  4. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies

    Czech Academy of Sciences Publication Activity Database

    Akhoundi, M.; Kuhls, K.; Cannet, A.; Votýpka, Jan; Marty, P.; Delaunay, P.; Sereno, D.

    2016-01-01

    Roč. 10, č. 3 (2016), č. článku e0004349. ISSN 1935-2735 Institutional support: RVO:60077344 Keywords : phlebotomine sand flies * human cutaneous leishmaniasis * North-Central Venezuela * visceral leishmaniasis * genus Leishmania * diptera-psychodidae * old world * causative agent * kinetoplastida trypanosomatidae Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.834, year: 2016

  5. BALB/c Mice Vaccinated with Leishmania major Ribosomal Proteins Extracts Combined with CpG Oligodeoxynucleotides Become Resistant to Disease Caused by a Secondary Parasite Challenge

    Directory of Open Access Journals (Sweden)

    Laura Ramírez

    2010-01-01

    Full Text Available Leishmaniasis is an increasing public health problem and effective vaccines are not currently available. We have previously demonstrated that vaccination with ribosomal proteins extracts administered in combination of CpG oligodeoxynucleotides protects susceptible BALB/c mice against primary Leishmania major infection. Here, we evaluate the long-term immunity to secondary infection conferred by this vaccine. We show that vaccinated and infected BALB/c mice were able to control a secondary Leishmania major challenge, since no inflammation and very low number of parasites were observed in the site of reinfection. In addition, although an increment in the parasite burden was observed in the draining lymph nodes of the primary site of infection we did not detected inflammatory lesions at that site. Resistance against reinfection correlated to a predominant Th1 response against parasite antigens. Thus, cell cultures established from spleens and the draining lymph node of the secondary site of infection produced high levels of parasite specific IFN-γ in the absence of IL-4 and IL-10 cytokine production. In addition, reinfected mice showed a high IgG2a/IgG1 ratio for anti-Leishmania antibodies. Our results suggest that ribosomal vaccine, which prevents pathology in a primary challenge, in combination with parasite persistence might be effective for long-term maintenance of immunity.

  6. Phenotypic characterization of Leishmania spp. causing cutaneous leishmaniasis in the lower Amazon region, western Pará state, Brazil, reveals a putative hybrid parasite, Leishmania (Viannia guyanensis × Leishmania (Viannia shawi shawi

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    Jennings Yara Lins

    2014-01-01

    Full Text Available We phenotypically characterized 43 leishmanial parasites from cutaneous leishmaniasis by isoenzyme electrophoresis and the indirect immunofluorescence antibody test (23 McAbs. Identifications revealed 11 (25.6% strains of Leishmania (V. braziliensis, 4 (9.3% of L. (V. shawi shawi, 7 (16.3% of L. (V. shawi santarensis, 6 (13.9% of L. (V. guyanensis and L. (V. lainsoni, 2 (4.7% of L. (L. amazonensis, and 7 (16.3% of a putative hybrid parasite, L. (V. guyanensis/L. (V. shawi shawi. McAbs detected three different serodemes of L. (V. braziliensis: I-7, II-1, and III-3 strains. Among the strains of L. (V. shawi we identified two populations: one (7 strains expressing the B19 epitope that was previously considered to be species-specific for L. (V. guyanensis. We have given this population sub-specific rank, naming it L. (V. s. santarensis. The other one (4 strains did not express the B19 epitope like the L. (V. shawi reference strain, which we now designate as L. (V. s. shawi. For the first time in the eastern Brazilian Amazon we register a putative hybrid parasite (7 strains, L. (V. guyanensis/L. (V. s. shawi, characterized by a new 6PGDH three-band profile at the level of L. (V. guyanensis. Its PGM profile, however, was very similar to that of L. (V. s. shawi. These results suggest that the lower Amazon region – western Pará state, Brazil, represents a biome where L. (V. guyanensis and L. (V. s. shawi exchange genetic information.

  7. Phenotypic characterization of Leishmania spp. causing cutaneous leishmaniasis in the lower Amazon region, western Pará state, Brazil, reveals a putative hybrid parasite, Leishmania (Viannia) guyanensis × Leishmania (Viannia) shawi shawi

    Science.gov (United States)

    Jennings, Yara Lins; de Souza, Adelson Alcimar Almeida; Ishikawa, Edna Aoba; Shaw, Jeffrey; Lainson, Ralph; Silveira, Fernando

    2014-01-01

    We phenotypically characterized 43 leishmanial parasites from cutaneous leishmaniasis by isoenzyme electrophoresis and the indirect immunofluorescence antibody test (23 McAbs). Identifications revealed 11 (25.6%) strains of Leishmania (V.) braziliensis, 4 (9.3%) of L. (V.) shawi shawi, 7 (16.3%) of L. (V.) shawi santarensis, 6 (13.9%) of L. (V.) guyanensis and L. (V.) lainsoni, 2 (4.7%) of L. (L.) amazonensis, and 7 (16.3%) of a putative hybrid parasite, L. (V.) guyanensis/L. (V.) shawi shawi. McAbs detected three different serodemes of L. (V.) braziliensis: I-7, II-1, and III-3 strains. Among the strains of L. (V.) shawi we identified two populations: one (7 strains) expressing the B19 epitope that was previously considered to be species-specific for L. (V.) guyanensis. We have given this population sub-specific rank, naming it L. (V.) s. santarensis. The other one (4 strains) did not express the B19 epitope like the L. (V.) shawi reference strain, which we now designate as L. (V.) s. shawi. For the first time in the eastern Brazilian Amazon we register a putative hybrid parasite (7 strains), L. (V.) guyanensis/L. (V.) s. shawi, characterized by a new 6PGDH three-band profile at the level of L. (V.) guyanensis. Its PGM profile, however, was very similar to that of L. (V.) s. shawi. These results suggest that the lower Amazon region – western Pará state, Brazil, represents a biome where L. (V.) guyanensis and L. (V.) s. shawi exchange genetic information. PMID:25083790

  8. Epidemiologic profile of oriental sore caused by Leishmania parasites in a new endemic focus of cutaneous leishmaniasis, southern Iran.

    Science.gov (United States)

    Khosravani, Moosa; Moemenbellah-Fard, Mohammad Djaefar; Sharafi, Mehdi; Rafat-Panah, Azam

    2016-09-01

    Cutaneous leishmaniasis (CL) is the first and most important vector-borne zoonotic disease transmitted by sand flies in Iran. As a parasitic disease in the Old World, it is a complex zoonosis with multiple vertebrate hosts and arthropod vectors of pathogenic flagellate protozoan in the genus of Leishmania in different parts of its range. Phlebotomine sand flies are proven as vectors of this parasite which can be transmitted through the bite of an infected female sand fly distributed in almost all parts of Iran. This research performed on all CL patients as that were registered into special forms by physicians and experts during the study period 2006-2013 in the county town of Fasa, Iran. Data were analyzed by Chi square test using SPSS 17 statistics software. Overall, 1,908 patients (59.18 %) lived in rural and 1,316 (40.82 %) lived in urban areas. All ages were between 1 and ≥30 year. The most frequent age group was ≥20 years (54.6 %). Sex ratio of patients was almost 1:1 (1,561; 48.42 % male vs. 1,663; 51.58 % female). Most of them (66.84 %) had wet lesions and those with dry lesions were less frequent (33.16 %). There was a significant difference between the frequencies of these two groups (P counties in Iran showed that it was most likely an endemic disease in this region.

  9. Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

    Science.gov (United States)

    Chadha, Sanya; Mallampudi, N Arjunreddy; Mohapatra, Debendra K; Madhubala, Rentala

    2017-01-01

    Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase ( Ld LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. Ld LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized Ld LysRS-1. Recombinant Ld LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The Ld LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. Ld LysRS-1 appears to be an essential gene, as a chromosomal knockout of Ld LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC 50 ], 4.19 µM) and intracellular amastigotes (IC 50 , 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using Ld LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant Ld LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for

  10. Evaluation of a Microculture Method for Isolation of Leishmania Parasites from Cutaneous Lesions of Patients in Peru▿

    Science.gov (United States)

    Boggild, Andrea K.; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E.

    2007-01-01

    Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-μl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture. PMID:17881557

  11. Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru.

    Science.gov (United States)

    Boggild, Andrea K; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E

    2007-11-01

    Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.

  12. Experimental treatment with sodium stibogluconate of hamsters infected with Leishmania (Leishmania) chagasi and Leishmania (Leishmania) amazonensis Tratamento experimental com stibogluconato de sódio em hamsters infectados com Leishmania (Leishmania) chagasi e Leishmania (Leishmania) amazonensis

    OpenAIRE

    Elizabeth M. de Figueiredo; Jaime Costa e Silva; Reginaldo P. Brazil

    1999-01-01

    The present paper reports the experimental treatment of hamsters infected with Leishmania chagasi and Leishmania amazonensis with sodium stibogluconate (20mg/kg/day x 20 days). Only with L. chagasi did the treatment result in the complete elimination of parasites from the spleen. However, no parasitological cure was achieved in hamsters infected with L. amazonensis.O presente trabalho é um relato do tratamento experimental de hamsters infectado com Leishmania chagasi e Leishmania amazonensis ...

  13. Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors.

    Science.gov (United States)

    Borja, Lairton Souza; Sousa, Orlando Marcos Farias de; Solcà, Manuela da Silva; Bastos, Leila Andrade; Bordoni, Marcelo; Magalhães, Jairo Torres; Larangeira, Daniela Farias; Barrouin-Melo, Stella Maria; Fraga, Deborah Bittencourt Mothé; Veras, Patrícia Sampaio Tavares

    2016-10-15

    The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as

  14. Optimization of microculture and evaluation of miniculture for the isolation of Leishmania parasites from cutaneous lesions in Peru.

    Science.gov (United States)

    Boggild, Andrea K; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Martinez-Medina, Dalila; Llanos-Cuentas, Alejandro; Low, Donald E

    2008-12-01

    Traditional culture of Leishmania parasites is labor-intensive and shows poor sensitivity. We evaluated microculture and novel miniculture methods for diagnosis of cutaneous leishmaniasis (CL). Consecutive patients who came to the Leishmaniasis Clinic, Hospital Nacional Cayetano Heredia, Lima, Peru, were enrolled. Lesion aspirates were cultured in traditional tubes containing Novy-MacNeal-Nicolle medium and in miniculture tubes (Eppendorf, Hamburg, Germany) and capillary tubes (microculture) containing RPMI 1640 medium containing 20% fetal bovine serum. The reference standard was positive results in two of four tests (smear, culture, polymerase chain reaction, or leishmanin skin test). Outcome measures were sensitivity and time to positivity. Fifty-five patients with 74 lesions were enrolled. Of 59 lesions that fulfilled reference criteria for CL, 50 were positive by microculture (sensitivity=84.7%; P=0.001), 45 by miniculture (sensitivity=76.3%; P=0.042), and 35 by traditional culture (sensitivity=59.3%). Median time to positivity was three days by microculture and miniculture and five days by traditional culture (PMicroculture and miniculture are sensitive and efficient means of diagnosing CL.

  15. Programmed cell death in trypanosomatids and other unicellular organisms.

    Science.gov (United States)

    Debrabant, Alain; Lee, Nancy; Bertholet, Sylvie; Duncan, Robert; Nakhasi, Hira L

    2003-03-01

    In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms. However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms. The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway. Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.

  16. The NAD+ metabolism of Leishmania, notably the enzyme nicotinamidase involved in NAD+ salvage, offers prospects for development of anti-parasite chemotherapy.

    Science.gov (United States)

    Michels, Paul A M; Avilán, Luisana

    2011-10-01

    NAD+ plays multiple, essential roles in the cell. As a cofactor in many redox reactions it is key in the cellular energy metabolism and as a substrate it participates in many reactions leading to a variety of covalent modifications of enzymes with major roles in regulation of expression and metabolism. Cells may have the ability to produce this metabolite either via alternative de novo synthesis pathways and/or by different salvage pathways. In this issue of Molecular Microbiology, Gazanion et al. (2011) demonstrate that Leishmania species can only rely on the salvage of NAD+ building blocks. One of the enzymes involved, nicotinamidase, is absent from human cells. The enzyme is important for growth of Leishmania infantum and essential for establishing an infection. The crystal structure of the parasite protein has been solved and shows prospects for design of inhibitors to be used as leads for development of new drugs. Indeed, NAD+ metabolism is currently being considered as a promising drug target in various diseases and the vulnerability of Leishmania for interference of this metabolism has been proved in previous work by the same group, by showing that administration of NAD+ precursors has detrimental effect on the pathogenic, amastigote stage of this parasite. © 2011 Blackwell Publishing Ltd.

  17. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum-naturally infected dogs.

    Science.gov (United States)

    Cavalcanti, Amanda S; Ribeiro-Alves, Marcelo; Pereira, Luiza de O R; Mestre, Gustavo Leandro; Ferreira, Anna Beatriz Robottom; Morgado, Fernanda N; Boité, Mariana C; Cupolillo, Elisa; Moraes, Milton O; Porrozzi, Renato

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) shares many aspects with the human disease and dogs are considered the main urban reservoir of L. infantum in zoonotic VL. Infected dogs develop progressive disease with a large clinical spectrum. A complex balance between the parasite and the genetic/immunological background of the host are decisive for infection evolution and clinical outcome. This study comprised 92 Leishmania infected mongrel dogs of various ages from Mato Grosso, Brazil. Spleen samples were collected for determining parasite load, humoral response, cytokine mRNA expression and histopathology alterations. By real-time PCR for the ssrRNA Leishmania gene, two groups were defined; a low (lowP, n = 46) and a high parasite load groups (highP, n = 42). When comparing these groups, results show variable individual humoral immune response with higher specific IgG production in infected animals but with a notable difference in CVL rapid test optical densities (DPP) between highP and lowP groups. Splenic architecture disruption was characterized by disorganization of white pulp, more evident in animals with high parasitism. All cytokine transcripts in spleen were less expressed in highP than lowP groups with a large heterogeneous variation in response. Individual correlation analysis between cytokine expression and parasite load revealed a negative correlation for both pro-inflammatory cytokines: IFNγ, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and TGFβ. TNF showed the best negative correlation (r2 = 0.231; pdogs with high parasite load associated with a structural modification in the splenic lymphoid micro-architecture. We also discuss the possible mechanism responsible for the uncontrolled parasite growth and clinical outcome.

  18. Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishma-nia major Infected Mouse

    Directory of Open Access Journals (Sweden)

    Somayeh GHOTLOO

    2015-12-01

    Full Text Available Background:Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate para­site burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumera­tion of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification.Methods:The SYBR Green based real- time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homoge­nates were prepared in the Schneider medium and incubated at 26°C.After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software.Results:Spearman's correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (Pvalue = 0.008.Conclusion:Real-time PCR assay is an appropriate replacement to existing limit­ing dilution assay in quantifying parasite burden in the experimental model of Leishma­nia infection.

  19. Automatic segmentation of Leishmania parasite in microscopic images using a modified CV level set method

    Science.gov (United States)

    Farahi, Maria; Rabbani, Hossein; Talebi, Ardeshir; Sarrafzadeh, Omid; Ensafi, Shahab

    2015-12-01

    Visceral Leishmaniasis is a parasitic disease that affects liver, spleen and bone marrow. According to World Health Organization report, definitive diagnosis is possible just by direct observation of the Leishman body in the microscopic image taken from bone marrow samples. We utilize morphological and CV level set method to segment Leishman bodies in digital color microscopic images captured from bone marrow samples. Linear contrast stretching method is used for image enhancement and morphological method is applied to determine the parasite regions and wipe up unwanted objects. Modified global and local CV level set methods are proposed for segmentation and a shape based stopping factor is used to hasten the algorithm. Manual segmentation is considered as ground truth to evaluate the proposed method. This method is tested on 28 samples and achieved 10.90% mean of segmentation error for global model and 9.76% for local model.

  20. Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development

    Science.gov (United States)

    Zimara, Nicole; Chanyalew, Menberework; Aseffa, Abraham; van Zandbergen, Ger; Lepenies, Bernd; Schmid, Maximilian; Weiss, Richard; Rascle, Anne; Wege, Anja Kathrin; Jantsch, Jonathan; Schatz, Valentin; Brown, Gordon D.; Ritter, Uwe

    2018-01-01

    Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1−/− BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore

  1. Cyclobenzaprine Raises ROS Levels in Leishmania infantum and Reduces Parasite Burden in Infected Mice.

    Directory of Open Access Journals (Sweden)

    Edézio Ferreira Cunha-Júnior

    2017-01-01

    Full Text Available The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity.In vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1μM and an IC90 of 74.5±1.2 μM in promastigotes and an IC50 of 12.6±1.05 μM and an IC90 of 28.7±1.3 μM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 μM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages co-culture.To the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to

  2. Myeloid-derived suppressor cell functionality and interaction with Leishmania major parasites differ in C57BL/6 and BALB/c mice.

    Science.gov (United States)

    Schmid, Maximilian; Zimara, Nicole; Wege, Anja Kathrin; Ritter, Uwe

    2014-11-01

    Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of CD11b+ cells. According to the surface molecules Ly6G and Ly6C (where Ly6G and Ly6C are lymphocyte antigen 6, locus G and C, respectively), MDSCs are further divided into monocytic (Mo-MDSCs, CD11b+ /Ly6C(high) /Ly6G-) and polymorphonucleated suppressor cells (PMN-MDSCs, CD11b+ /Ly6C(int) /Ly6G+). Most published manuscripts focus on the suppressive role of MDSCs in cancer, whereas their impact on adaptive immunity against obligatory intracellular parasites is not well understood. Furthermore, it is not clear how the genetic background of mice influences MDSC functionality. Therefore, we implemented an experimental model of leishmaniasis, and analyzed MDSC maturation and the impact of MDSCs on the parasite-specific T-cell responses in resistant C57BL/6 and susceptible BALB/c mice. This experimental setup demonstrated the impaired ability of BALB/c mice to produce Mo-MDSCs when compared with C57BL/6 mice. This phenotype is detectable after subcutaneous infection with parasites and is specifically represented by a reduced accumulation of Mo-MDSCs at the site of infection in BALB/c mice. Moreover, infected C57BL/6-derived MDSCs were able to suppress Leishmania-specific CD4+ -cell proliferation, whereas BALB/c-derived MDSCs harboring parasites lost this suppressive function. In conclusion, we demonstrate that (i) genetic background defines MDSC differentiation; and (ii) Leishmania major parasites are able to modulate the suppressive effect of MDSCs in a strain-dependent manner. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Leishmania infantum proteophosphoglycans regurgitated by the bite of its natural sand fly vector, Lutzomyia longipalpis, promote parasite establishment in mouse skin and skin-distant tissues.

    Science.gov (United States)

    Rogers, Matthew Edward; Corware, Karina; Müller, Ingrid; Bates, Paul Andrew

    2010-10-01

    We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10³) and high (10⁵) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis. Copyright © 2010. Published by Elsevier SAS.

  4. Characterization of Leishmania Soluble Exo-Antigen

    National Research Council Canada - National Science Library

    Cui, Liwang

    2003-01-01

    .... Vaccine development is the ultimate solution for this problem. Our previous research indicates that Leishmania parasites secrete, excrete, or shed antigens into the medium during in vitro culture...

  5. Autophagy in unicellular eukaryotes

    NARCIS (Netherlands)

    Kiel, J.A.K.W.

    2010-01-01

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components

  6. Inflammatory response, parasite load and AgNOR expression in ear skin of symptomatic and asymptomatic Leishmania (Leishmania chagasi infected dogs

    Directory of Open Access Journals (Sweden)

    Verçosa BLA

    2011-01-01

    Full Text Available The skin has an important role in the transmission of visceral leishmaniasis (VL as the infection pathway in dogs. To better characterize the inflammatory response of intact skin in VL, sixty infected dogs (30 symptomatic and 30 asymptomatic and six non-infected controls were studied. Diagnosis of visceral leishmaniasis was confirmed by RIFI and ELISA; direct visualization of the parasite in bone marrow aspirate; imprints of popliteal lymph nodes, spleen, liver and skin; culture in NNN-phase liquid Schneider's medium; and PCR (performed only in the ear skin. Amastigote forms of the parasite in intact skin were found only in symptomatic dogs. Inflammatory infiltrates were observed in all groups, varying from intense and/or moderate in symptomatic to discrete and/or negligible in asymptomatic and control animals. Parasite load was associated with the intensity of the inflammatory response and with clinical manifestations in canine visceral leishmaniasis. AgNOr as active transcription markers were expressed in inflammatory cells and within apoptotic bodies in all groups, including controls, with no statistical difference. Therefore, cell activation and transcription do occur in both symptomatic and asymptomatic canine visceral leishmaniasis and may result in more necrosis and inflammation or in apoptosis and less symptoms, depending on the parasite load.

  7. Molecular detection of Leishmania parasites and host blood meal identification in wild sand flies from a new endemic rural region, south of Iran.

    Science.gov (United States)

    Azizi, Kourosh; Askari, Mohammad Bagher; Kalantari, Mohsen; Moemenbellah-Fard, Mohammad Djaefar

    Zoonotic Cutaneous Leishmaniosis (ZCL) remains the most crucial vector-borne public health disease particularly in endemic rural parts of Iran. The main aim of this study is to identify wild sand flies (Diptera: Psychodidae), determine their infection rate, and differentiate their host blood meal sources using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Sand fly populations were caught with sticky paper traps from 10 different villages in the county of Darab, Fars province, southern Iran. Following their species identification, they were used in one step PCR to determine their infection with Leishmania spp. parasites. They were then subjected to PCR-RFLP protocol to identify and differentiate their blood meal sources. Two genera of Phlebotomus and Sergentomyia comprising 13 species of sand flies were identified in this region. From a total of 150 parous female sand flies, encompassing 4 different medically important species, 7 specimens (4.7%) including 6 Phlebotomus papatasi and 1 Phlebotomus bergeroti were infected with Leishmania major. Molecular data indicated that about 32% of female sand flies fed on man, while nearly 43% fed on rodent and canine hosts. Molecular detection is an efficient way of differentiating the source of blood meals in female sand flies feeding on different vertebrate hosts. It is suggested that P. papatasi is not highly anthropophagic and appears to be an opportunistic feeder on man. This species is, however, the primary vector of ZCL in this region.

  8. Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L. Smith in protozoan parasite Leishmania donovani due to oxidative stress

    Directory of Open Access Journals (Sweden)

    Debarati Mukherjee

    2016-01-01

    Full Text Available In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmania infection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L. Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovani infection and provided the basis for future research on the application of transitional medicinal plants.

  9. High density of Leishmania major and rarity of other mammals' Leishmania in zoonotic cutaneous leishmaniasis foci, Iran.

    Science.gov (United States)

    Bordbar, Ali; Parvizi, Parviz

    2014-03-01

    Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously. Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites. Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies. Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans. © 2014 John Wiley & Sons Ltd.

  10. Molecular basis of Trypanosoma cruzi and Leishmania interaction with their host(s): exploitation of immune and defense mechanisms by the parasite leading to persistence and chronicity, features reminiscent of immune system evasion strategies in cancer diseases.

    Science.gov (United States)

    Ouaissi, Ali; Ouaissi, Mehdi

    2005-01-01

    A number of features occurring during host-parasite interactions in Chagas disease caused by the protozoan parasite, Trypanosoma cruzi, and Leishmaniasis, caused by a group of kinetoplastid protozoan parasites are reminiscent of those observed in cancer diseases. In fact,although the cancer is not a single disease, and that T.cruzi and Leishmania are sophisticated eukaryotic parasites presenting a high level of genotypic variability the growth of the parasites in their host and that of cancer cells share at least one common feature, that is their mutual capacity for rapid cell division. Surprisingly, the parasitic diseases and cancers share some immune evasion strategies. Consideration of these immunological alterations must be added to the evaluation of the pathogenic processes. The molecular and functional characterization of virulence factors and the study of their effect on the arms of the immune system have greatly improved understanding of the regulation of immune effectors functions. The purpose of this review is to analyze some of the current data related to the regulatory components or processes originating from the parasite that control or interfere with host cell physiology. Attempts are also made to delineate some similarities between the immune evasion strategies that parasites and tumors employ. The elucidation of the mode of action of parasite virulence factors toward the host cell allow not only provide us with a more comprehensive view of the host-parasite relationships but may also represent a step forward in efforts aimed to identify new target molecules for therapeutic intervention.

  11. Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

    Science.gov (United States)

    Oliveira, Valéria da Costa; Boechat, Viviane Cardoso; Mendes Junior, Artur Augusto Velho; Madeira, Maria de Fátima; Ferreira, Luiz Claudio; Figueiredo, Fabiano Borges; Campos, Monique Paiva; de Carvalho Rodrigues, Francisco das Chagas; Carvalhaes de Oliveira, Raquel de Vasconcellos; Amendoeira, Maria Regina Reis; Menezes, Rodrigo Caldas

    2017-01-01

    Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

  12. Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

    Directory of Open Access Journals (Sweden)

    Valéria da Costa Oliveira

    Full Text Available Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%, followed by the spinal cord (96%, spleen (95%, and CSF (50%. The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

  13. LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites

    Science.gov (United States)

    Carvalho, Sandra; da Silva, Rosa Barreira; Shawki, Ali; Castro, Helena; Lamy, Márcia; Eide, David; Costa, Vítor; Mackenzie, Bryan; Tomás, Ana M.

    2016-01-01

    Summary Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn2+ was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis. PMID:25644708

  14. Innate Immunity against Leishmania Infections

    Science.gov (United States)

    Gurung, Prajwal; Kanneganti, Thirumala-Devi

    2015-01-01

    Leishmaniasis is a major health problem that affects more than 300 million people throughout the world. The morbidity associated with the disease causes serious economic burden in Leishmania endemic regions. Despite the morbidity and economic burden associated with Leishmaniasis, this disease rarely gets noticed and is still categorized under neglected tropical diseases. The lack of research combined with the ability of Leishmania to evade immune recognition has rendered our efforts to design therapeutic treatments or vaccines challenging. Herein, we review the literature on Leishmania from innate immune perspective and discuss potential problems as well as solutions and future directions that could aid in identifying novel therapeutic targets to eliminate this parasite. PMID:26249747

  15. Optical tweezers force measurements to study parasites chemotaxis

    Science.gov (United States)

    de Thomaz, A. A.; Pozzo, L. Y.; Fontes, A.; Almeida, D. B.; Stahl, C. V.; Santos-Mallet, J. R.; Gomes, S. A. O.; Feder, D.; Ayres, D. C.; Giorgio, S.; Cesar, C. L.

    2009-07-01

    In this work, we propose a methodology to study microorganisms chemotaxis in real time using an Optical Tweezers system. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of taxis response of cells and microorganisms with high sensitivity to capture instantaneous responses to a given stimulus. Forces involved in the movement of unicellular parasites are very small, in the femto-pico-Newton range, about the same order of magnitude of the forces generated in an Optical Tweezers. We applied this methodology to investigate the Leishmania amazonensis (L. amazonensis) and Trypanossoma cruzi (T. cruzi) under distinct situations.

  16. Parasites

    Centers for Disease Control (CDC) Podcasts

    2010-05-06

    In this podcast, a listener wants to know what to do if he thinks he has a parasite or parasitic disease.  Created: 5/6/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 5/6/2010.

  17. Papel de la vacuola parasitófora de macrófagos de ratón infectados por Leishmania amazonensis en la adquisición de moléculas

    Directory of Open Access Journals (Sweden)

    Tania M. Cortázar

    2006-10-01

    Full Text Available Introducción. Leishmania son parásitos intracelulares de macrófagos, confinados encompartimentos denominados vacuolas parasitóforas. La permeabilidad de este compartimentodepende de su interacción con el tráfico vesicular y transportadores presentes en su membrana. Objetivo. En este trabajo se estudió la permeabilidad de la membrana de la vacuola parasitóforaen la línea celular J774.A1 infectada con Leishmania amazonensis, in situ y en compartimentosaislados. Materiales y métodos. El aislamiento de vacuolas parasitóforas se hizo por gradiente dedensidad. La permeabilidad de la membrana de estas se valoró por distribución de sondasfluorescentes y electrofisiología. Para establecer indirectamente el transporte de protones seusó naranja de acridina. La presencia de transportadores ABC sensibles a probenecid seestableció con amarillo lucifer y calceína. Por primera vez con la técnica de patch-clamp seregistraron corrientes en la membrana de este compartimento aislado. Resultados. La vacuola parasitófora colorea de rojo con naranja de acridina indicando un pHácido. Concentra amarillo lucifer a través de un transportador sensible a probenecid, peroexcluye la sonda calceína. Vacuolas aisladas se marcan de rojo con naranja de acridina yconcentran amarillo lucifer a través de un transportador sensible a probenecid. Estas vacuolasexcluyeron calceína y presentaron en su membrana una corriente iónica que se activa adiferencias de potencial cercanas a 60 mV, con una conductancia de 46 ± 3 pS. Conclusiones. Se pueden aislar vacuolas parasitóforas con propiedades de permeabilidadque preservan mecanismos de transporte similares a los encontrados in situ. Se registra porprimera vez la presencia de una corriente iónica poco selectiva en la membrana de estecompartimiento.

  18. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters.

    Directory of Open Access Journals (Sweden)

    Jacqueline Araújo Fiuza

    2016-01-01

    Full Text Available Visceral leishmaniasis (VL is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-, a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani.To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution, antibody production (ELISA and cytokine expression (qPCR in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection.Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

  19. Screening For Inhibitors Of Essential Leishmania Glucose Transporters

    Science.gov (United States)

    2011-07-01

    parasite life cycle and, unlike he amastigote form that lives inside mammalian macrophages, s viable provided that an alternative energy source such as pro...glucose transporters havebeenvalidated asnewdrug targets for proto- zoan parasites including Plasmodium falciparum, Leishmania mexicana and Trypanosoma...such as Leishmania species, Trypanosoma rucei, and Plasmodium falciparum, the causative agents of leish- aniasis, African sleeping sickness, and malaria

  20. Leishmania Hijacks Myeloid Cells for Immune Escape

    Directory of Open Access Journals (Sweden)

    María Martínez-López

    2018-05-01

    Full Text Available Protozoan parasites of the Leishmania genus are the causative agents of leishmaniasis, a group of neglected tropical diseases whose clinical manifestations vary depending on the infectious Leishmania species but also on host factors. Recognition of the parasite by host myeloid immune cells is a key to trigger an effective Leishmania-specific immunity. However, the parasite is able to persist in host myeloid cells by evading, delaying and manipulating host immunity in order to escape host resistance and ensure its transmission. Neutrophils are first in infiltrating infection sites and could act either favoring or protecting against infection, depending on factors such as the genetic background of the host or the parasite species. Macrophages are the main host cells where the parasites grow and divide. However, macrophages are also the main effector population involved in parasite clearance. Parasite elimination by macrophages requires the priming and development of an effector Th1 adaptive immunity driven by specific subtypes of dendritic cells. Herein, we will provide a comprehensive outline of how myeloid cells regulate innate and adaptive immunity against Leishmania, and the mechanisms used by the parasites to promote their evasion and sabotage. Understanding the interactions between Leishmania and the host myeloid cells may lead to the development of new therapeutic approaches and improved vaccination to leishmaniases, an important worldwide health problem in which current therapeutic or preventive approaches are limited.

  1. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  2. Peptone-yeast autolysate-fetal bovine serum 10, a simple, inexpensive liquid medium for cultivation of Leishmania spp.

    OpenAIRE

    Palomino, J C

    1982-01-01

    A simple liquid medium for the cultivation of Leishmania parasites is described. Leishmania brasiliensis and Leishmania peruviana cultured in this medium reached cell densities greater than 10(7) promastigotes per ml within 7 days. This medium compares very favorably with the more complex media used to cultivate Leishmania spp. and other hemoflagellates.

  3. Mitochondrial uncoupling proteins in unicellular eukaryotes.

    Science.gov (United States)

    Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

    2010-01-01

    Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

  4. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

    Science.gov (United States)

    Raymond, Frédéric; Boisvert, Sébastien; Roy, Gaétan; Ritt, Jean-François; Légaré, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques

    2012-01-01

    The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage. PMID:21998295

  5. Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

    Science.gov (United States)

    Hajjaran, Homa; Mahdi, Maryam; Mohebali, Mehdi; Samimi-Rad, Katayoun; Ataei-Pirkooh, Angila; Kazemi-Rad, Elham; Naddaf, Saied Reza; Raoofian, Reza

    2016-12-01

    Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

  6. Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador

    Science.gov (United States)

    Cevallos, Varsovia; Morales, Diego; Baldeón, Manuel E; Cárdenas, Paúl; Rojas-Silva, Patricio; Ponce, Patricio

    2017-01-01

    Abstract The detection and identification of natural infections in sand flies by Leishmania protozoan species in endemic areas is a key factor in assessing the risk of leishmaniasis and in designing prevention and control measures for this infectious disease. In this study, we analyzed the Leishmania DNA using nuclear ribosomal internal transcript spacer (ITS) sequences. Parasite DNA was extracted from naturally infected, blood-fed sand flies collected in nine localities considered leishmaniasis-endemic foci in Ecuador. The species of parasites identified in sand flies were Leishmania major-like, Leishmania naiffi, Leishmania mexicana, Leishmania lainsoni, and “Leishmania sp. siamensis”. Sand fly specimens of Brumptomyia leopoldoi, Mycropigomyia cayennensis, Nyssomyia yuilli yuilli, Nyssomyia trapidoi, Pressatia triacantha, Pressatia dysponeta, Psychodopygus carrerai carrerai, Psychodopygus panamensis, and Trichophoromyia ubiquitalis were found positive for Leishmania parasite. These findings contribute to a better understanding of the epidemiology and transmission dynamics of the disease in high-risk areas of Ecuador. PMID:28981860

  7. Involvement of Leishmania donovani major surface glycoprotein ...

    Indian Academy of Sciences (India)

    The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were ...

  8. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples

    NARCIS (Netherlands)

    Adams, Emily R.; Schoone, Gerard J.; Ageed, Al Farazdag; El Safi, Sayda; Schallig, Henk D. F. H.

    2010-01-01

    Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved

  9. Leishmania hijacking of the macrophage intracellular compartments.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Loiseau, Philippe M

    2016-02-01

    Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

  10. Trophic strategies of unicellular plankton

    DEFF Research Database (Denmark)

    Chakraborty, Subhendu; Nielsen, Lasse Tor; Andersen, Ken Haste

    2017-01-01

    . To this end, we develop and calibrate a trait-based model for unicellular planktonic organisms characterized by four traits: cell size and investments in phototrophy, nutrient uptake, and phagotrophy. We use the model to predict how optimal trophic strategies depend on cell size under various environmental...... unicellulars are colimited by organic carbon and nutrients, and only large photoautotrophs and smaller mixotrophs are nutrient limited; (2) trophic strategy is bottom-up selected by the environment, while optimal size is top-down selected by predation. The focus on cell size and trophic strategies facilitates......Unicellular plankton employ trophic strategies ranging from pure photoautotrophs over mixotrophy to obligate heterotrophs (phagotrophs), with cell sizes from 10-8 to 1 μg C. A full understanding of how trophic strategy and cell size depend on resource environment and predation is lacking...

  11. Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

    Science.gov (United States)

    Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

    2017-08-01

    Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

  12. Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation Inibição do crescimento de Leishmania mexicana mexicana por Leishmania mexicana amazonensis durante o co-cultivo "in vitro"

    Directory of Open Access Journals (Sweden)

    Raquel S. Pacheco

    1987-12-01

    Full Text Available Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.Inhibição do crescimento de um subespécie de Leishmania por exometabólitos de outra subespécie, um fenômeno ainda não notificado, é sugerido em nossas recentes observações em experimentos de clonagem celular com Leishmania mexicana mexicana e Leishmania mexicana amazonensis. Os clones foram identificados usando a técnica de análise de esquizodemas. O fenômeno observado é claramente relevante em estudos de isolamento parasitário, metabolismo, imunidade cruzada e quimioterapia.

  13. The first suicides: a legacy inherited by parasitic protozoans from prokaryote ancestors.

    Science.gov (United States)

    Taylor-Brown, Emilie; Hurd, Hilary

    2013-04-18

    It is more than 25 years since the first report that a protozoan parasite could die by a process resulting in a morphological phenotype akin to apoptosis. Since then these phenotypes have been observed in many unicellular parasites, including trypanosomatids and apicomplexans, and experimental evidence concerning the molecular pathways that are involved is growing. These observations support the view that this form of programmed cell death is an ancient one that predates the evolution of multicellularity. Here we review various hypotheses that attempt to explain the origin of apoptosis, and look for support for these hypotheses amongst the parasitic protists as, with the exception of yeast, most of the work on death mechanisms in unicellular organisms has focussed on them. We examine the role that addiction modules may have played in the original eukaryote cell and the part played by mitochondria in the execution of present day cells, looking for examples from Leishmania spp. Trypanosoma spp. and Plasmodium spp. In addition, the expanding knowledge of proteases, nucleases and other molecules acting in protist execution pathways has enabled comparisons to be made with extant Archaea and bacteria and with biochemical pathways that evolved in metazoans. These comparisons lend support to the original sin hypothesis but also suggest that present-day death pathways may have had multifaceted beginnings.

  14. Human cutaneous leishmaniasis caused by Leishmania (Viannia braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes Leishmaniose cutânea humana causada por Leishmania (Viannia braziliensis na Província de Santiago del Estero, Argentina: identificação dos parasitas por anticorpos monoclonais e isoenzimas

    Directory of Open Access Journals (Sweden)

    C.A. Cuba Cuba

    1996-12-01

    Full Text Available Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30 did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia braziliensis marker disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.Estudos de diagnóstico, caracterização parasitária e identificação foram conduzidos em

  15. Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus.

    Science.gov (United States)

    Coughlan, Simone; Taylor, Ali Shirley; Feane, Eoghan; Sanders, Mandy; Schonian, Gabriele; Cotton, James A; Downing, Tim

    2018-04-01

    The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. ( Viannia ) braziliensis and L. ( V. ) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. ( V. ) naiffi and L. ( V. ) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia : aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia , there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni , L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3' end of chromosome 34 . This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

  16. Seroprevalence rates of antibodies against Leishmania infantum and other protozoan and rickettsial parasites in dogs Soroprevalência de anticorpos contra Leishmania infantum e outras espécies de protozoários e rickettsia em cães

    Directory of Open Access Journals (Sweden)

    Silvana de Cássia Paulan

    2013-03-01

    Full Text Available Canine visceral leishmaniasis (CVL is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT to analyze L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii and Neospora caninum infection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.Leishmaniose Visceral Canina (LVC é causada pelo protozoário Leishmania infantum, podendo infectar cães e humanos em várias regiões do Brasil. O presente estudo teve por objetivo realizar a reação de imunofluorescência indireta (RIFI para analisar os índices de infecção parasitária para L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii e Neospora caninum, em 93 amostras de soro de cães de um assentamento rural no município de Ilha Solteira, SP, Brasil. A taxa de soroprevalência de cães com anticorpos anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii e anti-N. caninum foi de 37,6%, 75,3%, 72%, 47,3% e 6

  17. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    NARCIS (Netherlands)

    Barrera, P.; Sulsen, V.P.; Lozano, E.; Rivera, M.; Beer, M.F.; Tonn, C.; Martino, V.S.; Sosa, M.A.

    2013-01-01

    Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on

  18. First evidence of Leishmania infection in European brown hare (Lepus europaeus) in Greece: GIS analysis and phylogenetic position within the Leishmania spp.

    Science.gov (United States)

    Tsokana, C N; Sokos, C; Giannakopoulos, A; Mamuris, Z; Birtsas, P; Papaspyropoulos, K; Valiakos, G; Spyrou, V; Lefkaditis, M; Chatzopoulos, D C; Kantere, M; Manolakou, K; Touloudi, A; Burriel, A Rodi; Ferroglio, E; Hadjichristodoulou, C; Billinis, C

    2016-01-01

    Although the existence of a sylvatic transmission cycle of Leishmania spp., independent from the domestic cycle, has been proposed, data are scarce on Leishmania infection in wild mammals in Greece. In this study, we aimed to investigate the presence of Leishmania infection in the European brown hare in Greece, to infer the phylogenetic position of the Leishmania parasites detected in hares in Greece, and to identify any possible correlation between Leishmania infection in hares with environmental parameters, using the geographical information system (GIS). Spleen samples from 166 hares were tested by internal transcribed spacer-1 (ITS-1)-nested PCR for the detection of Leishmania DNA. Phylogenetic analysis was performed on Leishmania sequences from hares in Greece in conjunction with Leishmania sequences from dogs in Greece and 46 Leishmania sequences retrieved from GenBank. The Leishmania DNA prevalence in hares was found to be 23.49 % (95 % confidence interval (CI) 17.27-30.69). The phylogenetic analysis confirmed that the Leishmania sequences from hares in Greece belong in the Leishmania donovani complex. The widespread Leishmania infection in hares should be taken into consideration because under specific circumstances, this species can act as a reservoir host. This study suggests that the role of wild animals, including hares, in the epidemiology of Leishmania spp. in Greece deserves further elucidation.

  19. A draft genome of the honey bee trypanosomatid parasite Crithidia mellificae.

    Directory of Open Access Journals (Sweden)

    Charles Runckel

    Full Text Available Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. Crithidia mellificae is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. C. mellificae is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (e.g., the bumble bee pathogen Crithidia bombi, as well as species that infect both invertebrate and vertebrate hosts including human pathogens (e.g.,Trypanosoma cruzi, T. brucei, and Leishmania spp.. To better characterize C. mellificae, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species Leishmania major. We estimate that C. mellificae encodes over 8,300 genes, the majority of which are orthologs of genes encoded by L. major and other Leishmania or Trypanosoma species. Genes unique to C. mellificae, including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of C. mellificae infection on honey bee health and provide insight into the evolution of this unique family.

  20. Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

    Science.gov (United States)

    Badirzadeh, A; Taheri, T; Abedi-Astaneh, F; Taslimi, Y; Abdossamadi, Z; Montakhab-Yeganeh, H; Aghashahi, M; Niyyati, M; Rafati, S

    2017-09-01

    Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL. © 2017 John Wiley & Sons Ltd.

  1. First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis.

    Science.gov (United States)

    Soares, Isabel R; Silva, Soraia O; Moreira, Filipe Moraghi; Prado, Luan Gavião; Fantini, Priscila; Maranhão, Renata de Pino Albuquerque; da Silva Filho, José Monteiro; Melo, Maria Norma; Palhares, Maristela S

    2013-11-08

    This study reports the first evidence of infection by Leishmania infantum in Equus caballus in Americas and the first mixed infection of L. infantum/Leishmania braziliensis on this mammalian species in the world. The diagnoses was based on presence of parasites in lesions and bone marrow aspirates, their identification by using specific primers for L. infantum and L. braziliensis complexes and also serological methods IFAT and ELISA. The analysis of the PCR products suggested mixed infection in three animals. Further studies involving equine leishmaniasis are carrying out in order to clarify the dynamic of Leishmania sp. in this mammalian specie and their role in the transmission of those parasites in urban endemic area of Belo Horizonte, Minas Gerais State, Brazil. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Acuña, Stephanie Maia; Aoki, Juliana Ide; Laranjeira-Silva, Maria Fernanda; Zampieri, Ricardo Andrade; Fernandes, Juliane Cristina Ribeiro; Muxel, Sandra Marcia; Floeter-Winter, Lucile Maria

    2017-01-01

    Arginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection. The transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount through the NOS activity inhibition. NO quantification was measured by DAF-FM labeling analysis in a flow cytometry. We described an arginase-dependent NOS-like activity in L. (L.) amazonensis and its role in the parasite growth. The increased detection of NO production in the mid-stationary and late-stationary growth phases of La-WT promastigotes could suggest that this production is an important factor to metacyclogenesis triggering. On the other hand, La-arg- showed an earlier increase in NO production compared to La-WT, suggesting that NO production can be arginase-dependent. Interestingly, La-WT and La-arg- axenic amastigotes produced higher levels of NO than those observed in promastigotes. As a conclusion, our work suggested that NOS-like is expressed in Leishmania in the stationary growth phase promastigotes and amastigotes, and could be correlated to metacyclogenesis and amastigotes growth in a dependent way to the internal pool of L-arginine and arginase activity.

  3. Progress towards a Leishmania vaccine.

    Science.gov (United States)

    Tabbara, Khaled S

    2006-07-01

    Leishmaniasis is a vector-born protozoan disease. Approximately 12 million individuals are affected worldwide with an estimated annual incidence of 1.5-2 million. Two clinical manifestations are recognized, cutaneous, and visceral, both of which are common in the Middle East. In both forms, infection is chronic, with potential deformities, persistence following cure, and lifelong risk of reactivation. Attempts to develop an effective human Leishmania vaccine have not yet succeeded. Leishmanization, a crude form of live vaccination historically originated in this part of the world. Experimental vaccination has been extensively studied in model animals in the past 2 decades. In this review, major human killed vaccine trials are surveyed, and modern trends in Leishmania vaccine development, including subunit vaccines, naked DNA vaccines, and transmission blocking vaccines are explored. Recent findings of a link between persistence of live parasites, and maintenance of long-term immunity suggest live vaccination with attenuated strains, as a future vaccination strategy.

  4. Serpins in unicellular Eukarya, Archaea, and Bacteria:

    DEFF Research Database (Denmark)

    Roberts, T.H.; Hejgaard, Jørn; Saunders, N.F.W

    2004-01-01

    , where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics...

  5. Leishmania infantum and Leishmania braziliensis: differences and similarities to evade the innate immune system

    Directory of Open Access Journals (Sweden)

    Sarah Athayde Couto Falcão

    2016-08-01

    Full Text Available Visceral Leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human dendritic cells biology to better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood’s healthy volunteers donors and infected with L. infantum or L. braziliensis for 24 hours. We observed similar rates of infection (around 40% as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of dendritic cells after 24 hours. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model.

  6. The current status of phlebotomine sandflies (Diptera: Psychodidae) in Tunisia and their role on Leishmania transmission: A review

    OpenAIRE

    Ahmed Tabbabi; Sajida Sboui; Jabeur Daaboub

    2017-01-01

    In Tunisia, the epidemiological situation of leishmaniasis is characterized by the coexistence in a rather circumscribed territory (165000 km2, including the Sahara) of 4 forms of leishmaniasis caused by 3 species: Leishmania infantum, Leishmania major and Leishmania tropica (L. tropica) (synonymous Leishmania killicki). One of the factors determining the clinical, epidemiological and immunological diversity of leishmanioses is certainly the existence of a vector-parasite specificity of of...

  7. Phylogenomic reconstruction supports supercontinent origins for Leishmania.

    Science.gov (United States)

    Harkins, Kelly M; Schwartz, Rachel S; Cartwright, Reed A; Stone, Anne C

    2016-03-01

    Leishmania, a genus of parasites transmitted to human hosts and mammalian/reptilian reservoirs by an insect vector, is the causative agent of the human disease complex leishmaniasis. The evolutionary relationships within the genus Leishmania and its origins are the source of ongoing debate, reflected in conflicting phylogenetic and biogeographic reconstructions. This study employs a recently described bioinformatics method, SISRS, to identify over 200,000 informative sites across the genome from newly sequenced and publicly available Leishmania data. This dataset is used to reconstruct the evolutionary relationships of this genus. Additionally, we constructed a large multi-gene dataset, using it to reconstruct the phylogeny and estimate divergence dates for species. We conclude that the genus Leishmania evolved at least 90-100 million years ago, supporting a modified version of the Multiple Origins hypothesis that we call the Supercontinent hypothesis. According to this scenario, separate Leishmania clades emerged prior to, and during, the breakup of Gondwana. Additionally, we confirm that reptile-infecting Leishmania are derived from mammalian forms and that the species that infect porcupines and sloths form a clade long separated from other species. Finally, we firmly place the guinea-pig infecting species, Leishmaniaenriettii, the globally dispersed Leishmaniasiamensis, and the newly identified Australian species from a kangaroo, as sibling species whose distribution arises from the ancient connection between Australia, Antarctica, and South America. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Sand fly-Leishmania interactions: long relationships are not necessarily easy

    OpenAIRE

    Ramalho-Ortigao, Marcelo; Saraiva, Elvira M.; Traub-Csekö, Yara M.

    2010-01-01

    Sand fly and Leishmania are one of the best studied vector-parasite models. Much is known about the development of these parasites within the sand fly, and how transmission to a suitable vertebrate host takes place. Various molecules secreted by the vector assist the establishment of the infection in a vertebrate, and changes to the vector are promoted by the parasites in order to facilitate or enhance transmission. Despite a generally accepted view that sand flies and Leishmania are also one...

  9. Distinctive cellular immunity in genetically susceptible BALB/c mice recovered from Leishmania major infection or after subcutaneous immunization with killed parasites

    International Nuclear Information System (INIS)

    Liew, F.Y.; Dhaliwal, J.S.

    1987-01-01

    Genetically susceptible BALB/c mice are refractory to further infection after recovery from Leishmania major infection after a sublethal dose of gamma-irradiation. In contrast, mice immunized with killed promastigotes s.c. develop exacerbated lesions after infection. Both groups of mice produce only a low level of specific antibody and no detectable cytotoxic T cells, but do have a strong antigen-specific DTH, which is adoptively transferable with Lyt-1+2-, L3T4+ T cells. Kinetic and histological studies revealed that mice immunized s.c. developed Jones-Mote hypersensitivity, peaking at 15 hr. with little mononuclear cell infiltration at the site of antigen administration; whereas mice that had recovered from infection developed tuberculin-type of reactivity, peaking at 24 to 48 hr, with intense mononuclear cell infiltration. Splenic T cells from recovered mice, when injected into the footpads of normal recipients together with live promastigotes, were able to retard lesion development; whereas T cells from s.c. immunized mice, when similarly transferred, accelerated disease progression. Antigen-specific culture supernatant of spleen cells from recovered mice also activated normal resident peritoneal macrophages to kill intracellular L. major amastigotes and tumor cells. Culture supernatants of spleen cells from s.c. immunized or normal mice were devoid of such activities. Part of the macrophage-activating potential can be inhibited by antibody specific for IFN-gamma. These results therefore demonstrate that whereas the Jones-Mote reaction is correlated with disease exacerbation, the tuberculin-type of DTH may be protective. Furthermore, in vivo immunity is directly related to the capacity of T cells to produce macrophage-activating factor

  10. Natural infection of bats with Leishmania in Ethiopia.

    Science.gov (United States)

    Kassahun, Aysheshm; Sadlova, Jovana; Benda, Petr; Kostalova, Tatiana; Warburg, Alon; Hailu, Asrat; Baneth, Gad; Volf, Petr; Votypka, Jan

    2015-10-01

    The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador.

    Science.gov (United States)

    Kato, Hirotomo; Gomez, Eduardo A; Yamamoto, Yu-ichi; Calvopiña, Manuel; Guevara, Angel G; Marco, Jorge D; Barroso, Paola A; Iwata, Hiroyuki; Hashiguchi, Yoshihisa

    2008-09-01

    Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.

  12. TRANSCRIPTIONAL INHIBITION OF INTERLEUKIN-12 PROMOTER ACTIVITY IN LEISHMANIA SPP.-INFECTED MACROPHAGES

    Science.gov (United States)

    Jayakumar, Asha; Widenmaier, Robyn; Ma, Xiaojing; McDowell, Mary Ann

    2009-01-01

    To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12. PMID:18372625

  13. Bystander effects in unicellular organisms

    International Nuclear Information System (INIS)

    DeVeaux, Linda C.; Durtschi, Lynn S.; Case, Jonathan G.; Wells, Douglas P.

    2006-01-01

    Radiation-induced bystander effects have been seen in mammalian cells from diverse origins. These effects can be transmitted through the medium to cells not present at the time of irradiation. We have developed an assay for detecting bystander effects in the unicellular eukaryote, the fission yeast Schizosaccharomyces pombe. This assay allows maximal exposure of unirradiated cells to cells that have received electron beam irradiation. S. pombe cells were irradiated with 16-18 MeV electrons from a pulsed electron LINAC. When survival of the irradiated cells decreased to approximately 50%, forward-mutation to 2-deoxy-D-glucose resistance increased in the unirradiated bystander cells. Further increase in dose had no additional effect on this increase. In order to detect this response, it was necessary for the irradiated cell/unirradiated cell ratio to be high. Other cellular stresses, such as heat treatment, UV irradiation, and bleomycin exposure, also caused a detectable response in untreated cells grown with the treated cells. We discuss evolutionary implications of these results

  14. Bystander effects in unicellular organisms

    Energy Technology Data Exchange (ETDEWEB)

    DeVeaux, Linda C. [Idaho Accelerator Center, Campus Box 8263, Idaho State University, Pocatello, ID 83209 (United States) and Department of Biological Sciences, Campus Box 8007, Idaho State University, Pocatello, ID 83209 (United States)]. E-mail: develind@isu.edu; Durtschi, Lynn S. [Department of Biological Sciences, Campus Box 8007, Idaho State University, Pocatello, ID 83209 (United States); Case, Jonathan G. [Department of Physics, Campus Box 8106, Idaho State University, Pocatello, ID 83209 (United States); Wells, Douglas P. [Department of Physics, Campus Box 8106, Idaho State University, Pocatello, ID 83209 (United States)

    2006-05-11

    Radiation-induced bystander effects have been seen in mammalian cells from diverse origins. These effects can be transmitted through the medium to cells not present at the time of irradiation. We have developed an assay for detecting bystander effects in the unicellular eukaryote, the fission yeast Schizosaccharomyces pombe. This assay allows maximal exposure of unirradiated cells to cells that have received electron beam irradiation. S. pombe cells were irradiated with 16-18 MeV electrons from a pulsed electron LINAC. When survival of the irradiated cells decreased to approximately 50%, forward-mutation to 2-deoxy-D-glucose resistance increased in the unirradiated bystander cells. Further increase in dose had no additional effect on this increase. In order to detect this response, it was necessary for the irradiated cell/unirradiated cell ratio to be high. Other cellular stresses, such as heat treatment, UV irradiation, and bleomycin exposure, also caused a detectable response in untreated cells grown with the treated cells. We discuss evolutionary implications of these results.

  15. Bystander effects in unicellular organisms.

    Science.gov (United States)

    DeVeaux, Linda C; Durtschi, Lynn S; Case, Jonathan G; Wells, Douglas P

    2006-05-11

    Radiation-induced bystander effects have been seen in mammalian cells from diverse origins. These effects can be transmitted through the medium to cells not present at the time of irradiation. We have developed an assay for detecting bystander effects in the unicellular eukaryote, the fission yeast Schizosaccharomyces pombe. This assay allows maximal exposure of unirradiated cells to cells that have received electron beam irradiation. S. pombe cells were irradiated with 16-18 MeV electrons from a pulsed electron LINAC. When survival of the irradiated cells decreased to approximately 50%, forward-mutation to 2-deoxy-d-glucose resistance increased in the unirradiated bystander cells. Further increase in dose had no additional effect on this increase. In order to detect this response, it was necessary for the irradiated cell/unirradiated cell ratio to be high. Other cellular stresses, such as heat treatment, UV irradiation, and bleomycin exposure, also caused a detectable response in untreated cells grown with the treated cells. We discuss evolutionary implications of these results.

  16. Understanding serine proteases implications on Leishmania spp lifecycle.

    Science.gov (United States)

    Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

    2018-01-01

    Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes.

    Science.gov (United States)

    Petri e Silva, Simone Carolina Soares; Palace-Berl, Fanny; Tavares, Leoberto Costa; Soares, Sandra Regina Castro; Lindoso, José Angelo Lauletta

    2016-04-01

    Leishmaniasis is an overlooked tropical disease affecting approximately 1 million people in several countries. Clinical manifestation depends on the interaction between Leishmania and the host's immune response. Currently available treatment options for leishmaniasis are limited and induce severe side effects. In this research, we tested nitro-heterocyclic compounds (BSF series) as a new alternative against Leishmania. Its activity was measured in Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes using MTT colorimetric assay. Additionally, we assessed the phosphatidylserine exposure by promastigotes, measured by flow cytometry, as well as nitric oxide production, measured by Griess' method. The nitro-heterocyclic compounds (BSF series) showed activity against L. (L.) infantum promastigotes, inducting the phosphatidylserine exposition by promastigotes, decreasing intracellular amastigotes and increasing oxide nitric production. The selectivity index was more prominent to Leishmania than to macrophages. Compared to amphotericin b, our compounds presented higher IC50, however the selectivity index was more specific to parasite than to amphotericin b. In conclusion, these nitro-heterocyclic compounds showed to be promising as an anti-Leishmania drug, in in vitro studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Molecular crosstalks in Leishmania-sandfly-host relationships

    Directory of Open Access Journals (Sweden)

    Volf P.

    2008-09-01

    Full Text Available Sandflies (Diptera: Phlebotominae are vectors of Leishmania parasites, causative agents of important human and animal diseases with diverse manifestations. This review summarizes present knowledge about the vectorial part of Leishmania life cycle and parasite transmission to the vertebrate host. Particularly, it focuses on molecules that determine the establishment of parasite infection in sandfly midgut. It describes the concept of specific versus permissive sandfly vectors, explains the epidemiological consequences of broad susceptibility of permissive sandflies and demonstrates that genetic exchange may positively affect Leishmania fitness in the vector. Last but not least, the review describes recent knowledge about circulating antibodies produced by hosts in response to sandfly bites. Studies on specificity and kinetics of antibody response revealed that anti-saliva IgG could be used as a marker of host exposure to sandflies, i.e. as a useful tool for evaluation of vector control.

  19. Cell migration induced by Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis into the peritoneal cavity of BALB/c mice

    OpenAIRE

    DT Wakimoto; KV Gaspareto; TGV Silveira; MVC Lonardoni; SMA Aristides

    2010-01-01

    In American cutaneous leishmaniasis, the initial infection phase is characterized by recruitment of neutrophils and monocytes. The migration of these cells in response to the presence of Leishmania in the peritoneum of affected animals remains unclear. The objective of this study was to investigate cell migration to the peritoneum of BALB/c mice after infection with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) major. Initially, Leishmania ...

  20. Molecular detection of Leishmania infection due to Leishmania major and Leishmania turanica in the vectors and reservoir host in Iran.

    Science.gov (United States)

    Rassi, Yavar; Oshaghi, Mohammad Ali; Azani, Sadegh Mohammadi; Abaie, Mohammad Reza; Rafizadeh, Sina; Mohebai, Mehdi; Mohtarami, Fatemeh; Zeinali, Mohammad kazem

    2011-02-01

    An epidemiological study was carried out on the vectors and reservoirs of cutaneous leishmaniasis in rural areas of Damghan district, Semnan province, central Iran, during 2008-2009. Totally, 6110 sand flies were collected using sticky papers and were subjected to molecular methods for detection of Leishmania parasite. Phlebotomus papatasi Scopoli was the common species in outdoor and indoor resting places. Polymerase chain reaction technique showed that 24 out of 218 P. papatasi (11%) and 4 out of 62 Phlebotomus caucasicus Marzinovskyi (6.5%) were positive for parasites Leishmania major Yakimoff and Schokhor. Twenty-one rodent reservoir hosts captured using Sherman traps were identified as Rhombomys opimus Lichtenstein (95%) and Meriones libycus Lichtenstein (5%). Microscopic investigation on blood smear of the animals for amastigote parasites revealed 8 (40%) rodents infected with R. opimus. L. major infection in these animals was then confirmed by polymerase chain reaction against internal transcribed spacer ribosomal DNA (rDNA) loci of the parasite followed by restriction fragment length polymorphism. Further, sequence analysis of 297 bp of ITS1-rDNA loci revealed the presence of L. major and Leishmania turanica in P. papatasi, and L. major in R. opimus. This is the first molecular report of L. major infection in both vectors (P. papatasi and P. caucasicus) and reservoir host (R. opimus) in this region. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and P. caucasicus could be considered as a secondary vector. Further, our study showed that R. opimus is the most important host reservoir for maintenance of the parasite source in the area.

  1. LR1: a candidate RNA virus of Leishmania.

    OpenAIRE

    Tarr, P I; Aline, R F; Smiley, B L; Scholler, J; Keithly, J; Stuart, K

    1988-01-01

    Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately ...

  2. Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador.

    Science.gov (United States)

    Quiroga, Cristina; Cevallos, Varsovia; Morales, Diego; Baldeón, Manuel E; Cárdenas, Paúl; Rojas-Silva, Patricio; Ponce, Patricio

    2017-11-07

    The detection and identification of natural infections in sand flies by Leishmania protozoan species in endemic areas is a key factor in assessing the risk of leishmaniasis and in designing prevention and control measures for this infectious disease. In this study, we analyzed the Leishmania DNA using nuclear ribosomal internal transcript spacer (ITS) sequences. Parasite DNA was extracted from naturally infected, blood-fed sand flies collected in nine localities considered leishmaniasis-endemic foci in Ecuador.The species of parasites identified in sand flies were Leishmania major-like, Leishmania naiffi, Leishmania mexicana, Leishmania lainsoni, and "Leishmania sp. siamensis". Sand fly specimens of Brumptomyia leopoldoi, Mycropigomyia cayennensis, Nyssomyia yuilli yuilli, Nyssomyia trapidoi, Pressatia triacantha, Pressatia dysponeta, Psychodopygus carrerai carrerai, Psychodopygus panamensis, and Trichophoromyia ubiquitalis were found positive for Leishmania parasite. These findings contribute to a better understanding of the epidemiology and transmission dynamics of the disease in high-risk areas of Ecuador. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  3. Dihydrotestosterone enhances growth and infectivity of Leishmania Mexicana.

    Science.gov (United States)

    Sánchez-García, L; Wilkins-Rodriguez, A; Salaiza-Suazo, N; Morales-Montor, J; Becker, I

    2018-03-01

    A strong sex-associated susceptibility towards Leishmania has been reported in males, yet little is known on the effect of hormones in Leishmania physiopathogenicity. Due to the enhanced susceptibility of males to Leishmania mexicana infections, we were interested in analysing the effect exerted by the main androgen produced in males (DHT) on L. mexicana promastigotes. Thus, the aim of this study was to assess the regulation exerted by dihydrotestosterone (DHT) on L. mexicana replication, infectivity, survival and development of tissue lesions. Experiments included growth curves of L. mexicana promastigotes incubated with different doses of DHT, their infection rate, intracellular survival and lesion development in BALB/c mice. Our data show that DHT significantly enhances parasite replication, infection rate and survival in bone marrow-derived macrophages (BMMФ). Promastigotes in the presence of DHT produced significantly larger lesions in BALB/c earlobes. These results suggest that DHT probably plays a critical role during L. mexicana infections, and the higher susceptibility of males possibly relates to benefits gained by the parasite from host-derived hormones. Our data shed new light on the physiopathology of Leishmania infections and are the first attempt to understand the direct interaction between Leishmania and androgens, particularly DHT. Understanding this trans-regulation process employed by parasites to exploit host molecules sheds new light on L. mexicana physiopathogenesis and opens a possible field for studies on drug development. © 2017 John Wiley & Sons Ltd.

  4. Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania

    DEFF Research Database (Denmark)

    Chen, M; Christensen, S B; Blom, J

    1993-01-01

    Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies...... killing of the parasite. These data show that intracellular Leishmania amastigotes are more susceptible than promastigotes to licochalcone A. Results of studies on the site of action of licochalcone A indicate that the target organelle appears to be the parasite mitochondria. These findings demonstrate...

  5. Leishmania and its quest for iron: An update and overview.

    Science.gov (United States)

    Zaidi, Amir; Singh, Krishn Pratap; Ali, Vahab

    2017-01-01

    Parasites of genus Leishmania are the causative agents of complex neglected diseases called leishmaniasis and continue to be a significant health concern globally. Iron is a vital nutritional requirement for virtually all organisms, including pathogenic trypanosomatid parasites, and plays a crucial role in many facets of cellular metabolism as a cofactor of several enzymes. Iron acquisition is essential for the survival of parasites. Yet parasites are also vulnerable to the toxicity of iron and reactive oxygen species. The aim of this review is to provide an update on the current knowledge about iron acquisition and usage by Leishmania species. We have also discussed about host strategy to modulate iron availability and the strategies deployed by Leishmania parasites to overcome iron withholding defences and thus favour parasite growth within host macrophages. Since iron plays central roles in the host's response and parasite metabolism, a comprehensive understanding of the iron metabolism is beneficial to identify potential viable therapeutic opportunities against leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Arginine and Polyamines Fate in Leishmania Infection

    Science.gov (United States)

    Muxel, Sandra M.; Aoki, Juliana I.; Fernandes, Juliane C. R.; Laranjeira-Silva, Maria F.; Zampieri, Ricardo A.; Acuña, Stephanie M.; Müller, Karl E.; Vanderlinde, Rubia H.; Floeter-Winter, Lucile M.

    2018-01-01

    Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg-). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection. PMID:29379478

  7. Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.

    Science.gov (United States)

    Laurenti, Márcia Dalastra; Rossi, Claudio Nazaretian; da Matta, Vânia Lúcia Ribeiro; Tomokane, Thaise Yumie; Corbett, Carlos Eduardo Pereira; Secundino, Nágila Francinete Costa; Pimenta, Paulo Filemon Paulocci; Marcondes, Mary

    2013-09-23

    We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody

  8. Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania.

    Science.gov (United States)

    Chan, Marion Man-Ying; Adapala, Naga Suresh; Fong, Dunne

    2005-04-01

    Upon Leishmania infection, macrophages are activated to produce nitrogen and oxygen radicals simultaneously. It is well established that the infected host cells rely on nitric oxide (NO) as the major weapon against the intracellular parasite. In India where leishmaniasis is endemic, the spice turmeric is used prolifically in food and for insect bites. Curcumin, the active principle of turmeric, is a scavenger of NO. This report shows that curcumin protects promastigotes and amastigotes of the visceral species, Leishmania donovani, and promastigotes of the cutaneous species, L. major, against the actions of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETANONOate, which release NO, 3-morpholino-sydnonimine hydrochloride (SIN-1), which releases NO and superoxide, and peroxynitrite, which is formed from the reaction of NO with superoxide. Thus, curcumin, as an antioxidant, is capable of blocking the action of both NO and NO congeners on the Leishmania parasite.

  9. Generation of species-specific DNA probes for Leishmania aethiopica

    NARCIS (Netherlands)

    Laskay, T.; Kiessling, R.; Rinke deWit, T. F.; Wirth, D. F.

    1991-01-01

    We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within

  10. Recognition of Leishmania antigens by T lymphocytes from nonexposed individuals

    DEFF Research Database (Denmark)

    Kemp, M; Hansen, M B; Theander, T G

    1992-01-01

    Crude antigen preparations of Leishmania promastigote sonicates were found to induce in vitro proliferation and gamma interferon production in peripheral blood mononuclear cells (PBMC) from individuals without known exposure to the parasite. The proliferating cells were mainly CD2-positive T cell...

  11. Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging

    Science.gov (United States)

    Fatoux-Ardore, Marie; Peysselon, Franck; Weiss, Anthony; Bastien, Patrick; Pratlong, Francine

    2014-01-01

    We have set up an assay to study the interactions of live pathogens with their hosts by using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have used this assay to characterize the interactions of Leishmania promastigotes with ∼70 mammalian host biomolecules (extracellular proteins, glycosaminoglycans, growth factors, cell surface receptors). We have identified, in total, 27 new partners (23 proteins, 4 glycosaminoglycans) of procyclic promastigotes of six Leishmania species and 18 partners (15 proteins, 3 glycosaminoglycans) of three species of stationary-phase promastigotes for all the strains tested. The diversity of the interaction repertoires of Leishmania parasites reflects their dynamic and complex interplay with their mammalian hosts, which depends mostly on the species and strains of Leishmania. Stationary-phase Leishmania parasites target extracellular matrix proteins and glycosaminoglycans, which are highly connected in the extracellular interaction network. Heparin and heparan sulfate bind to most Leishmania strains tested, and 6-O-sulfate groups play a crucial role in these interactions. Numerous Leishmania strains bind to tropoelastin, and some strains are even able to degrade it. Several strains interact with collagen VI, which is expressed by macrophages. Most Leishmania promastigotes interact with several regulators of angiogenesis, including antiangiogenic factors (endostatin, anastellin) and proangiogenic factors (ECM-1, VEGF, and TEM8 [also known as anthrax toxin receptor 1]), which are regulated by hypoxia. Since hypoxia modulates the infection of macrophages by the parasites, these interactions might influence the infection of host cells by Leishmania. PMID:24478075

  12. A atividade da azitromicina contra a Leishmania (Viannia) braziliensis e a Leishmania (Leishmania) amazonensis no modelo golden hamster

    OpenAIRE

    Sinagra, Ángel; Luna, Concepción; Abraham, David; Iannella, Maria del Carmen; Riarte, Adelina; Krolewiecki, Alejandro J.

    2007-01-01

    New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania) major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with unt...

  13. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the golden hamster model

    OpenAIRE

    Sinagra,Ángel; Luna,Concepción; Abraham,David; Iannella,Maria del Carmen; Riarte,Adelina; Krolewiecki,Alejandro J.

    2007-01-01

    New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania) major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with unt...

  14. Leishmania chagasi/infantum : further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

    NARCIS (Netherlands)

    Campos Ponce, M.; Ponce, C.; Ponce, E; Maingon, R.D.

    2005-01-01

    In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran

  15. Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America.

    NARCIS (Netherlands)

    Campos Ponce, M.; Ponce, C.; Ponce, E.; Maingon, R.D.

    2005-01-01

    In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran

  16. Detection and characterization of Leishmania (Leishmania and Leishmania (Viannia by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

    Directory of Open Access Journals (Sweden)

    Marcello Ceccarelli

    Full Text Available Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1, whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2. The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania and Leishmania (Viannia using the qPCR2 assay followed by melting or High Resolution Melt (HRM analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania and Leishmania (Viannia subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L. infantum WHO international reference strain (MHOM/TN/80/IPT1, highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical

  17. Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

    Directory of Open Access Journals (Sweden)

    B Bastrenta

    2003-03-01

    Full Text Available Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39 were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6% presented a mixed infection Leishmania complex species, 17 (58.6% a mixed infection Leishmania-T. cruzi, and 4 (13.8% a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%. The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V. braziliensis, L. (L. chagasi and L. (L. mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V. braziliensis-L. (L. mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

  18. Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections.

    Science.gov (United States)

    Craig, Evan; Huyghues-Despointes, Charles-Eugene; Yu, Chun; Handy, Emma L; Sello, Jason K; Kima, Peter E

    2017-05-01

    In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.

  19. Leishmania (Viannia) guyanensis in tegumentary leishmaniasis.

    Science.gov (United States)

    Borges, Arissa Felipe; Gomes, Rodrigo Saar; Ribeiro-Dias, Fátima

    2018-06-01

    Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.

  20. Aging and immortality in unicellular species.

    Science.gov (United States)

    Florea, Michael

    2017-10-01

    It has been historically thought that in conditions that permit growth, most unicellular species do not to age. This was particularly thought to be the case for symmetrically dividing species, as such species lack a clear distinction between the soma and the germline. Despite this, studies of the symmetrically dividing species Escherichia coli and Schizosaccharomyces pombe have recently started to challenge this notion. They indicate that E. coli and S. pombe do age, but only when subjected to environmental stress. If true, this suggests that aging may be widespread among microbial species in general, and that studying aging in microbes may inform other long-standing questions in aging. This review examines the recent evidence for and against replicative aging in symmetrically dividing unicellular organisms, the mechanisms that underlie aging, why aging evolved in these species, and how microbial aging fits into the context of other questions in aging. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Macroecology of unicellular organisms - patterns and processes.

    Science.gov (United States)

    Soininen, Janne

    2012-02-01

    Macroecology examines the relationship between organisms and their environment at large spatial (and temporal) scales. Typically, macroecologists explain the large-scale patterns of abundance, distribution and diversity. Despite the difficulties in sampling and characterizing microbial diversity, macroecologists have recently also been interested in unicellular organisms. Here, I review the current advances made in microbial macroecology, as well as discuss related ecosystem functions. Overall, it seems that microorganisms suit surprisingly well to known species abundance distributions and show positive relationship between distribution and adundance. Microbial species-area and distance-decay relationships tend to be weaker than for macroorganisms, but nonetheless significant. Few findings on altitudinal gradients in unicellular taxa seem to differ greatly from corresponding findings for larger taxa, whereas latitudinal gradients among microorganisms have either been clearly evident or absent depending on the context. Literature also strongly emphasizes the role of spatial scale for the patterns of diversity and suggests that patterns are affected by species traits as well as ecosystem characteristics. Finally, I discuss the large role of local biotic and abiotic variables driving the community assembly in unicellular taxa and eventually dictating how multiple ecosystem processes are performed. Present review highlights the fact that most microorganisms may not differ fundamentally from larger taxa in their large-scale distribution patterns. Yet, review also shows that many aspects of microbial macroecology are still relatively poorly understood and specific patterns depend on focal taxa and ecosystem concerned. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  2. Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction.

    Science.gov (United States)

    Côrtes, Luzia Mc; Silva, Roger Mm; Pereira, Bernardo As; Guerra, Camila; Zapata, Angela C; Bello, Felio J; Finkelstein, Léa C; Madeira, Maria F; Brazil, Reginaldo P; Côrte-Real, Suzana; Alves, Carlos R

    2011-11-14

    Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

  3. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

    Directory of Open Access Journals (Sweden)

    Sameh Rabhi

    Full Text Available Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

  4. Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

    Directory of Open Access Journals (Sweden)

    Norris-Mullins Brianna

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

  5. The past, present, and future of Leishmania genomics and transcriptomics

    Science.gov (United States)

    Cantacessi, Cinzia; Dantas-Torres, Filipe; Nolan, Matthew J.; Otranto, Domenico

    2015-01-01

    It has been nearly 10 years since the completion of the first entire genome sequence of a Leishmania parasite. Genomic and transcriptomic analyses have advanced our understanding of the biology of Leishmania, and shed new light on the complex interactions occurring within the parasite–host–vector triangle. Here, we review these advances and examine potential avenues for translation of these discoveries into treatment and control programs. In addition, we argue for a strong need to explore how disease in dogs relates to that in humans, and how an improved understanding in line with the ‘One Health’ concept may open new avenues for the control of these devastating diseases. PMID:25638444

  6. The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

    Science.gov (United States)

    Real, Fernando; Vidal, Ramon Oliveira; Carazzolle, Marcelo Falsarella; Mondego, Jorge Maurício Costa; Costa, Gustavo Gilson Lacerda; Herai, Roberto Hirochi; Würtele, Martin; de Carvalho, Lucas Miguel; e Ferreira, Renata Carmona; Mortara, Renato Arruda; Barbiéri, Clara Lucia; Mieczkowski, Piotr; da Silveira, José Franco; Briones, Marcelo Ribeiro da Silva; Pereira, Gonçalo Amarante Guimarães; Bahia, Diana

    2013-01-01

    We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment. PMID:23857904

  7. Nitric oxide production by Peromyscus yucatanicus (Rodentia infected with Leishmania (Leishmania mexicana

    Directory of Open Access Journals (Sweden)

    Elsy Nalleli Loría-Cervera

    2013-04-01

    Full Text Available Peromyscus yucatanicus (Rodentia: Cricetidae is a primary reservoir of Leishmania (Leishmania mexicana (Kinetoplastida: Trypanosomatidae. Nitric oxide (NO generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L. mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001 in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L. mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

  8. Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani

    DEFF Research Database (Denmark)

    Chen, M; Christensen, S B; Theander, T G

    1994-01-01

    This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion...... development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen...... consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases....

  9. Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

    Science.gov (United States)

    Montalvo Alvarez, Ana Margarita; Nodarse, Jorge Fraga; Goodridge, Ivón Montano; Fidalgo, Lianet Monzote; Marin, Marcel; Van Der Auwera, Gert; Dujardin, Jean-Claude; Bernal, Iván Darío Velez; Muskus, Carlos

    2010-05-01

    Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

  10. Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology.

    Science.gov (United States)

    Baneth, Gad; Yasur-Landau, Daniel; Gilad, Matan; Nachum-Biala, Yaarit

    2017-03-13

    Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick. Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative. Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This

  11. Parasitic infections of the external eye.

    Science.gov (United States)

    Pahuja, Shivani; Puranik, Charuta; Jelliti, Bechir; Khairallah, Moncef; Sangwan, Virender S

    2013-08-01

    To review the published literature on parasitic infections of external eye. Published articles and case reports on parasitic infections of external eye were reviewed and relevant information was collected. Parasitic infections of the eye are rare. However, being more commonly seen in developing nations, they require active measures for screening, diagnosis, and therapy. Parasites of importance causing external ocular disease are protozoan parasites, such as Leishmania; metazoans, such as nematodes (roundworms), cestodes (tapeworms), and trematodes (flatworms); or ectoparasites, such as Phthirus pubis and Demodex.

  12. Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

    Directory of Open Access Journals (Sweden)

    Luiza de Oliveira Ramos Pereira

    2013-08-01

    Full Text Available Leishmania RNA virus (LRV has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ, no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V. guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V. brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

  13. Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae

    Directory of Open Access Journals (Sweden)

    MICHALSKY Érika M.

    2002-01-01

    Full Text Available DNA amplification by the polymerase chain reaction (PCR was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei.

  14. A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Kárita Cláudia Freitas-Lidani

    2014-07-01

    Full Text Available The aim of the present study was to detect natural infection by Leishmania (Leishmania infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA, the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

  15. The Gut Microbiome of the Vector Lutzomyia longipalpis Is Essential for Survival of Leishmania infantum.

    Science.gov (United States)

    Kelly, Patrick H; Bahr, Sarah M; Serafim, Tiago D; Ajami, Nadim J; Petrosino, Joseph F; Meneses, Claudio; Kirby, John R; Valenzuela, Jesus G; Kamhawi, Shaden; Wilson, Mary E

    2017-01-17

    The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission. Leishmania infantum, a parasitic protozoan causing fatal visceral leishmaniasis, is transmitted to humans through the bite of the sand fly Lutzomyia longipalpis Development of the parasite to its virulent metacyclic state occurs in the sand fly gut. In this study, the microbiota within the Lu. longipalpis midgut was delineated by 16S ribosomal DNA (rDNA) sequencing, revealing a highly diverse community composition that lost diversity as parasites developed to their metacyclic state and increased in abundance in infected flies. Perturbing sand fly gut microbiota with an antibiotic cocktail, which alone had no effect on either the parasite or the fly, arrested both the development of virulent parasites and parasite expansion

  16. The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

    Science.gov (United States)

    Di-Blasi, Tatiana; Lobo, Amanda R; Nascimento, Luanda M; Córdova-Rojas, Jose L; Pestana, Karen; Marín-Villa, Marcel; Tempone, Antonio J; Telleria, Erich L; Ramalho-Ortigão, Marcelo; McMahon-Pratt, Diane; Traub-Csekö, Yara M

    2015-03-01

    Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

  17. Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

    Science.gov (United States)

    Atteia, Ariane; van Lis, Robert; Tielens, Aloysius G M; Martin, William F

    2013-02-01

    Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Gluconeogenesis in Leishmania mexicana

    Science.gov (United States)

    Rodriguez-Contreras, Dayana; Hamilton, Nicklas

    2014-01-01

    Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. PMID:25288791

  19. Apoptosis in unicellular organisms: mechanisms and evolution.

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    Gordeeva, A V; Labas, Y A; Zvyagilskaya, R A

    2004-10-01

    Data about the programmed death (apoptosis) in unicellular organisms, from bacteria to ciliates, are discussed. Firstly apoptosis appeared in lower eukaryotes, but its mechanisms in these organisms are different from the classical apoptosis. During evolution, the apoptotic process has been improving gradually, with reactive oxygen species and Ca2+ playing an essential role in triggering apoptosis. All eukaryotic organisms have apoptosis inhibitors, which might be introduced by viruses. In the course of evolution, caspases and apoptosis-inducing factor appeared before other apoptotic proteins, with so-called death receptors being the last among them. The functional analogs of eukaryotic apoptotic proteins take parts in the programmed death of bacteria.

  20. DSFL database: A hub of target proteins of Leishmania sp. to combat leishmaniasis

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    Ameer Khusro

    2017-07-01

    Full Text Available Leishmaniasis is a vector-borne chronic infectious tropical dermal disease caused by the protozoa parasite of the genus Leishmania that causes high mortality globally. Among three different clinical forms of leishmaniasis, visceral leishmaniasis (VL or kala-azar is a systemic public health disease with high morbidity and mortality in developing countries, caused by Leishmania donovani, Leishmania infantum or Leishmania chagasi. Unfortunately, there is no vaccine available till date for the treatment of leishmaniasis. On the other hand, the therapeutics approved to treat this fatal disease is expensive, toxic, and associated with serious side effects. Furthermore, the emergence of drug-resistant Leishmania parasites in most endemic countries due to the incessant utilization of existing drugs is a major concern at present. Drug Search for Leishmaniasis (DSFL is a unique database that involves 50 crystallized target proteins of varied Leishmania sp. in order to develop new drugs in future by interacting several antiparasitic compounds or molecules with specific protein through computational tools. The structure of target protein from different Leishmania sp. is available in this database. In this review, we spotlighted not only the current global status of leishmaniasis in brief but also detailed information about target proteins of various Leishmania sp. available in DSFL. DSFL has created a new expectation for mankind in order to combat leishmaniasis by targeting parasitic proteins and commence a new era to get rid of drug resistance parasites. The database will substantiate to be a worthwhile project for further development of new, non-toxic, and cost-effective antileishmanial drugs as targeted therapies using in vitro/in vivo assays.

  1. Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

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    McConville, Malcolm J; Saunders, Eleanor C; Kloehn, Joachim; Dagley, Michael J

    2015-01-01

    A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments.  How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.

  2. Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis.

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    Chaouch, Melek; Fathallah-Mili, Akila; Driss, Mehdi; Lahmadi, Ramzi; Ayari, Chiraz; Guizani, Ikram; Ben Said, Moncef; Benabderrazak, Souha

    2013-03-01

    Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research. The PCR products of the cpb gene and the N-acetylglucosamine-1-phosphate transferase (nagt) Leishmania gene were sequenced and aligned. Phylogenetic trees of Leishmania based cpb and nagt sequences are close in topology and present the classic distribution of Leishmania in the Old World. The phylogenetic analysis has enabled the characterization and identification of different strains, using both multicopy (cpb) and single copy (nagt) genes. Indeed, the cpb phylogenetic analysis allowed us to identify the Tunisian Leishmania killicki species, and a group which gathers the least evolved isolates of the Leishmania donovani complex, that was originated from East Africa. This clustering confirms the African origin for the visceralizing species of the L. donovani complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

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    Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

    2015-10-19

    DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

  4. Identification of Leishmania tropica from micro-foci of cutaneous leishmaniasis in the Kenyan Rift Valley.

    Science.gov (United States)

    Odiwuor, Samwel; Muia, Alfred; Magiri, Charles; Maes, Ilse; Kirigi, George; Dujardin, Jean-Claude; Wasunna, Monique; Mbuchi, Margaret; Auwera, Gert Van der

    2012-07-01

    We performed diagnosis and species identification of parasites in lesion samples from suspected cutaneous leishmaniasis patients in four villages, three of which are in a known Leishmania tropica endemic region in Kenya. Samples were analyzed both by microscopy and PCR for Leishmania, and typed by an assay using four ribosomal DNA-based species-identification PCRs. The lesions were demonstrated to be caused by L. tropica, which confirms the re-emergence of cutaneous leishmaniasis from this species after a period of reduced incidence in the endemic zone. Our report highlights the importance of an intervention and sustained Leishmania control program.

  5. Leishmania (L.) mexicana Infected Bats in Mexico: Novel Potential Reservoirs

    Science.gov (United States)

    Berzunza-Cruz, Miriam; Rodríguez-Moreno, Ángel; Gutiérrez-Granados, Gabriel; González-Salazar, Constantino; Stephens, Christopher R.; Hidalgo-Mihart, Mircea; Marina, Carlos F.; Rebollar-Téllez, Eduardo A.; Bailón-Martínez, Dulce; Balcells, Cristina Domingo; Ibarra-Cerdeña, Carlos N.; Sánchez-Cordero, Víctor; Becker, Ingeborg

    2015-01-01

    Leishmania (Leishmania) mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L.) mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L.) mexicana DNA. We found that 41 bats (9.77%), belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus), and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L.) mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L.) mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology. PMID:25629729

  6. Leishmania (L. mexicana infected bats in Mexico: novel potential reservoirs.

    Directory of Open Access Journals (Sweden)

    Miriam Berzunza-Cruz

    2015-01-01

    Full Text Available Leishmania (Leishmania mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L. mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L. mexicana DNA. We found that 41 bats (9.77%, belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus, and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L. mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L. mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology.

  7. Differences in species richness patterns between unicellular and multicellular organisms.

    Science.gov (United States)

    Hillebrand, Helmut; Watermann, Frank; Karez, Rolf; Berninger, Ulrike-G

    2001-01-01

    For unicellular organisms, a lack of effects of local species richness on ecosystem function has been proposed due to their locally high species richness and their ubiquitous distribution. High dispersal ability and high individual numbers may enable unicellular taxa to occur everywhere. Using our own and published data sets on uni- and multicellular organisms, we conducted thorough statistical analyses to test whether (1) unicellular taxa show higher relative local species richness compared to multicellular taxa, (2) unicellular taxa show lower slopes of the species:area relationships and species:individuals relationships, and (3) the species composition of unicellular taxa is less influenced by geographic distance compared to multicellular taxa. We found higher local species richness compared to the global species pool for unicellular organisms than for metazoan taxa. The difference was significant if global species richness was conservatively estimated but not if extrapolated, and therefore higher richness estimates were used. Both microalgae and protozoans showed lower slopes between species richness and sample size (area or individuals) compared to macrozoobenthos, also indicating higher local species richness for unicellular taxa. The similarity of species composition of both benthic diatoms and ciliates decreased with increasing geographic distance. This indicated restricted dispersal ability of protists and the absence of ubiquity. However, a steeper slope between similarity and distance was found for polychaetes and corals, suggesting a stronger effect of distance on the dispersal of metazoans compared to unicellular taxa. In conclusion, we found partly different species richness patterns among uni- and multicellular eukaryotes, but no strict ubiquity of unicellular taxa. Therefore, the effect of local unicellular species richness on ecosystem function has to be reanalyzed. Macroecological patterns suggested for multicellular organisms may differ in

  8. Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

    Science.gov (United States)

    Atayde, Vanessa Diniz; Hassani, Kasra; da Silva Lira Filho, Alonso; Borges, Andrezza Raposo; Adhikari, Anupam; Martel, Caroline; Olivier, Martin

    2016-11-01

    Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

    Science.gov (United States)

    Chauhan, Indira Singh; Kaur, Jaspreet; Krishna, Shagun; Ghosh, Arpita; Singh, Prashant; Siddiqi, Mohammad Imran; Singh, Neeloo

    2015-11-21

    Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information

  10. In vivo and in vitro phagocytosis of Leishmania (Leishmania) amazonensis promastigotes by B-1 cells.

    Science.gov (United States)

    Geraldo, M M; Costa, C R; Barbosa, F M C; Vivanco, B C; Gonzaga, W F K M; Novaes E Brito, R R; Popi, A F; Lopes, J D; Xander, P

    2016-06-01

    Leishmaniasis is caused by Leishmania parasites that infect several cell types. The promastigote stage of Leishmania is internalized by phagocytic cells and transformed into the obligate intracellular amastigote form. B-1 cells are a subpopulation of B cells that are able to differentiate in vitro and in vivo into mononuclear phagocyte-like cells with phagocytic properties. B-1 cells use several receptors for phagocytosis, such as the mannose receptor and third complement receptor. Leishmania binds to the same receptors on macrophages. In this study, we demonstrated that phagocytes derived from B-1 cells (B-1 CDP) were able to internalize promastigotes of L. (L.) amazonensis in vitro. The internalized promastigotes differentiated into amastigotes. Our results showed that the phagocytic index was higher in B-1 CDP compared to peritoneal macrophages and bone marrow-derived macrophages. The in vivo phagocytic ability of B-1 cells was also demonstrated. Parasites were detected inside purified B-1 cells after intraperitoneal infection with L. (L.) amazonensis promastigotes. Intraperitoneal stimulation with the parasites led to an increase in both IL-10 and TNF-α. These results highlight the importance of studying B-1 CDP cells as phagocytic cells that can participate and contribute to immunity to parasites. © 2016 John Wiley & Sons Ltd.

  11. immune response in human leishmania infections Respuesta inmune en infecciones humanas por Leishmania spp

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    Sara María Robledo Restrepo

    2000-03-01

    Full Text Available This review summarizes relevant information about the immune response triggered during leishmaniosis, a disease of great importance from the epidemiological point of view, since it is endemic in Colombia and other countries. We emphasize on human leishmaniosis; nevertheless, some important findings in the murine model are also mentioned. This information allows to conclude that Leishmania infection is a complex and coordinated process, which includes adhesion and entrance of the parasite into the host cells and its survival inside them. Events that mediate the infection process may influence its result in terms of elimination of the parasite or development of the disease, through induction or not of an effective specific immune response which involves host cell activation and parasite destruction. La presente revisión tiene como objetivo resumir la información más relevante acerca de la respuesta inmune que se desencadena durante la leishmaniosis, una enfermedad de gran importancia desde el punto de vista epidemiológico dado que es endémica en Colombia y otros países. Aunque la respuesta inmune en la leishmaniosis es un tema que se ha estudiado ampliamente en las infecciones por especies de Leishmania del Viejo Mundo, particularmente Leishmania major y Leishmania donovani y en el modelo murino, la presente revisión hace énfasis en la leishmaniosis humana. Algunos hallazgos importantes en el modelo murino también se mencionan. La información contenida en la revisión, en su mayoría, proviene de publicaciones derivadas de investigaciones, las cuales se seleccionaron con base en la calidad del trabajo realizado y en los aportes de sus resultados en el avance del conocimiento sobre las infecciones en humanos. La síntesis de la información seleccionada nos permite concluir que la infección por Leishmania es un proceso complejo y coordinado que incluye la adherencia y entrada del parásito a la célula hospedera y su posterior

  12. The Capsaspora genome reveals a complex unicellular prehistory of animals.

    Science.gov (United States)

    Suga, Hiroshi; Chen, Zehua; de Mendoza, Alex; Sebé-Pedrós, Arnau; Brown, Matthew W; Kramer, Eric; Carr, Martin; Kerner, Pierre; Vervoort, Michel; Sánchez-Pons, Núria; Torruella, Guifré; Derelle, Romain; Manning, Gerard; Lang, B Franz; Russ, Carsten; Haas, Brian J; Roger, Andrew J; Nusbaum, Chad; Ruiz-Trillo, Iñaki

    2013-01-01

    To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans' unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans.

  13. Unraveling the genetic diversity and phylogeny of Leishmania RNA virus 1 strains of infected Leishmania isolates circulating in French Guiana.

    Science.gov (United States)

    Tirera, Sourakhata; Ginouves, Marine; Donato, Damien; Caballero, Ignacio S; Bouchier, Christiane; Lavergne, Anne; Bourreau, Eliane; Mosnier, Emilie; Vantilcke, Vincent; Couppié, Pierre; Prevot, Ghislaine; Lacoste, Vincent

    2017-07-01

    Leishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now. We subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%-23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates. Here, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.

  14. Methodology optimizing SAGE library tag-to-gene mapping: application to Leishmania

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    Smandi Sondos

    2012-01-01

    Full Text Available Abstract Background Leishmaniasis are widespread parasitic-diseases with an urgent need for more active and less toxic drugs and for effective vaccines. Understanding the biology of the parasite especially in the context of host parasite interaction is a crucial step towards such improvements in therapy and control. Several experimental approaches including SAGE (Serial analysis of gene expression have been developed in order to investigate the parasite transcriptome organisation and plasticity. Usual SAGE tag-to-gene mapping techniques are inadequate because almost all tags are normally located in the 3'-UTR outside the CDS, whereas most information available for Leishmania transcripts is restricted to the CDS predictions. The aim of this work is to optimize a SAGE libraries tag-to-gene mapping technique and to show how this development improves the understanding of Leishmania transcriptome. Findings The in silico method implemented herein was based on mapping the tags to Leishmania genome using BLAST then mapping the tags to their gene using a data-driven probability distribution. This optimized tag-to-gene mappings improved the knowledge of Leishmania genome structure and transcription. It allowed analyzing the expression of a maximal number of Leishmania genes, the delimitation of the 3' UTR of 478 genes and the identification of biological processes that are differentially modulated during the promastigote to amastigote differentiation. Conclusion The developed method optimizes the assignment of SAGE tags in trypanosomatidae genomes as well as in any genome having polycistronic transcription and small intergenic regions.

  15. Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection.

    Science.gov (United States)

    Araújo-Santos, Théo; Prates, Deboraci Brito; França-Costa, Jaqueline; Luz, Nívea F; Andrade, Bruno B; Miranda, José Carlos; Brodskyn, Claudia I; Barral, Aldina; Bozza, Patrícia T; Borges, Valéria Matos

    2014-12-20

    Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.

  16. An agent-based model for Leishmania major infection

    Science.gov (United States)

    Dancik, Garrett M.; Jones, Douglas E.; Dorman, Karin S.

    Leishmania are protozoan parasites transmitted by bites of infected sandflies. Over 20 species of Leishmania, endemic in 88 countries, are capable of causing human disease. Disease is either cutaneous, where skin ulcers occur on exposed surfaces of the body, or visceral, with near certain mortality if untreated. C3HeB/FeJ mice are resistant to L. major, but develop chronic cutaneous lesions when infected with another species L. amazonensis. The well-characterized mechanism of resistance to L. major depends on a CD4+ Thl immune response, macrophage activation, and elimination of the parasite [Sacks 2002]. The factors that account for host susceptibility to L. Amazonensis, however, are not completely understood, despite being generally attributed to a weakened Th1 response [Vanloubbeck 2004].

  17. Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

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    Kelly Ben L

    2010-02-01

    Full Text Available Abstract Background Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen Leishmania has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of Leishmania-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease. Methods We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of Leishmania using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of Leishmania uptake on LPS-induced cytokine expression with uptake of inert latex beads. Results Whilst Leishmania uptake alone did not induce significant levels of any cytokine analysed in this study, Leishmania uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, L. amazonensis was generally more suppressive than L. major. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during Leishmania uptake, in a parasite-specific manner. Conclusions During uptake by macrophages, Leishmania evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, Leishmania suppresses certain proinflammatory cytokine responses in a parasite

  18. Increased transmission potential of Leishmania major/Leishmania infantum hybrids

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    Volf, Petr; Benkova, Ivana; Myskova, Jitka; Sadlova, Jovana; Campino, Lenea; Ravel, Christophe

    2007-01-01

    Development of Leishmania infantum/Leishmania major hybrids was studied in two sand fly species. In Phlebotomus papatasi, which supported development of L. major but not L. infantum, the hybrids produced heavy late-stage infections with high numbers of metacyclic promastigotes. In the permissive vector Lutzomyia longipalpis, all Leishmania strains included in this study developed well. Hybrids were found to express L. major lipophosphoglycan, apparently enabling them to survive in P. papatasi...

  19. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-01-01

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

  20. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-12-11

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

    Science.gov (United States)

    Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

    2015-01-01

    Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

  2. Thrichomys laurentius (Rodentia; Echimyidae as a putative reservoir of Leishmania infantum and L. braziliensis: patterns of experimental infection.

    Directory of Open Access Journals (Sweden)

    André Luiz Rodrigues Roque

    Full Text Available The importance of the genus Thrichomys in the retention of infection and transmission of Leishmania species is supported by previous studies that describe an ancient interaction between caviomorphs and trypanosomatids and report the natural infection of Thrichomys spp. Moreover, these rodents are widely dispersed in Brazil and recognized as important hosts of other tripanosomatids. Our main purpose was to evaluate the putative role of Thrichomys laurentius in the retention of infection and amplification of the transmission cycle of Leishmania infantum and L. braziliensis. Male and female T. laurentius (n = 24 born in captivity were evaluated for the retention of infection with these Leishmania species and followed up by parasitological, serological, hematological, biochemical, histological, and molecular assays for 3, 6, 9, or 12 months post infection (mpi. T. laurentius showed its competence as maintenance host for the two inoculated Leishmania species. Four aspects should be highlighted: (i re-isolation of parasites 12 mpi; (ii the low parasitic burden displayed by T. laurentius tissues; (iii the early onset and maintenance of humoral response, and (iv the similar pattern of infection by the two Leishmania species. Both Leishmania species demonstrated the ability to invade and maintain itself in viscera and skin of T. laurentius, and no rodent displayed any lesion, histological changes, or clinical evidence of infection. We also wish to point out the irrelevance of the adjective dermotropic or viscerotropic to qualify L. braziliensis and L. infantum, respectively, when these species are hosted by nonhuman hosts. Our data suggest that T. laurentius may act at least as a maintenance host of both tested Leishmania species since it maintained long-lasting infections. Moreover, it cannot be discarded that Leishmania spp. infection in free-ranging T. laurentius could result in higher parasite burden due the more stressing conditions in the wild

  3. Histopatologia da forma localizada de leishmaniose cutânea por Leishmania (Leishmania amazonensis Histopathology of the localized form of cutaneous leishmaniasis due to Leishmania (Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Mário A. P. Moraes

    1994-10-01

    to Leishmania (Leishmania amazonensis are reported. In this form, less known than the diffuse one caused by the same species, the clinical manifestations are identical to those produced by other Leishmania species of the subgenus Viannia. There is, however, in the localized infection by L (L. amazonensis, a peculiar feature, only recently discovered: about 50% of the affected individuals are Montenegro-negatives. The main histologic change observed in the skin sections was the presence of groups of macrophages with a large vacuole in the cytoplasm, containing many amastigotes. The microscopic picture is similar to that found in the diffuse form of the disease, the difference being only quantitative. When in large numbers, the macrophages suffers necrosis, which generally starts at the center of the groups. First, in this process, the membrane of the parasitized cells ruptures, and the amastigotes become free; later, both cells and parasites are destroyed. The picture can be seen either in Montenegro-negative or in Montenegro-positive patients. The macrophages with amastigotes may persist in tissues for as long as 6-7 months, while in the infections due to L (V. braziliensis the parasites usually disappear in a few weeks.

  4. Serological evidence of Leishmania donovani infection in apparently healthy dogs using direct agglutination test (DAT) and rk39 dipstick tests in Kafta Humera, north-west Ethiopia

    NARCIS (Netherlands)

    Kalayou, S.; Tadelle, H.; Bsrat, A.; Abebe, N.; Haileselassie, M.; Schallig, H. D. F. H.

    2011-01-01

    Leishmania (Kinetoplastida: Trypanosomatidae) are protozoan parasites of significant medical and veterinary importance. Over the last decade, visceral leishmaniasis (VL) has emerged as a major opportunistic infection associated with HIV/AIDS in North Western Ethiopia. This paper reports on

  5. LeishCyc: a biochemical pathways database for Leishmania major

    Directory of Open Access Journals (Sweden)

    Doyle Maria A

    2009-06-01

    Full Text Available Abstract Background Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. Description The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2, based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute. Conclusion The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania

  6. Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Isabel M. Vincent

    2015-10-01

    Full Text Available Antimony (SbIII and miltefosine (MIL are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.

  7. Chemotherapeutic targets in parasites: contemporary strategies

    National Research Council Canada - National Science Library

    Mansour, Tag E; Mansour, Joan MacKinnon

    2002-01-01

    ... identify effective antiparasitic agents. An introduction to the early development of parasite chemotherapy is followed by an overview of biophysical techniques and genomic and proteomic analyses. Several chapters are devoted to specific types of chemotherapeutic agents and their targets in malaria, trypanosomes, leishmania, and amitochondrial...

  8. Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones

    DEFF Research Database (Denmark)

    Chen, M; Zhai, L; Christensen, S B

    2001-01-01

    Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity...... of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major...... promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major...

  9. Activity evaluation from different native or irradiated with 60 Co gamma rays snake venoms and their inhibitory effect on Leishmania (Leishmania) amazonensis

    International Nuclear Information System (INIS)

    Lourenco, Cecilia de Oliveira

    2000-01-01

    Cutaneous leishmaniasis is a disease, caused by Leishmania parasites, that occurs frequently in tropical and sub-tropical regions of the world. Skin lesions that could results in disfiguring aspect characterize it. The treatment is based on few drugs as antimony salts or pentamidine that are toxic with increasing resistance by the parasite. Alternative forms of disease treatment are in constant search, including natural components as snake venoms. Previous studies demonstrate that some components of snake venoms have an inhibitory effect against those parasites, including Leishmania species. Although snake venoms presented high toxicity, several methods have been described to detoxify most or some of their toxic components, with favorable results by the use of gamma irradiation. In this report we tested several native and irradiated snake venoms for inhibitory effect against Leishmania (Leishmania) amazonensis parasite and LLCMK 2 mammalian cells, with enzymatic tests and electrophoresis. There are significant activity in Acanthophis antarcticus, Agkistrodon bilineatus, Bothrops moojeni, Bothrops jararaca, Hoplocephalus stephensi, Naja melanoleuca, Naja mossambica, Pseudechis australis, Pseudechis colletti, Pseudechis guttatus and Pseudechis porphyriacus, venom being inactive Pseudonaja textilis, Notechis ater niger, Notechis scutatus. Oxyuranus microlepidotus and Oxyuranus scutellatus venoms. After 2 KGy of 60 Co irradiation most venom loses significantly their activity. Venoms with antileishmanial activity presented L-amino acid oxidase (L-AO) activity and showed common protein with a molecular weight about 60kDa in SDS-PAGE. These results indicate that L-AO activity in those venoms are probably related with antileishmanial effect. (author)

  10. Metacyclic promastigotes of Leishmania amazonensis selection using gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco C.; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Centro de Biologia Molecular]. E-mail: fbonetti@usp.br; Andrade Junior, Heitor Franco de [Instituto de Medicina Tropical de Sao Paulo, SP (Brazil). Lab. de Protozoologia

    2005-07-01

    Leishmania spp. causes a spectrum of human diseases, ranging from self-healing skin lesions to severe and lethal visceral disease. In previous work we demonstrated that the protein and nucleic acid metabolism and oxidative respiration were severely affected by irradiation, in a dose response way, but a small but representative fractions are relatively radio resistant, surviving after 800 Gy of {sup 60}Co irradiation. The best explanation could be a selection of metacyclic promastigotes. In these forms, the G0 state allows the adequate correction of DNA repair after the irradiation insult. In this work, we are looking for the ideal radiation dose to select the higher proportion of metacyclic forms of L.. (L.) amazonensis in culture. Parasites were grown in RPMI 1640 medium, plus 20% fetal calf serum, than they were irradiated with different doses ranging between 25 and 400 Gy. Parasites irradiated at 400 Gy infected, proportionally, more cells than parasites irradiated at other doses. To confirm this metacyclogenesis, a complement lysis assay was performed with 5, 10 and 20% of male guinea pig blood serum at 20 deg C for 3 hours, and parasites counted. Guinea pig serum a 10% promotes more lysis, with 200 Gy irradiated parasites being less affected, probably due to metacyclic selection. These preliminary results suggests that the ionizing radiation, specially between 200 and 400 Gy, could be a alternative tool for the selection of metacyclic forms of Leishmania amazonensis in culture. (a0011uth.

  11. Reproduction in Leishmania: A focus on genetic exchange.

    Science.gov (United States)

    Rougeron, V; De Meeûs, T; Bañuls, A-L

    2017-06-01

    One key process of the life cycle of pathogens is their mode of reproduction. Indeed, this fundamental biological process conditions the multiplication and the transmission of genes and thus the propagation of diseases in the environment. Reproductive strategies of protozoan parasites have been a subject of debate for many years, principally due to the difficulty in making direct observations of sexual reproduction (i.e. genetic recombination). Traditionally, these parasites were considered as characterized by a preeminent clonal structure. Nevertheless, with the development of elaborate culture experiments, population genetics and evolutionary and population genomics, several studies suggested that most of these pathogens were also characterized by constitutive genetic recombination events. In this opinion, we focused on Leishmania parasites, pathogens responsible of leishmaniases, a major public health issue. We first discuss the evolutionary advantages of a mixed mating reproductive strategy, then we review the evidence of genetic exchange, and finally we detail available tools to detect naturally occurring genetic recombination in Leishmania parasites and more generally in protozoan parasites. Copyright © 2016. Published by Elsevier B.V.

  12. Metacyclic promastigotes of Leishmania amazonensis selection using gamma irradiation

    International Nuclear Information System (INIS)

    Bonetti, Franco C.; Nascimento, Nanci do; Andrade Junior, Heitor Franco de

    2005-01-01

    Leishmania spp. causes a spectrum of human diseases, ranging from self-healing skin lesions to severe and lethal visceral disease. In previous work we demonstrated that the protein and nucleic acid metabolism and oxidative respiration were severely affected by irradiation, in a dose response way, but a small but representative fractions are relatively radio resistant, surviving after 800 Gy of 60 Co irradiation. The best explanation could be a selection of metacyclic promastigotes. In these forms, the G0 state allows the adequate correction of DNA repair after the irradiation insult. In this work, we are looking for the ideal radiation dose to select the higher proportion of metacyclic forms of L.. (L.) amazonensis in culture. Parasites were grown in RPMI 1640 medium, plus 20% fetal calf serum, than they were irradiated with different doses ranging between 25 and 400 Gy. Parasites irradiated at 400 Gy infected, proportionally, more cells than parasites irradiated at other doses. To confirm this metacyclogenesis, a complement lysis assay was performed with 5, 10 and 20% of male guinea pig blood serum at 20 deg C for 3 hours, and parasites counted. Guinea pig serum a 10% promotes more lysis, with 200 Gy irradiated parasites being less affected, probably due to metacyclic selection. These preliminary results suggests that the ionizing radiation, specially between 200 and 400 Gy, could be a alternative tool for the selection of metacyclic forms of Leishmania amazonensis in culture. (author)

  13. Introduction of New Parasites in Denmark

    DEFF Research Database (Denmark)

    Enemark, Heidi L.

    examples of such parasites/parasitic diseases: Setaria tundra, a mosquito-borne filarioid nematode which was detected for the first time in Danish deer in 2010. This parasite is usually considered harmless but is capable of causing peritonitis and mortality in ungulates. The newly detected parasite...... was genetically very similar to previously published isolates from France and Italy, and may have been spread to Denmark from southern Europe. Giardia spp. a zoonotic, unicellular parasite (protozoa) well known in Danish livestock but recently found in extremely high numbers in Danish deer with chronic diarrhea...... for the first time in Denmark approximately 10 years ago in 3 foxes from the Copenhagen area. Since then, no systematic surveillance has been performed, and therefore the current prevalence among wildlife and pets is unknown. So far the parasite has not been found in intermediate hosts (rodents) in Denmark...

  14. Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

    Science.gov (United States)

    Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

    2016-05-10

    Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

  15. Role of pro-inflammatory cytokine IL-17 in Leishmania pathogenesis and in protective immunity by Leishmania vaccines.

    Science.gov (United States)

    Banerjee, Antara; Bhattacharya, Parna; Joshi, Amritanshu B; Ismail, Nevien; Dey, Ranadhir; Nakhasi, Hira L

    2016-11-01

    The clinical outcome of Leishmania pathogenesis ranges from active skin lesions to fatal visceral dissemination and severely impaired T cell immunity. It is well established that a strong Th1 immune response is protective against cutaneous forms of the disease, however a mixed Th1/Th2 response is most commonly observed against visceral infections as evident from previous studies. Aside from Th1/Th2 cytokines, the pro-inflammatory IL-17 cytokine family plays an important role in the clearance of intracellular pathogens. In Leishmania induced skin lesions, IL-17 produced by Th17 cells is shown to exacerbate the disease, suggesting a role in pathogenesis. However, a protective role for IL-17 is indicated by the expansion of IL-17 producing cells in vaccine-induced immunity. In human visceral leishmaniasis (VL) it has been demonstrated that IL-17 and IL-22 are associated with protection against re-exposure to Leishmania, which further suggests the involvement of IL-17 in vaccine induced protective immunity. Although there is no vaccine against any form of leishmaniasis, the development of genetically modified live attenuated parasites as vaccine candidates prove to be promising, as they successfully induce a robust protective immune response in various animal models. However, the role of IL-17 producing cells and Th17 cells in response to these vaccine candidates remains unexplored. In this article, we review the role of IL-17 in Leishmania pathogenesis and the potential impact on vaccine induced immunity, with a special focus on live attenuated Leishmania parasites. Published by Elsevier Inc.

  16. Chromosome organizaton in simple and complex unicellular organisms.

    Science.gov (United States)

    O'Sullivan, Justin M

    2011-01-01

    The genomes of unicellular organisms form complex 3-dimensional structures. This spatial organization is hypothesized to have a significant role in genomic function. Spatial organization is not limited solely to the three-dimensional folding of the chromosome(s) in genomes but also includes genome positioning, and the folding and compartmentalization of any additional genetic material (e.g. episomes) present within complex genomes. In this comment, I will highlight similarities in the spatial organization of eukaryotic and prokaryotic unicellular genomes.

  17. Microbial stasis of Leishmania enriettii in activated guinea pig macrophages

    International Nuclear Information System (INIS)

    Groocock, C.M.; Soulsby, E.J.L.

    1980-01-01

    Peritoneal exudate cells (PEC) from Leishmania-sensitized guinea pigs were cultured in vitro in the presence (activated) or absence (non-activated) of leishmanial antigen for 24 or 48 hours. These were then labelled with 51 Cr and challenged with 125 I-labelled promastigotes. The changing relationship between the macrophage and the parasite was monitored by observing changes in the ratio of the cell-associated isotopes. Highly significant differences in the ratio change developed during culture. These differences were a result of the activated cultures showing a higher release of 51 Cr and a lower release of 125 I when compared with the non-activated cells, at 12 hours the percentage release of 125 I from the parasite within the activated macrophage was fourfold less than that released by parasites within non-activated cells (9.2% versus 38.3%) and tenfold less than that released from glutaraldehyde-killed organisms phagocytosed by activated macrophages (91.6%). These studies indicate that stasis rather than killing of leishmaniae occurs in the activated macrophage in vitro. Parallel experiments evaluated by the visual counting of leishmaniae within the macrophages support these data. PEC from tuberculin-sensitized guinea pigs activated in vitro by purified protein derivative showed little or no activity against leishmaniae, indicating a specific requirement for this microbial stasis by activated macrophages. As a corollary of this, peritoneal exudate lymphocytes separated from the same preparations of PEC were shown to be specifically reactive to leishmanial antigen by transformation and incorporation of 3 H-thymidine. (author)

  18. Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis.

    Science.gov (United States)

    DaMata, Jarina Pena; Mendes, Bárbara Pinheiro; Maciel-Lima, Kátia; Menezes, Cristiane Alves Silva; Dutra, Walderez Ornelas; Sousa, Lirlândia Pires; Horta, Maria Fátima

    2015-01-01

    Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

  19. Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania amazonensis, but Not by Leishmania (Viannia guyanensis.

    Directory of Open Access Journals (Sweden)

    Jarina Pena DaMata

    Full Text Available Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6, whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

  20. LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania

    Science.gov (United States)

    González-Rey, Elena; Delgado, Mario; Castanys, Santiago; Pérez-Victoria, José M.; Gamarro, Francisco

    2013-01-01

    Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite. PMID:23638200

  1. Downsides and benefits of unicellularity in budding yeast

    Science.gov (United States)

    Balazsi, Gabor; Chen, Lin; Kuzdzal-Fick, Jennie

    Yeast cells that do not separate after cell division form clumps. Clumping was shown to aid utilization of certain sugars, but its effects in stressful conditions are unknown. Generally speaking, what are the costs and benefits of unicellularity versus clumping multicellularity in normal and stressful conditions? To address this question, we evolved clumping yeast towards unicellularity by continuously propagating only those cells that remain suspended in liquid culture after settling. Whole-genome sequencing indicated that mutations in the AMN1 (antagonist of mitotic exit network) gene underlie the changes from clumping to unicellular phenotypes in these evolved yeast cells. Simple models predict that clumping should hinder growth in normal conditions while being protective in stress. Accordingly, we find experimentally that yeast clumps are more resistant to freeze/thaw, hydrogen peroxide, and ethanol stressors than their unicellular counterparts. On the other hand, unicellularity seems to be advantageous in normal conditions. Overall, these results reveal the downsides and benefits of unicellularity in different environmental conditions and uncover its genetic bases in yeast. This research was supported by the NIH Director's New Innovator Award Program (1DP2 OD006481-01), by NSF/IOS 1021675 and the Laufer Center for Physical & Quantitative Biology.

  2. Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population.

    Science.gov (United States)

    Rogers, Matthew B; Downing, Tim; Smith, Barbara A; Imamura, Hideo; Sanders, Mandy; Svobodova, Milena; Volf, Petr; Berriman, Matthew; Cotton, James A; Smith, Deborah F

    2014-01-01

    Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

  3. Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

    OpenAIRE

    Vasudevan, Gayatri; Ullman, Buddy; Landfear, Scott M.

    2001-01-01

    Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. do...

  4. Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil.

    Science.gov (United States)

    Dorval, Maria Elizabeth Cavalheiros; Alves, Tulia Peixoto; Cristaldo, Geucira; Rocha, Hilda Carlos da; Alves, Murilo Andrade; Oshiro, Elisa Teruya; Oliveira, Alessandra Gutierrez de; Brazil, Reginaldo Peçanha; Galati, Eunice Aparecida Bianchi; Cunha, Rivaldo Venancio da

    2010-01-01

    The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3%) and Bi flaviscutellata (41.4%), present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania) amazonensis. Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.

  5. Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae: Phlebotominae) in the Lençóis Maranhenses National Park, Brazil.

    Science.gov (United States)

    Pereira-Filho, Adalberto Alves; Fonteles, Raquel Silva; Bandeira, Maria da Conceição Abreu; Moraes, Jorge Luiz Pinto; Rebêlo, José Manuel Macário; Melo, Maria Norma

    2018-02-20

    Sand flies are very common in the region of Lençóis Maranhenses National Park, an important tourist attraction in Brazil. However, the role of some species and their relative importance locally in Leishmania Ross 1903 transmission is unclear. The objective of this study was to identify Leishmania infection in phlebotomine sand flies collected around the Lençóis Maranhenses National Park, an important conservation area and popular international/national tourist destination with a high incidence of leishmaniasis. Sand flies were collected in peridomiciliary areas on the tourist route from September 2012 to August 2013. The captured females were subjected to molecular analyses for the detection of Leishmania DNA. Sand flies were infected with four Leishmania species: Leishmania (Viannia) braziliensis (Vianna, 1911) was found in Lutzomyia whitmani (Antunes and Coutinho, 1939) (2.1%) and Lutzomyia longipalpis (Lutz and Neiva, 1912) (1.7%); Leishmania (Leishmania) infantum (Nicole, 1908) infected Lutzomyia wellcomei (Fraiha, Shaw, and Lainson, 1971) (20%), Lutzomyia sordellii (Shannon and Del Ponte, 1927) (4.3%), Lu. longipalpis (3.7%), and Lu. whitmani (0.8%); Leishmania (Leishmania) amazonensis (Lainson & Shaw, 1972) was found in Lu. whitmani (0.58%), while Leishmania (Viannia) lainsoni infected Lutzomyia evandroi (Costa Lima and Antunes, 1936) (3.4%), Lu. longipalpis (1.06%), and Lu. whitmani (0.29%). The occurrence of these parasites requires control measures to reduce the incidence of cutaneous leishmaniasis and to contain a possible epidemic of visceral leishmaniasis, the most severe form of the disease.

  6. Leishmania promastigotes lack phosphatidylserine but bind annexin V upon permeabilization or miltefosine treatment.

    Directory of Open Access Journals (Sweden)

    Adrien Weingärtner

    Full Text Available The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31P nuclear magnetic resonance (NMR spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

  7. Genetic Diversity in Natural Populations of New World Leishmania

    Directory of Open Access Journals (Sweden)

    Cupolillo Elisa

    1998-01-01

    Full Text Available Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can not be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of transmission of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and follow Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

  8. Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

    Science.gov (United States)

    de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

    2016-06-01

    Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

  9. Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

    Directory of Open Access Journals (Sweden)

    Tamara Salloum

    2016-06-01

    Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ method. The results were statistically correlated with the parasitic index (PI. The DNA storage in formalin-fixed paraffin embedded (FFPE tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica, each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.

  10. Activity evaluation from different native or irradiated with {sup 60} Co gamma rays snake venoms and their inhibitory effect on Leishmania (Leishmania) amazonensis; Avaliacao da atividade de diferentes venenos de serpentes, nativos ou irradiados, com radiacao gama de {sup 60} Co, quanto ao poder inibitorio do crescimento de Leishmania (Leishmania) amazonensis

    Energy Technology Data Exchange (ETDEWEB)

    Lourenco, Cecilia de Oliveira

    2000-07-01

    Cutaneous leishmaniasis is a disease, caused by Leishmania parasites, that occurs frequently in tropical and sub-tropical regions of the world. Skin lesions that could results in disfiguring aspect characterize it. The treatment is based on few drugs as antimony salts or pentamidine that are toxic with increasing resistance by the parasite. Alternative forms of disease treatment are in constant search, including natural components as snake venoms. Previous studies demonstrate that some components of snake venoms have an inhibitory effect against those parasites, including Leishmania species. Although snake venoms presented high toxicity, several methods have been described to detoxify most or some of their toxic components, with favorable results by the use of gamma irradiation. In this report we tested several native and irradiated snake venoms for inhibitory effect against Leishmania (Leishmania) amazonensis parasite and LLCMK{sub 2} mammalian cells, with enzymatic tests and electrophoresis. There are significant activity in Acanthophis antarcticus, Agkistrodon bilineatus, Bothrops moojeni, Bothrops jararaca, Hoplocephalus stephensi, Naja melanoleuca, Naja mossambica, Pseudechis australis, Pseudechis colletti, Pseudechis guttatus and Pseudechis porphyriacus, venom being inactive Pseudonaja textilis, Notechis ater niger, Notechis scutatus. Oxyuranus microlepidotus and Oxyuranus scutellatus venoms. After 2 KGy of {sup 60}Co irradiation most venom loses significantly their activity. Venoms with antileishmanial activity presented L-amino acid oxidase (L-AO) activity and showed common protein with a molecular weight about 60kDa in SDS-PAGE. These results indicate that L-AO activity in those venoms are probably related with antileishmanial effect. (author)

  11. Serological survey of dogs from Egypt for antibodies to Leishmania spp.

    Science.gov (United States)

    Leishmaniasis is an insect-transmitted parasitic disease with worldwide distribution. Leishmania spp. infections cause a broad spectrum of clinical signs ranging from skin lesions to fatal visceral disease. Dogs are a major reservoir host for visceral leishmaniasis in humans. Leishmaniasis is endemi...

  12. Mapping the genes for susceptibility and response to Leishmania tropica in mouse

    Czech Academy of Sciences Publication Activity Database

    Sohrabi, Yahya; Havelková, Helena; Kobets, Tetyana; Šíma, Matyáš; Volkova, Valeriya; Grekov, Igor; Jarošíková, T.; Kurey, Irina; Vojtíšková, Jarmila; Svobodová, M.; Demant, P.; Lipoldová, Marie

    2013-01-01

    Roč. 7, č. 7 (2013), s. 1-17 ISSN 1935-2735 R&D Projects: GA ČR GA310/08/1697; GA MŠk LH12049 Institutional support: RVO:68378050 Keywords : Leishmania tropica * gene controlling susceptibility * host-parasite interactions * leishmaniasis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.489, year: 2013

  13. Antibodies to Toxoplasma gondii and Leishmania spp. in domestic cats from Luanda, Angola

    Science.gov (United States)

    Toxoplasma gondii and Leishmania spp. are zoonotic agents of importance to public health, with domestic cats as potential reservoirs for both protozoal infections. The present study aimed at assessing for the first time the seroprevalence of these zoonotic parasites in a domestic feline population l...

  14. Evaluation of Leishmania Species Reactivity in Human Serologic Diagnosis of Leishmaniasis

    NARCIS (Netherlands)

    Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

    2009-01-01

    The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using, sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better

  15. Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

    Science.gov (United States)

    Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

    2016-07-01

    Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

  16. Antibodies to Toxoplasma gondii and Leishmania spp. in domestic cats from Luanda, Angola

    NARCIS (Netherlands)

    Lopes, Ana Patrícia; Oliveira, Ana Cristina; Granada, Sara; Rodrigues, Filipa T.; Papadopoulos, Elias; Schallig, Henk; Dubey, Jitender P.; Cardoso, Luís

    2017-01-01

    Toxoplasma gondii and Leishmania spp. are zoonotic protozoa of importance to animal and public health. The present study aimed to assess for the first time the seroprevalence of these zoonotic parasites in a domestic feline population living in Luanda, Angola. One hundred and two cats were sampled

  17. Evaluating the Role of Host AMPK in Leishmania Burden.

    Science.gov (United States)

    Moreira, Diana; Estaquier, Jérôme; Cordeiro-da-Silva, Anabela; Silvestre, Ricardo

    2018-01-01

    The study of host AMP-activated protein kinase (AMPK) activation during Leishmania infection imposes distinct types of techniques to measure protein expression and activation, as well as to quantify, at transcription and translational levels, its downstream targets. The investigation of host AMPK protein modulation during Leishmania infection should primarily be assessed during in vitro infections using as a host murine bone marrow-derived macrophages (BMMos). The infection outcome is assessed measuring the percentage of infected cells in the context of BMMos. To evaluate AMPK activity during infection, the expression of AMPK phosphorylated at Thr172 as well as the transcription and translational levels of its downstream targets are evaluated by quantitative PCR and immunoblotting. The modulation of AMPK activity in vivo is determined specifically in sorted splenic macrophages harboring Leishmania parasites recovered from infected mice using fluorescent-labeled parasites in the infectious inocolum. The modulation of AMPK activity was assessed by AMPK activators and inhibitors and also using AMPK, SIRT1, or LKB1 KO mice models. The infection outcome in BMMos and in vivo was further determined using these two different approaches. To finally understand the metabolic impact of AMPK during infection, in vitro metabolic assays in infected BMMos were measured in the bioenergetic profile using an extracellular flux analyzer.

  18. Effect of ionizing radiation on the morphology, physiology and growth of Leishmania ssp; Acao da radiacao ionizante sobre a morfologia, fisiologia e crescimento da Leishmania spp

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco C.; Spencer, Patrick J.; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Junior A, Heitor F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Instituto de Medicina Tropical

    2000-07-01

    The Leishmania spp is a pathogenic protozoan, which cause different diseases in man. The human diseases, in America, caused by this group of protozoa are divided in cutaneous or tegumentar and visceral, known as kala-azar. In this work, our principal study object was the specie that causes tegumentar leishmaniasis, in Brazil. Metabolic studies of cellular respiration and proteins and nucleic acids synthesis were accomplished using radiation as a form of sterilizing the parasites without however affecting their immunogenic capacity The promastigotes forms of irradiated Leishmania spp were totally sterilized with the dose of 1500 Gy, with their reproductive and nucleic acids, as well as protein synthesis capacity blocked. (author)

  19. Infección de fibroblastos de piel de animales con distinto grado de susceptibilidad a Leishmania infantum y Leishmania mexicana (Kinetoplastida: Trypanosomatidae

    Directory of Open Access Journals (Sweden)

    Miguel Angel Minero

    2004-03-01

    Full Text Available En este estudio se presenta un modelo in vitro de cultivo de fibroblastos de piel de hámster, ratón y rata hecho con el propósito de determinar diferencias en cuanto a la susceptibilidad a la infección por dos especies del género Leishmania (Kinetoplastida: Trypanosomatidae. Se realizó además un estudio ultraestructural por microscopía electrónica de transmisión con el fin de establecer si las formas intracelulares observadas correspondían a multiplicación interna o a fagocitosis múltiple. Se estudió la multiplicación de los parásitos en los fibroblastos de las tres especies de roedores infectados tanto por Leishmania infantum como por L. mexicana (cepa OCR y las diferencias entre las tres fueron estadísticamente significativas (pInfection and multiplication of Leishmania infantum and L. mexicana inside of skin fibroblasts from hamsters, mice and rats was achieved. This process was demonstrated either by counting parasites inside the stained cells or by electronic microscopy studies. In addition multiplication rate differences in the cells from these rodent species were determined, for L. infantum as well as for L. mexicana. Parasite development in hamsters and mice fibroblasts was evident but there was not multiplication in rat cells showing that apparently they are refractory to Leishmania infection. These results suggest that the parasite affinity for each animal, as well as any intracellular environment resistance, could involve genetic factors in the parasite multiplication. On the other hand, presence of amastigote multiplication inside of parasitophorus vacuole, showed by electronic microscopy images, probes a true parasite transformation. Therefore it is suggested that fibroblasts could work as host cells for parasite survival and permanency in the infected animals

  20. Lutzomyia migonei is a permissive vector competent for Leishmania infantum.

    Science.gov (United States)

    Guimarães, Vanessa Cristina Fitipaldi Veloso; Pruzinova, Katerina; Sadlova, Jovana; Volfova, Vera; Myskova, Jitka; Filho, Sinval Pinto Brandão; Volf, Petr

    2016-03-17

    Leishmania infantum is the most widespread etiological agent of visceral leishmaniasis (VL) in the world, with significant mortality rates in human cases. In Latin America, this parasite is primarily transmitted by Lutzomyia longipalpis, but the role of Lutzomyia migonei as a potential vector for this protozoan has been discussed. Laboratory and field investigations have contributed to this hypothesis; however, proof of the vector competence of L. migonei has not yet been provided. In this study, we evaluate for the first time the susceptibility of L. migonei to L. infantum. Females of laboratory-reared L. migonei were fed through a chick-skin membrane on rabbit blood containing L. infantum promastigotes, dissected at 1, 5 and 8 days post-infection (PI) and checked microscopically for the presence, intensity and localisation of Leishmania infections. In addition, morphometric analysis of L. infantum promastigotes was performed. High infection rates of both L. infantum strains tested were observed in L. migonei, with colonisation of the stomodeal valve already on day 5 PI. At the late-stage infection, most L. migonei females had their cardia and stomodeal valve colonised by high numbers of parasites, and no significant differences were found compared to the development in L. longipalpis. Metacyclic forms were found in all parasite-vector combinations since day 5 PI. We propose that Lutzomyia migonei belongs to sand fly species permissive to various Leishmania spp. Here we demonstrate that L. migonei is highly susceptible to the development of L. infantum. This, together with its known anthropophily, abundance in VL foci and natural infection by L. infantum, constitute important evidence that L. migonei is another vector of this parasite in Latin America.

  1. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    Directory of Open Access Journals (Sweden)

    Linder Markus

    2006-03-01

    Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

  2. Post-Genomics and Vaccine Improvement for Leishmania

    Science.gov (United States)

    Seyed, Negar; Taheri, Tahereh; Rafati, Sima

    2016-01-01

    Leishmaniasis is a parasitic disease that primarily affects Asia, Africa, South America, and the Mediterranean basin. Despite extensive efforts to develop an effective prophylactic vaccine, no promising vaccine is available yet. However, recent advancements in computational vaccinology on the one hand and genome sequencing approaches on the other have generated new hopes in vaccine development. Computational genome mining for new vaccine candidates is known as reverse vaccinology and is believed to further extend the current list of Leishmania vaccine candidates. Reverse vaccinology can also reduce the intrinsic risks associated with live attenuated vaccines. Individual epitopes arranged in tandem as polytopes are also a possible outcome of reverse genome mining. Here, we will briefly compare reverse vaccinology with conventional vaccinology in respect to Leishmania vaccine, and we will discuss how it influences the aforementioned topics. We will also introduce new in vivo models that will bridge the gap between human and laboratory animal models in future studies. PMID:27092123

  3. Leishmania (V.) braziliensis infecting bats from Pantanal wetland, Brazil: First records for Platyrrhinus lineatus and Artibeus planirostris.

    Science.gov (United States)

    de Castro Ferreira, Eduardo; Pereira, Agnes Antônio Sampaio; Silveira, Maurício; Margonari, Carina; Marcon, Glaucia Elisete Barbosa; de Oliveira França, Adriana; Castro, Ludiele Souza; Bordignon, Marcelo Oscar; Fischer, Erich; Tomas, Walfrido Moraes; Dorval, Maria Elizabeth Cavalheiros; Gontijo, Célia Maria Ferreira

    2017-08-01

    In the New World genus Leishmania parasites are etiological agents of neglected zoonoses known as leishmaniasis. Its epidemiology is very complex due to the participation of several species of sand fly vectors and mammalian hosts, and man is an accidental host. Control is very difficult because of the different epidemiological patterns of transmission observed. Studies about Leishmania spp. infection in bats are so scarce, which represents a large gap in knowledge about the role of these animals in the transmission cycle of these pathogens, especially when considering that Chiroptera is one of the most abundant and diverse orders among mammals. Leishmaniasis in Mato Grosso do Sul, Brazil are remarkably frequent, probably due to the abundance of its regional mastofauna. The recent record of L. braziliensis in bats from this state indicates the need to clarify the role of these mammals in the transmission cycle. In this study we evaluated the presence of Leishmania parasites in the skin of different species of bats, using PCR directed to Leishmania spp. kDNA for screening followed by PCR/RFLP analysis of the hsp70 gene for the identification of parasite species. Leishmania species identification was confirmed by PCR directed to the G6PD gene of L. braziliensis, followed by sequencing of the PCR product. Samples from 47 bats were processed, of which in three specimens (6.38%) was detected the presence of Leishmania sp. kDNA. PCR/RFLP and sequencing identified the species involved in the infection as L. braziliensis in all of them. This is the first report of Leishmania braziliensis in bats from Pantanal ecosystem and the first record of this species in Platyrrhinus lineatus and Artibeus planirostris, bats with a wide distribution in South America. These results reinforce the need to deepen the knowledge about the possibility of bats act as reservoirs of Leishmania spp. especially considering their ability of dispersion and occupation of anthropic environments

  4. Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

    Science.gov (United States)

    Myšková, Jitka; Dostálová, Anna; Pěničková, Lucie; Halada, Petr; Bates, Paul A; Volf, Petr

    2016-07-25

    Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

  5. Detection of Leishmania spp. in Bats from an Area of Brazil Endemic for Visceral Leishmaniasis.

    Science.gov (United States)

    de Rezende, M B; Herrera, H M; Carvalho, C M E; Carvalho Anjos, E A; Ramos, C A N; de Araújo, F R; Torres, J M; de Oliveira, C E

    2017-12-01

    The multihost parasites Leishmania spp. infect a broad range of wild mammalian species including bats. Several species of bats have adapted to a variety of food resources and shelters in urban areas. This study aimed to detect Leishmania spp. DNA in bats present in forest fragments located in metropolitan areas endemic for leishmaniasis in Campo Grande, Mato Grosso do Sul (MS), Brazil. Blood samples were obtained from 80 individuals, including eight species of Phyllostomidae and one species of Vespertilionidae. Thirty of the 80 bats were positive for Leishmania spp. using conventional PCR, all belonging to the family Phyllostomidae. Eighteen samples tested by real-time PCR (qPCR) using specific primers for the kDNA of Leishmania infantum were positive. To the best of our knowledge, this is the first report detecting Leishmania spp. in Platyrrhinus incarum in addition to being the first reported detection of L. infantum in the bat species Phyllostomus discolor, Platyrrhinus lineatus, Artibeus planirostris and Artibeus lituratus. Our results show that bats can host Leishmania spp. in areas endemic for leishmaniasis, which must be taken into account in disease control operations by public health authorities. © 2017 Blackwell Verlag GmbH.

  6. Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report.

    Science.gov (United States)

    Cavalcanti, Amanda; Lobo, Rogério; Cupolillo, Elisa; Bustamante, Fábio; Porrozzi, Renato

    2012-12-01

    Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

    Science.gov (United States)

    Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

    2016-06-15

    Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

  8. T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia braziliensis

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    Rita C Bittar

    2007-08-01

    Full Text Available Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma production in comparison to CCL patients. On the other hand, interleukin (IL-10 production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma and regulatory mechanisms (IL-10. The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10 and effector cytokines (IFN-gamma, leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

  9. Molecular analysis of the replication program in unicellular model organisms.

    Science.gov (United States)

    Raghuraman, M K; Brewer, Bonita J

    2010-01-01

    Eukaryotes have long been reported to show temporal programs of replication, different portions of the genome being replicated at different times in S phase, with the added possibility of developmentally regulated changes in this pattern depending on species and cell type. Unicellular model organisms, primarily the budding yeast Saccharomyces cerevisiae, have been central to our current understanding of the mechanisms underlying the regulation of replication origins and the temporal program of replication in particular. But what exactly is a temporal program of replication, and how might it arise? In this article, we explore this question, drawing again on the wealth of experimental information in unicellular model organisms.

  10. What do unicellular organisms teach us about DNA methylation?

    Science.gov (United States)

    Harony, Hala; Ankri, Serge

    2008-05-01

    DNA methylation is an epigenetic hallmark that has been studied intensively in mammals and plants. However, knowledge of this phenomenon in unicellular organisms is scanty. Examining epigenetic regulation, and more specifically DNA methylation, in these organisms represents a unique opportunity to better understand their biology. The determination of their methylation status is often complicated by the presence of several differentiation stages in their life cycle. This article focuses on some recent advances that have revealed the unexpected nature of the epigenetic determinants present in protozoa. The role of the enigmatic DNA methyltransferase Dnmt2 in unicellular organisms is discussed.

  11. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis.

    Science.gov (United States)

    Ceccarelli, Marcello; Galluzzi, Luca; Diotallevi, Aurora; Andreoni, Francesca; Fowler, Hailie; Petersen, Christine; Vitale, Fabrizio; Magnani, Mauro

    2017-05-16

    Leishmaniasis 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. DNA 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. The 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 C q values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C q values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples

  12. Leishmania diagnostic and identification py using 32P labelled DNA probes

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Melo, Maria Norma de

    1999-10-01

    P 32 labelled DNA probes are valious instruments for the parasitic diseases by using hybridization reaction. In this paper we describe the methodology and present the foundations for the radioactive probes production, based on the kinetoplast DNA (kDNA), for the Leishmania diagnostic an identification. We also describe the kDNA purification protocol from Leishmania reference cepa, the process of P 32 labelling of the kDNA by using the nick translation method, gathering, sample preparation and treatment, the optimum conditions for the hybridization reaction and the procedures for the autoradiography

  13. Effect of ionizing radiation on the morphology, physiology and growth of Leishmania ssp

    International Nuclear Information System (INIS)

    Bonetti, Franco C.; Spencer, Patrick J.; Nascimento, Nanci do; Junior A, Heitor F.

    2000-01-01

    The Leishmania spp is a pathogenic protozoan, which cause different diseases in man. The human diseases, in America, caused by this group of protozoa are divided in cutaneous or tegumentar and visceral, known as kala-azar. In this work, our principal study object was the specie that causes tegumentar leishmaniasis, in Brazil. Metabolic studies of cellular respiration and proteins and nucleic acids synthesis were accomplished using radiation as a form of sterilizing the parasites without however affecting their immunogenic capacity The promastigotes forms of irradiated Leishmania spp were totally sterilized with the dose of 1500 Gy, with their reproductive and nucleic acids, as well as protein synthesis capacity blocked. (author)

  14. Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

    Science.gov (United States)

    Rosales-Chilama, Mariana; Gongora, Rafael E; Valderrama, Liliana; Jojoa, Jimena; Alexander, Neal; Rubiano, Luisa C; Cossio, Alexandra; Adams, Emily R; Saravia, Nancy G; Gomez, María Adelaida

    2015-12-01

    The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof

  15. Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

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    Mariana Rosales-Chilama

    2015-12-01

    Full Text Available The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking.We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed.Persistent Leishmania infection was demonstrated in 40% (46 of 114 of leishmanin skin test (LST positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total. Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia subgenus.Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping

  16. Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference

    Science.gov (United States)

    2013-01-01

    Background Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis. Methods We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods. Results Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution. Conclusions The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have

  17. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

    OpenAIRE

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have bee...

  18. Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background.

    Directory of Open Access Journals (Sweden)

    Saskia Decuypere

    Full Text Available The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L. donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread

  19. Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

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    Pinto Aldair JW

    2011-12-01

    Full Text Available Abstract Background The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT, including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania. Methods Twenty mongrel dogs naturally infected with Leishmania (Leishmania infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania. The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs. Results The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT. Conclusion The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance.

  20. Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

    Science.gov (United States)

    Mandal, Goutam; Mandal, Srotoswati; Sharma, Mansi; Charret, Karen Santos; Papadopoulou, Barbara; Bhattacharjee, Hiranmoy; Mukhopadhyay, Rita

    2015-01-01

    Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species. PMID:25714343

  1. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. AFLP polymorphisms allow high resolution genetic analysis of American Tegumentary Leishmaniasis agents circulating in Panama and other members of the Leishmania genus.

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    Carlos M Restrepo

    Full Text Available American Tegumentary Leishmaniasis is caused by parasites of the genus Leishmania, and causes significant health problems throughout the Americas. In Panama, Leishmania parasites are endemic, causing thousands of new cases every year, mostly of the cutaneous form. In the last years, the burden of the disease has increased, coincident with increasing disturbances in its natural sylvatic environments. The study of genetic variation in parasites is important for a better understanding of the biology, population genetics, and ultimately the evolution and epidemiology of these organisms. Very few attempts have been made to characterize genetic polymorphisms of parasites isolated from Panamanian patients of cutaneous leishmaniasis. Here we present data on the genetic variability of local isolates of Leishmania, as well as specimens from several other species, by means of Amplified Fragment Length Polymorphisms (AFLP, a technique seldom used to study genetic makeup of parasites. We demonstrate that this technique allows detection of very high levels of genetic variability in local isolates of Leishmania panamensis in a highly reproducible manner. The analysis of AFLP fingerprints generated by unique selective primer combinations in L. panamensis suggests a predominant clonal mode of reproduction. Using fluorescently labeled primers, many taxon-specific fragments were identified which may show potential as species diagnostic fragments. The AFLP permitted a high resolution genetic analysis of the Leishmania genus, clearly separating certain groups among L. panamensis specimens and highly related species such as L. panamensis and L. guyanensis. The phylogenetic networks reconstructed from our AFLP data are congruent with established taxonomy for the genus Leishmania, even when using single selective primer combinations. Results of this study demonstrate that AFLP polymorphisms can be informative for genetic characterization in Leishmania parasites, at

  3. Disseminated feline leishmaniosis due to Leishmania infantum in Southern France.

    Science.gov (United States)

    Ozon, C; Marty, P; Pratlong, F; Breton, C; Blein, M; Lelièvre, A; Haas, P

    1998-02-28

    A fortuitously discovered case of feline leishmaniosis is reported. The parasites were found in the skin and the bone marrow of a domestic female cat that spontaneously died after a few weeks of evolution. Serological tests for FeLV, FIV and PIF virus detection gave negative results. By using Western blot serology, a characteristic pattern of leishmaniosis was obtained and by performing an isoenzyme electrophoresis, a Leishmania infantum MON-1 strain was identified. The same zymodeme is implicated in most of the canine and human leishmaniosis in Southern Europe. A study on the prevalence of asymptomatic feline leismaniosis is foreseen.

  4. Detection of different Leishmania spp. and Trypanosoma cruzi antibodies in cats from the Yucatan Peninsula (Mexico) using an iron superoxide dismutase excreted as antigen.

    Science.gov (United States)

    Longoni, Silvia S; López-Cespedes, Angeles; Sánchez-Moreno, Manuel; Bolio-Gonzalez, Manuel E; Sauri-Arceo, Carlos H; Rodríguez-Vivas, Roger I; Marín, Clotilde

    2012-09-01

    Although human leishmaniasis has been reported in 20 states in Mexico, no case of leishmaniasis has been reported in cats to date. In the Yucatan Peninsula, it has been found that dogs may act as reservoirs for at least three Leishmania species (Leishmania mexicana, Leishmania braziliensis, and Leishmania panamensis). In this study we identified specific antibodies against these three Leishmania spp. and Trypanosoma cruzi in the sera from 95 cats from two States on the Yucatan Peninsula, namely Quintana Roo and Yucatan, by ELISA and Western blot techniques using whole extract and an iron superoxide dismutase excreted by the parasites as antigens. As well as demonstrating the presence of trypanosomatid antibodies in the feline population on the Yucatan Peninsula, we were also able to confirm the high sensitivity and specificity of the iron superoxide dismutase antigen secreted by them, which may prove to be very useful in epidemiological studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Comparison of Protein Extracts from Various Unicellular Green Sources

    NARCIS (Netherlands)

    Teuling, Emma; Wierenga, Peter A.; Schrama, Johan W.; Gruppen, Harry

    2017-01-01

    Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation

  6. Compositional patterns in the genomes of unicellular eukaryotes.

    Science.gov (United States)

    Costantini, Maria; Alvarez-Valin, Fernando; Costantini, Susan; Cammarano, Rosalia; Bernardi, Giorgio

    2013-11-05

    The genomes of multicellular eukaryotes are compartmentalized in mosaics of isochores, large and fairly homogeneous stretches of DNA that belong to a small number of families characterized by different average GC levels, by different gene concentration (that increase with GC), different chromatin structures, different replication timing in the cell cycle, and other different properties. A question raised by these basic results concerns how far back in evolution the compartmentalized organization of the eukaryotic genomes arose. In the present work we approached this problem by studying the compositional organization of the genomes from the unicellular eukaryotes for which full sequences are available, the sample used being representative. The average GC levels of the genomes from unicellular eukaryotes cover an extremely wide range (19%-60% GC) and the compositional patterns of individual genomes are extremely different but all genomes tested show a compositional compartmentalization. The average GC range of the genomes of unicellular eukaryotes is very broad (as broad as that of prokaryotes) and individual compositional patterns cover a very broad range from very narrow to very complex. Both features are not surprising for organisms that are very far from each other both in terms of phylogenetic distances and of environmental life conditions. Most importantly, all genomes tested, a representative sample of all supergroups of unicellular eukaryotes, are compositionally compartmentalized, a major difference with prokaryotes.

  7. Association of pro-inflammatory cytokines and iron regulatory protein 2 (IRP2 with Leishmania burden in canine visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Paulo Ricardo Porfírio do Nascimento

    Full Text Available Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2 were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.

  8. Leishmania amazonensis DNA in wild females of Lutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil.

    Science.gov (United States)

    Oliveira, Everton Falcão de; Casaril, Aline Etelvina; Mateus, Nathália Lopes Fontoura; Murat, Paula Guerra; Fernandes, Wagner Souza; Oshiro, Elisa Teruya; Oliveira, Alessandra Gutierrez de; Galati, Eunice Aparecida Bianchi

    2015-12-01

    Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmaniain wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruziwere dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

  9. In vitro and in vivo activity of an organic tellurium compound on Leishmania (Leishmania chagasi.

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    Isabella Aparecida Salerno Pimentel

    Full Text Available Tellurium compounds have shown several biological properties and recently the leishmanicidal effect of one organotellurane was demonstrated. These findings led us to test the effect of the organotellurium compound RF07 on Leishmania (Leishmania chagasi, the agent of visceral leishmaniasis in Latin America. In vitro assays were performed in L. (L. chagasi-infected bone marrow derived macrophages treated with different concentrations of RF07. In in vivo experiments Golden hamsters were infected with L. (L. chagasi and injected intraperitoneally with RF07 whereas control animals received either Glucantime or PBS. The effect of RF07 on cathepsin B activity of L. (L. chagasi amastigotes was assayed spectrofluorometrically using fluorogenic substrates. The main findings were: 1 RF07 showed significant leishmanicidal activity against intracellular parasites at submicromolar concentrations (IC50 of 529.7±26.5 nM, and the drug displayed 10-fold less toxicity to macrophages (CC50 of 5,426±272.8 nM; 2 kinetics assays showed an increasing leishmanicidal action of RF07 at longer periods of treatment; 3 one month after intraperitoneal injection of RF07 L. (L. chagasi-infected hamsters showed a reduction of 99.6% of parasite burden when compared to controls that received PBS; 4 RF07 inhibited the cathepsin B activity of L. (L. chagasi amastigotes. The present results demonstrated that the tellurium compound RF07 is able to destroy L. (L. chagasi in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support further study of the potential of RF07 as a possible alternative for the chemotherapy of visceral leishmaniasis.

  10. Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

    Science.gov (United States)

    Carvalho, G M L; Brazil, R P; Rêgo, F D; Ramos, M C N F; Zenóbio, A P L A; Andrade Filho, J D

    2017-01-01

    Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    Science.gov (United States)

    Isnard, Amandine; Shio, Marina T.; Olivier, Martin

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

  12. Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

    Science.gov (United States)

    da Silva, Edson R; Boechat, Nubia; Pinheiro, Luiz C S; Bastos, Monica M; Costa, Carolina C P; Bartholomeu, Juliana C; da Costa, Talita H

    2015-11-01

    Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking the glycosomal addressing signal and one in which the arginase-coding gene was knocked out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a potential drug target for Leishmania treatment. In an attempt to search for arginase inhibitors, 29 derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were tested against Leishmania (Leishmania) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold containing R1  = CF3 exhibited greater activity against the arginase rather than when the substituent R1  = CH3 in the 2-position. The novel compound 2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and IC50 values for arginase inhibition estimated to be 17 ± 1 μm and 16.5 ± 0.5 μm, respectively. These results can guide the development of new drugs against leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the arginase enzyme. © 2015 John Wiley & Sons A/S.

  13. Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania.

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    Frédéric Bringaud

    2007-09-01

    Full Text Available Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis, Trypanosoma brucei (sleeping sickness, and Trypanosoma cruzi (Chagas disease. Analysis of their recently completed genomes confirmed the presence of non-long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements--LmSIDER1 (785 copies and LmSIDER2 (1,073 copies--that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are approximately 70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3'-untranslated regions (3'UTRs of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3'UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

  14. Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

    Science.gov (United States)

    Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-11-01

    We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

  15. Transmissibility of Leishmania infantum from maned wolves (Chrysocyon brachyurus) and bush dogs (Speothos venaticus) to Lutzomyia longipalpis.

    Science.gov (United States)

    Mol, Juliana P S; Soave, Semíramis A; Turchetti, Andréia P; Pinheiro, Guilherme R G; Pessanha, Angela T; Malta, Marcelo C C; Tinoco, Herlandes P; Figueiredo, Luiza A; Gontijo, Nelder F; Paixão, Tatiane A; Fujiwara, Ricardo T; Santos, Renato L

    2015-09-15

    Leishmania (Leishmania) infantum is the cause of visceral leishmaniasis in the Americas. The disease is transmitted mostly through the bite of the invertebrate vector, the phlebotomine Lutzomyia longipalpis in the New World. Although the domestic dog is considered the most important reservoir of the disease, other mammalian, including wildlife, are susceptible to infection. The goal of this study was to perform xenodiagnosis to evaluate the capacity of naturally infected maned wolves (Chrysocyon brachyurus) and bush dogs (Speothos venaticus) to transmit Leishmania infantum to female sand flies (L. longipalpis). Xenodiagnoses were performed in February and August, 2013, when 77.7% (three maned wolves and four bush dogs) or 100% of the animals were positive, respectively. However, parasite loads in the engorged sand flies was low (longipalpis, although the parasite loads in engorged phlebotomines exposed to these animals were very low. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. The proliferation potential of promastigotes of the main Leishmania species of the old world in NNN culture medium prepared using blood of four different mammals.

    Science.gov (United States)

    Ladopoulos, Theodoros; Ntais, Pantelis; Tsirigotakis, Nikolaos; Dokianakis, Emmanouil; Antoniou, Maria

    2015-10-01

    The efficacy of the in vitro cultivation of promastigotes of four Leishmania spp. was tested in the biphasic Novy-MacNeal-Nicolle (NNN) medium prepared using blood from different animals (horse, donkey, goat and sheep). The aim was to test which NNN preparation gave the best yield in the shortest time for different parasite species, in order to obtain a large crop of promastigotes for experimental work and for antigen preparation. Promastigotes of Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania major, the four main parasite species occurring in the old world, were defrosted from -80 °C and placed, at equal numbers, in the 4 different NNN preparations. At the end of the 7th day, the NNN medium using horse blood produced the greatest number of promastigotes for all Leishmania spp. tested, whilst goat blood proved the poorest medium, providing culture results only for L. infantum. This finding may be explained by the fact that Leishmania is a nicotinamide adenine dinucleotide (NAD) auxotroph and horse erythrocytes support NAD-dependent microorganisms. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Natural infection of Didelphis aurita (Mammalia: Marsupialia with Leishmania infantum in Brazil

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    Carreira João Carlos

    2012-06-01

    Full Text Available Abstract Background The opossum Didelphis have been considered as natural hosts of Leishmania parasites in the New World, suggesting an important role in the epidemiology of Visceral Leishmaniasis (VL. Among six extant species that belong to the genus Didelphis, only two (D. marsupialis and D. albiventris, have been mentioned as natural hosts of Leishmania infantum in Brazil and Colombia. In the present paper, it is reported for the first time, the observation of intracellular parasites (amastigotes in tissues of Didelphis aurita naturally infected with Leishmania infantum in Brazil. We also discuss some aspects associated to the relationship between L. infantum and the geographical distribution of some species of the genus Didelphis. Methods The opossums studied were caught by wire traps (Tomahawk in Barra de Guaratiba, a peri-urban area in Rio de Janeiro. The opossums were killed with an overdose of Thiopental sodium.At necropsy, macroscopic alterations were examined and samples from liver, spleen, lymph nodes, ear, abdominal skin, scent glands and bone marrow were collected for parasitological and molecular diagnoses. Results Forty-eight opossums were captured in an AVL endemic region, 30 being caught in a mangrove area and eighteen animals in a forest area near to some residential-yards. Among the thirty opossums trapped in the mangrove area, all of them were negative by both imprint and sera samples assayed on Dipstick Tests, that is a test based on a combination of protein-A colloidal gold conjugate and rk39 Leishmania antigen to detect anti-Leishmania antibody in serum or plasma. At the macroscopic examination one out of eighteen opossums, caught close to the forest, presented alterations compatible with spleen hypertrophy and three were positive by Dipstick Tests (16.6% and presented amastigotes in the spleen and in one of them, the parasites were also observed in a submandibular lymph node. Leishmania infantum infections were confirmed

  18. Natural infection of Didelphis aurita (Mammalia: Marsupialia) with Leishmania infantum in Brazil.

    Science.gov (United States)

    Carreira, João Carlos Araujo; da Silva, Alba Valéria Machado; de Pita Pereira, Daniela; Brazil, Reginaldo Peçanha

    2012-06-07

    The opossum Didelphis have been considered as natural hosts of Leishmania parasites in the New World, suggesting an important role in the epidemiology of Visceral Leishmaniasis (VL). Among six extant species that belong to the genus Didelphis, only two (D. marsupialis and D. albiventris), have been mentioned as natural hosts of Leishmania infantum in Brazil and Colombia. In the present paper, it is reported for the first time, the observation of intracellular parasites (amastigotes) in tissues of Didelphis aurita naturally infected with Leishmania infantum in Brazil. We also discuss some aspects associated to the relationship between L. infantum and the geographical distribution of some species of the genus Didelphis. The opossums studied were caught by wire traps (Tomahawk) in Barra de Guaratiba, a peri-urban area in Rio de Janeiro. The opossums were killed with an overdose of Thiopental sodium.At necropsy, macroscopic alterations were examined and samples from liver, spleen, lymph nodes, ear, abdominal skin, scent glands and bone marrow were collected for parasitological and molecular diagnoses. Forty-eight opossums were captured in an AVL endemic region, 30 being caught in a mangrove area and eighteen animals in a forest area near to some residential-yards. Among the thirty opossums trapped in the mangrove area, all of them were negative by both imprint and sera samples assayed on Dipstick Tests, that is a test based on a combination of protein-A colloidal gold conjugate and rk39 Leishmania antigen to detect anti-Leishmania antibody in serum or plasma. At the macroscopic examination one out of eighteen opossums, caught close to the forest, presented alterations compatible with spleen hypertrophy and three were positive by Dipstick Tests (16.6%) and presented amastigotes in the spleen and in one of them, the parasites were also observed in a submandibular lymph node. Leishmania infantum infections were confirmed through dot blot hybridization using a L. infantum

  19. Natural infection of phlebotomines (Diptera: Psychodidae) by Leishmania (Leishmania) amazonensis in an area of ecotourism in Central-Western Brazil.

    Science.gov (United States)

    Brilhante, Andreia Fernandes; Nunes, Vânia Lúcia Brandão; Kohatsu, Kleber Augusto; Galati, Eunice Aparecida Bianchi; Rocca, Maria Elizabeth Ghizzi; Ishikawa, Edna Aoba Yassui

    2015-01-01

    Bonito municipality, known as an area of ecoturism, in Mato Grosso do Sul state, Brazil, is also a focus of visceral and cutaneous leishmaniases, with cases registered in both human and canine populations. This study sought to investigate natural infection by flagellate forms of Leishmania in phlebotomines of the urban area of Bonito. Sand flies were collected fortnightly from October 2005 to July 2006 with modified automatic light traps installed in peridomiciles and animal shelters in the center and on the outskirts of the city. The females were dissected and their guts observed under an optical microscope. A total of 1977 specimens were captured, Lutzomyia longipalpis (88.4 %) and Bichromomyia flaviscutelata (3.0 %) being the most frequent species. Bi. flaviscutellata was found infected by flagellates that were identified as Leishmania (Leishmania) amazonensis by indirect immunofluorescence reaction, employing monoclonal antibodies and the biotin-avidin system. This is the first report of natural infection by L. amazonensis in Bi. flaviscutellata in a Brazilian urban area. As Bi. flaviscutellata is only slightly attracted by humans, the transmission of L. amazonensis in the study area may have a zoonotic character; however, the sympatric occurrence of this parasite and Lu. longipalpis should be taken into consideration by the local health authorities since this sand fly has already been found with L. amazonensis DNA in a focus of canine visceral leishmaniasis in Bonito municipality.

  20. Direct detection of Leishmania from clinical samples.

    Science.gov (United States)

    Waitumbi, John N; Bast, Joshua; Nyakoe, Nancy; Magiri, Charles; Quintana, Miguel; Takhampunya, Ratree; Schuster, Anthony L; Van de Wyngaerde, Marshall T; McAvin, James C; Coleman, Russell E

    2017-01-01

    The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

  1. Phototoxic effects of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) on the viability of Leishmania major and Leishmania braziliensis promastigotes

    Science.gov (United States)

    Guerra Pinto, Juliana; Ferreira-Strixino, Juliana; Mittmann, Josane

    2016-06-01

    American cutaneous leishmaniasis (ACL) is an infectious disease caused by protozoans of the genus Leishmania. The treatment may consist of pentavalent antimonials or pentamidine and amphotericin. However, these treatments are extremely aggressive. Photodynamic antimicrobial chemotherapy (PACT) involves the same mechanism of photodynamic therapy which associates a photosensitizer with oxygen and a light source generating a photochemical reaction leading to cell death. The aim of this study was to verify the potential use of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) compound in photodynamic treatment through evaluation of its phototoxic effect in promastigotes of the genus Leishmania braziliensis and Leishmania major. Treatment with SiPc was able to drastically affect the viability of the parasites as well as affect their growth and morphology, after PACT treatment. The data shown in this study allows us to conclude that SiPc is a promising photosensitizer (PS) since it does not affect parasite growth and viability in the dark. After PACT with this phthalocyanine, over 99% of parasites were killed with the higher concentration and a light dose used. These results suggest that SiPc can be used in future to treat CL, however, further studies are necessary to determine whether the PS are toxic to mononuclear phagocytic cells and epithelial cells which will also be affected by therapy when applied topically.

  2. Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin.

    Science.gov (United States)

    Prestes, Suzane Ribeiro; Guerra, Jorge Augusto de Oliveira; Romero, Gustavo Adolfo Sierra; Magalhaes, Laylah Kelre Costa; Santana, Rosa Amelia Gonçalves; Maciel, Marcel Gonçalves; Custódio, Ana; Barbosa, Maria das Graças Vale; Silveira, Henrique

    2015-01-01

    In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species. We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses. Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis. The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

  3. Update on pathology of ocular parasitic disease.

    Science.gov (United States)

    Das, Dipankar; Ramachandra, Varsha; Islam, Saidul; Bhattacharjee, Harsha; Biswas, Jyotirmay; Koul, Akanksha; Deka, Panna; Deka, Apurba

    2016-11-01

    Parasites are a group of eukaryotic organisms that may be free-living or form a symbiotic or parasitic relationship with the hosts. Consisting of over 800,000 recognized species, parasites may be unicellular (Protozoa) or multicellular (helminths and arthropods). The association of parasites with human population started long before the emergence of civilization. Parasitic zoonotic diseases are prevalent worldwide including India. Appropriate epidemiological data are lacking on existing zoonotic parasitic diseases, and newer diseases are emerging in our scenario. Systemic diseases such as cysticercosis, paragonimiasis, hydatidosis, and toxoplasmosis are fairly common. Acquired Toxoplasma infections are rising in immune-deficient individuals. Amongst the ocular parasitic diseases, various protozoas such as Cystoidea, trematodes, tissue flagellates, sporozoas etc. affect humans in general and eyes in particular, in different parts of the world. These zoonoses seem to be a real health related problem globally. Recent intensification of research throughout the world has led to specialization in biological fields, creating a conducive situation for researchers interested in this subject. The basics of parasitology lie in morphology, pathology, and with recent updates in molecular parasitology, the scope has extended further. The current review is to address the recent update in ophthalmic parasites with special reference to pathology and give a glimpse of further research in this field.

  4. Update on pathology of ocular parasitic disease

    Directory of Open Access Journals (Sweden)

    Dipankar Das

    2016-01-01

    Full Text Available Parasites are a group of eukaryotic organisms that may be free-living or form a symbiotic or parasitic relationship with the hosts. Consisting of over 800,000 recognized species, parasites may be unicellular (Protozoa or multicellular (helminths and arthropods. The association of parasites with human population started long before the emergence of civilization. Parasitic zoonotic diseases are prevalent worldwide including India. Appropriate epidemiological data are lacking on existing zoonotic parasitic diseases, and newer diseases are emerging in our scenario. Systemic diseases such as cysticercosis, paragonimiasis, hydatidosis, and toxoplasmosis are fairly common. Acquired Toxoplasma infections are rising in immune-deficient individuals. Amongst the ocular parasitic diseases, various protozoas such as Cystoidea, trematodes, tissue flagellates, sporozoas etc. affect humans in general and eyes in particular, in different parts of the world. These zoonoses seem to be a real health related problem globally. Recent intensification of research throughout the world has led to specialization in biological fields, creating a conducive situation for researchers interested in this subject. The basics of parasitology lie in morphology, pathology, and with recent updates in molecular parasitology, the scope has extended further. The current review is to address the recent update in ophthalmic parasites with special reference to pathology and give a glimpse of further research in this field.

  5. Identification of six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay.

    Science.gov (United States)

    Hernández, Carolina; Alvarez, Catalina; González, Camila; Ayala, Martha Stella; León, Cielo Maritza; Ramírez, Juan David

    2014-11-14

    Leishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species. Herein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL. This is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

  6. Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    Science.gov (United States)

    Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

    2016-01-01

    The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

  7. Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

    Science.gov (United States)

    Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

    2015-01-01

    Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

  8. Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania amazonensis in the state of Mato Grosso do Sul, mid-western Brazil Capturas de flebotomíneos com armadilhas de Disney em área de ocorrência de Leishmania (Leishmania amazonensis no estado de Mato Grosso do Sul, região Centro-Oeste do Brasil

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    Maria Elizabeth Cavalheiros Dorval

    2010-10-01

    Full Text Available INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3% and Bi flaviscutellata (41.4%, present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania amazonensis. CONCLUSIONS: Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.INTRODUÇÃO: O estudo foi realizado com o objetivo de estudar a fauna de flebotomíneos (Diptera: Psychodidae e aspectos ligados à transmissão da leishmaniose tegumentar americana em uma área florestal com ocorrência de Leishmania (Leishmania amazonensis, situada no município de Bela Vista, Estado do Mato Grosso do Sul, Brasil. MÉTODOS: As capturas de flebotomíneos foram realizadas utilizando-se armadilhas tipo Disney modificadas, com isca roedor, Mesocricetus auratus, no período de maio de 2004 a janeiro de 2006. RESULTADOS: As coletas resultaram na identificação de 10 espécies de Phlebotominae

  9. Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease.

    Science.gov (United States)

    Scorza, Breanna M; Wacker, Mark A; Messingham, Kelly; Kim, Peter; Klingelhutz, Aloysius; Fairley, Janet; Wilson, Mary E

    2017-10-01

    All Leishmania species parasites are introduced into mammalian skin through a sand fly bite, but different species cause distinct clinical outcomes. Mouse studies suggest that early responses are critical determinants of subsequent adaptive immunity in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant cell in the epidermis. We hypothesized that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with Leishmania infantum or Leishmania major, which cause visceral or cutaneous leishmaniasis, respectively, elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of proinflammatory genes for IL-6, IL-8, tumor necrosis factor, and IL-1B, whereas keratinocytes exposed to several L. major isolates did not. Furthermore, keratinocyte-monocyte co-incubation studies across a 4 µM semipermeable membrane suggested that L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication (P Leishmania species that may affect the course of disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Total Leishmania antigens with Poly(I:C) induce Th1 protective response.

    Science.gov (United States)

    Sanchez, M V; Eliçabe, R J; Di Genaro, M S; Germanó, M J; Gea, S; García Bustos, M F; Salomón, M C; Scodeller, E A; Cargnelutti, D E

    2017-11-01

    Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries. © 2017 John Wiley & Sons Ltd.

  11. Mitochondrial associated ubiquitin fold modifier-1 mediated protein conjugation in Leishmania donovani.

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    Sreenivas Gannavaram

    2011-01-01

    Full Text Available In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1 and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2 specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

  12. Leishmania (Viannia) braziliensis infection in wild small mammals in ecotourism area of Brazil.

    Science.gov (United States)

    Tonelli, Gabriel Barbosa; Tanure, Aline; Rego, Felipe Dutra; Carvalho, Gustavo Mayr de Lima; Stumpp, Rodolfo; Ássimos, Gabriela Ribeiro; Campos, Aldenise Martins; Lima, Ana Cristina Viana Mariano da Rocha; Gontijo, Célia Maria Ferreira; Paz, Gustavo Fontes; Andrade Filho, José Dilermando

    2017-01-01

    Leishmaniases are parasitic diseases transmitted to mammalian hosts by sand fly vectors (Diptera: Psychodidae). Despite the increasing occurrence of visceral and cutaneous leishmaniasis cases in urban centers, their transmission still occur primarily in wild environments and may be associated with professional activities and recreation, such as ecotourism. The Reserva Particular do Patrimônio Natural Santuário do Caraça (RPPNSC) is one of the largest ecotourism attractions in the State of Minas Gerais, Brazil, and comprises an area of environmental preservation with 11,233 hectares presenting a transitional vegetation between Cerrado and Atlantic Forest. The present study describes the abundance of small mammals in RPPNSC, the isolation and identification of Leishmania in five wild animals. Small mammals were bimonthly trapped along 6 trails within the RPPNSC with 10 Tomahawk traps each. Two trails were located in peridomiciliary areas near tourist lodging facilities, and four trails were located at sites visited by tourists in forest areas. The most prevalent species were Akodon cursor, Cerradomys subflavus and Oligoryzomys nigripes. Six isolates of Leishmania were obtained from these animals and identified as Leishmania braziliensis through HSP70-PCR RFLP method. Leishmania spp. DNA was detected by kDNA-PCR method and isolated by biphasic culture. Studies point to some of the captured species as potential wild reservoirs of Leishmania, suggesting they may be involved in the transmission cycle in these wild environments.

  13. Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco.

    Science.gov (United States)

    Echchakery, Mohamed; Chicharro, Carmen; Boussaa, Samia; Nieto, Javier; Carrillo, Eugenia; Sheila, Ortega; Moreno, Javier; Boumezzough, Ali

    2017-10-02

    Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites' transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported. Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species. A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%). To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco.

  14. Differentiation of Leishmania species by FT-IR spectroscopy

    Science.gov (United States)

    Aguiar, Josafá C.; Mittmann, Josane; Ferreira, Isabelle; Ferreira-Strixino, Juliana; Raniero, Leandro

    2015-05-01

    Leishmaniasis is a parasitic infectious disease caused by protozoa that belong to the genus Leishmania. It is transmitted by the bite of an infected female Sand fly. The disease is endemic in 88 countries Desjeux (2001) [1] (16 developed countries and 72 developing countries) on four continents. In Brazil, epidemiological data show the disease is present in all Brazilian regions, with the highest incidences in the North and Northeast. There are several methods used to diagnose leishmaniasis, but these procedures have many limitations, are time consuming, have low sensitivity, and are expensive. In this context, Fourier Transform Infrared Spectroscopy (FT-IR) analysis has the potential to provide rapid results and may be adapted for a clinical test with high sensitivity and specificity. In this work, FT-IR was used as a tool to investigate the promastigotes of Leishmaniaamazonensis, Leishmaniachagasi, and Leishmaniamajor species. The spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. Results: cluster analysis found four specific regions that are able to identify the Leishmania species. The dendrogram representation clearly indicates the heterogeneity among Leishmania species. The band deconvolution done by the curve fitting in these regions quantitatively differentiated the polysaccharides, amide III, phospholipids, proteins, and nucleic acids. L. chagasi and L. major showed a greater biochemistry similarity and have three bands that were not registered in L. amazonensis. The L. amazonensis presented three specific bands that were not recorded in the other two species. It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. The high sensitivity and specificity of this technique opens up the possibilities for further studies about characterization of other microorganisms.

  15. Recent Advances in Vaccines Against Leishmania Based on Patent Applications.

    Science.gov (United States)

    Thomaz-Soccol, Vanete; Ferreira da Costa, Eduardo Scopel; Karp, Susan Grace; Junior Letti, Luiz Alberto; Soccol, Flavia Thomaz; Soccol, Carlos Ricardo

    2018-01-01

    Leishmaniasis is caused by parasites of the genus Leishmania, and represents a group of chronic diseases with an epidemiological and clinical diversity. The disease is endemic in tropical regions, being found in 98 countries, affecting around 12 million people, with an estimated increase of 1.5 million per year. The present review aims to analyze recent and most important patents regarding development of vaccines to improve immunization against leishmaniasis. For this purpose, the Web of Science - Derwent Innovations Index was consulted. There is also a short description of the licensed vaccines already on the market for commercialization, and a critical opinion on future developments. The data herein presented comprises national and international filings, thus considering the patent's country of origin, and can be used an indicator of a country's technological development regarding a specific field. Several types of vaccines against Leishmania were studied. The main classes comprise: vaccines using live cells (virulent or attenuated); dead cells; containing recombinant protein; using DNA of the parasite. United States (74 patents) leads the ranking of patent applications for vaccines against Leishmania, followed by Brazil (36 patents), which is an endemic region of leishmaniasis with 20,000 human cases of cutaneous leishmaniasis and over 3,000 cases of visceral form. This review showed that there is still a lot of space for development regarding the creation of a feasible, effective vaccine against leishmaniasis. The scientific community appears to be taking steps in the right direction, though. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Impact of Leishmania mexicana infection on dendritic cell signaling and functions.

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    Irazú Contreras

    2014-09-01

    Full Text Available Leishmania parasites have the ability to modify macrophage signaling pathways in order to survive and multiply within its mammalian host. They are also known to invade other cells including neutrophils, fibroblasts and dendritic cells (DCs. DCs have an important role in immunity as the link between innate and adaptive immunity, necessary for the development of an effective response; however, the impact of Leishmania mexicana infection on DCs has been poorly studied. Herein, we report that Leishmania infection rapidly induced DC protein tyrosine phosphatases activity, leading to MAP kinases inactivation. In line with this, L. mexicana was found to decrease the nuclear translocation of transcription factors such as AP-1 and NF-κB. Concomitantly, L. mexicana-infected DCs showed reduced expression of several surface antigen-presenting and co-stimulatory molecules upon LPS stimulation. Leishmania-induced interference on DC maturation was further reflected by their reduced capacity to present OVA antigen to OVA-specific T cells, as shown by abrogation of IL-2 production by the T cells. Collectively, our data revealed that DC infection by L. mexicana appears to affect the cellular and immunological mechanisms necessary for the development of an effective and protective immune response, therefore favouring the survival and propagation of the parasite within its host.

  17. Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection.

    Science.gov (United States)

    Glennie, Nelson D; Yeramilli, Venkata A; Beiting, Daniel P; Volk, Susan W; Weaver, Casey T; Scott, Phillip

    2015-08-24

    Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4+ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4+ TRM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells. © 2015 Glennie et al.

  18. Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil.

    Science.gov (United States)

    Carvalho, Gustavo M L; Andrade Filho, Jose D; Falcao, Alda L; Rocha Lima, Ana C V M; Gontijo, Celia M F

    2008-06-01

    In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.

  19. Parasites: Water

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    ... Consultations, and General Public. Contact Us Parasites Home Water Language: English Español (Spanish) Recommend on Facebook Tweet Share Compartir Parasites can live in natural water sources. When outdoors, treat your water before drinking ...

  20. Detection of Leishmania spp in silvatic mammals and isolation of Leishmania (Viannia braziliensis from Rattus rattus in an endemic area for leishmaniasis in Minas Gerais State, Brazil.

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    Agnes Antônia Sampaio Pereira

    Full Text Available Knowledge of potential reservoirs of Leishmania spp. in an anthropic environment is important so that surveillance and control measures can be implemented. The aim of this study was to investigate the infection by Leishmania in small mammals in an area located in Minas Gerais, Brazil, that undergoes changes in its natural environment and presents autochthonous human cases of cutaneous leishmaniasis (CL and visceral leishmaniasis (VL. For the capture of the animals, Sherman and Tomahawk traps were used and distributed in the peridomicile of houses with reports of autochthonous cases of CL or VL. Six catches were carried out on two consecutive nights with intervals of two months during one year and samples of spleen, liver, tail skin, ear skin and bone marrow of the animals were obtained. Parasitological and molecular methods were used to detect the infection. Identification of the Leishmania species was performed by PCR RFLPhsp70. Twenty five animals of four species were captured: ten Rattus rattus, nine Didelphis albiventris, five Cerradomys subflavus and one Marmosops incanus. In the PCR-hsp70, five animals were positive (20%. The Leishmania species identified in PCR-RFLPhsp70 were: Leishmania braziliensis in D. albiventris (2, C. subflavus (1 and R. rattus (1 and Leishmania infantum in R. rattus (1. The highest positivity rate for L. braziliensis was obtained in the liver samples. The spleen was the only tissue positive for L. infantum. It was isolated in culture medium L. braziliensis from two samples (liver and spleen of R. rattus. This is the first record of isolation of L. braziliensis from R. rattus in the southeastern region of Brazil. These results are relevant to the knowledge of the epidemiology of leishmaniasis in the region, mainly in the investigation of the presence of hosts and possible reservoirs of the parasite.

  1. Using the marine unicellular algae in biological monitoring

    OpenAIRE

    Kapkov V. I.; Shoshina E. V.; Belenikina O. A.

    2017-01-01

    The possibility of using marine unicellular algae from natural plankton community in biomonitoring of pollution by heavy metals has been investigated. Algae of different taxa from the Mediterranean Sea have been allocated to culture. In the laboratory the culture conditions – i. e. growth medium, temperature, photoperiod, level of artificial light and initial density – have been selected for every species. The impact of heavy metals (Hg, Cd, Cu, Pb) in the form of chloride salts on the growth...

  2. Molecular analysis of the replication program in unicellular model organisms

    OpenAIRE

    Raghuraman, M. K.; Brewer, Bonita J.

    2010-01-01

    Eukaryotes have long been reported to show temporal programs of replication, different portions of the genome being replicated at different times in S phase, with the added possibility of developmentally regulated changes in this pattern depending on species and cell type. Unicellular model organisms, primarily the budding yeast Saccharomyces cerevisiae, have been central to our current understanding of the mechanisms underlying the regulation of replication origins and the temporal program o...

  3. Persistence of Leishmania antigen in C57Bl/6j inbred mice infected with Leishmania (Leishmania amazonensis Persistência do antígeno da Leishmania no camundongo isogênico C57Bl/6j infectado com a Leishmania (Leishmania amazonensis

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    C. Vasconcellos

    1999-07-01

    Full Text Available PURPOSE. To develop an animal model for studying mucocutaneous leishmaniasis. METHODS. The hind footpad of C57Bl/6j inbred mice was experimentally infected with 10(7 Leishmania (Leishmania amazonensis promastigote and the skin was studied through light and electron transmission microscopy and immunohistochemistry (PAP techniques. RESULTS. There were morphological evidences of cellular immune mechanisms and hypersensitivity reaction after eight weeks of infection and metastasis and well shaped parasites at ultrastructural level by fifty-one weeks post infection. Relapse of infection with mucosa lesions occurred around the 50th week after inoculation. CONCLUSION. The use of this animal model in long term follow up could be an useful experimental model for human mucocutaneous leishmaniasis.OBJETIVO. Desenvolver um modelo experimental para o estudo da leishmaniose cutâneo-mucosa. MÉTODOS. O coxim plantar traseiro de camundongos isogênicos C57Bl/6j foi inoculado com 10(7 formas promastigotas da Leishmania (Leishmania amazonensis e a pele foi estudada através da microscopia óptica e eletrônica e de técnica imunohistoquímica (PAP. RESULTADOS. Ocorreram evidências morfológicas de mecanismos imunes mediados por células, concomitantemente ao de reação de hipersensibilidade, após a oitava semana de infecção e a presença de parasitas com ultraestrutura preservada na quinquagésima primeira semana após a infecção. Houve recidiva da infecção com surgimento de lesões mucosas por volta da 50a semana pós inoculação. CONCLUSÃO. Este modelo animal, com um período de tempo de seguimento prolongado, poderia ser empregado como modelo para o estudo experimental da leishmaniose cutâneo-mucosa.

  4. On the paradigm of altruistic suicide in the unicellular world.

    Science.gov (United States)

    Nedelcu, Aurora M; Driscoll, William W; Durand, Pierre M; Herron, Matthew D; Rashidi, Armin

    2011-01-01

    Altruistic suicide is best known in the context of programmed cell death (PCD) in multicellular individuals, which is understood as an adaptive process that contributes to the development and functionality of the organism. After the realization that PCD-like processes can also be induced in single-celled lineages, the paradigm of altruistic cell death has been extended to include these active cell death processes in unicellular organisms. Here, we critically evaluate the current conceptual framework and the experimental data used to support the notion of altruistic suicide in unicellular lineages, and propose new perspectives. We argue that importing the paradigm of altruistic cell death from multicellular organisms to explain active death in unicellular lineages has the potential to limit the types of questions we ask, thus biasing our understanding of the nature, origin, and maintenance of this trait. We also emphasize the need to distinguish between the benefits and the adaptive role of a trait. Lastly, we provide an alternative framework that allows for the possibility that active death in single-celled organisms is a maladaptive trait maintained as a byproduct of selection on pro-survival functions, but that could-under conditions in which kin/group selection can act-be co-opted into an altruistic trait. © 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.

  5. Multimodal sensorimotor system in unicellular zoospores of a fungus.

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    Swafford, Andrew J M; Oakley, Todd H

    2018-01-19

    Complex sensory systems often underlie critical behaviors, including avoiding predators and locating prey, mates and shelter. Multisensory systems that control motor behavior even appear in unicellular eukaryotes, such as Chlamydomonas , which are important laboratory models for sensory biology. However, we know of no unicellular opisthokonts that control motor behavior using a multimodal sensory system. Therefore, existing single-celled models for multimodal sensorimotor integration are very distantly related to animals. Here, we describe a multisensory system that controls the motor function of unicellular fungal zoospores. We found that zoospores of Allomyces arbusculus exhibit both phototaxis and chemotaxis. Furthermore, we report that closely related Allomyces species respond to either the chemical or the light stimuli presented in this study, not both, and likely do not share this multisensory system. This diversity of sensory systems within Allomyces provides a rare example of a comparative framework that can be used to examine the evolution of sensory systems following the gain/loss of available sensory modalities. The tractability of Allomyces and related fungi as laboratory organisms will facilitate detailed mechanistic investigations into the genetic underpinnings of novel photosensory systems, and how multisensory systems may have functioned in early opisthokonts before multicellularity allowed for the evolution of specialized cell types. © 2018. Published by The Company of Biologists Ltd.

  6. Metazoan-like signaling in a unicellular receptor tyrosine kinase

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    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  7. Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani.

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    Anjali Kumari

    Full Text Available Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl ethylenediamine (TPEN and Zinc Sulfate (ZnSO4. Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death

  8. A defined medium for Leishmania culture allows definition of essential amino acids.

    Science.gov (United States)

    Nayak, Archana; Akpunarlieva, Snezhana; Barrett, Michael; Burchmore, Richard

    2018-02-01

    Axenic culture of Leishmania is generally performed in rich, serum-supplemented media which sustain robust growth over multiple passages. The use of such undefined media, however, obscures proteomic analyses and confounds the study of metabolism. We have established a simple, defined culture medium that supports the sustained growth of promastigotes over multiple passages and which yields parasites that have similar infectivity to macrophages to parasites grown in a conventional semi-defined medium. We have exploited this medium to investigate the amino acid requirements of promastigotes in culture and have found that phenylalanine, tryptophan, arginine, leucine, lysine and valine are essential for viability in culture. Most of the 20 proteogenic amino acids promote growth of Leishmania promastigotes, with the exception of alanine, asparagine, and glycine. This defined medium will be useful for further studies of promastigote substrate requirements, and will facilitate future proteomic and metabolomic analyses. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth

    DEFF Research Database (Denmark)

    Lynn, Miriam A.; Kindrachuk, Jason; Marr, Alexandra K.

    2011-01-01

    Background: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under...... of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We...... with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model. Conclusions/Significance: Interestingly, D-BMAP-28 appears to be the most potent...

  10. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

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    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  11. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    Science.gov (United States)

    Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

    2016-02-01

    Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  12. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Zampieri

    2016-02-01

    Full Text Available Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.Exploring the High Resolution Melting (HRM dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR targeting heat-shock protein 70 coding gene (hsp70 revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania infantum chagasi, L. (L. amazonensis, L. (L. mexicana, L. (Viannia lainsoni, L. (V. braziliensis, L. (V. guyanensis, L. (V. naiffi and L. (V. shawi, and three species found in Eurasia and Africa, including L. (L. tropica, L. (L. donovani and L. (L. major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  13. Social Parasites

    Science.gov (United States)

    Lopez, Miguel A.; Nguyen, HoangKim T.; Oberholzer, Michael; Hill, Kent L.

    2011-01-01

    Summary of recent advances Protozoan parasites cause tremendous human suffering worldwide, but strategies for therapeutic intervention are limited. Recent studies illustrate that the paradigm of microbes as social organisms can be brought to bear on questions about parasite biology, transmission and pathogenesis. This review discusses recent work demonstrating adaptation of social behaviors by parasitic protozoa that cause African sleeping sickness and malaria. The recognition of social behavior and cell-cell communication as a ubiquitous property of bacteria has transformed our view of microbiology, but protozoan parasites have not generally been considered in this context. Works discussed illustrate the potential for concepts of sociomicrobiology to provide insight into parasite biology and should stimulate new approaches for thinking about parasites and parasite-host interactions. PMID:22020108

  14. Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

    Science.gov (United States)

    Bakhshi, H; Oshaghi, M A; Abai, M R; Rassi, Y; Akhavan, A A; Sheikh, Z; Mohtarami, F; Saidi, Z; Mirzajani, H; Anjomruz, M

    2013-10-01

    A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Antileishmanial activity and tubulin polymerization inhibition of podophyllotoxin derivatives on Leishmania infantum

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    José Miguel Escudero-Martínez

    2017-12-01

    Full Text Available Leishmania microtubules play an important role not only in cell division, but also in keeping the shape of the parasite and motility of its free-living stages. Microtubules result from the self-assembly of alpha and beta tubulins, two phylogenetically conserved and very abundant eukaryotic proteins in kinetoplastids. The colchicine binding domain has inspired the discovery and development of several drugs currently in clinical use against parasites. However, this domain is less conserved in kinetoplastids and may be selectively targeted by new compounds. This report shows the antileishmanial effect of several series of compounds (53, derived from podophyllotoxin (a natural cyclolignan isolated from rhizomes of Podophyllum spp. and podophyllic aldehyde, on a transgenic, fluorescence-emitting strain of Leishmania infantum. These compounds were tested on both promastigotes and amastigote-infected mouse splenocytes, and in mammalian – mouse non-infected splenocytes and liver HepG2 cells – in order to determine selective indexes of the drugs. Results obtained with podophyllotoxin derivatives showed that the hydroxyl group at position C-7α was a structural requisite to kill the parasites. On regards podophyllic aldehyde, derivatives with C9-aldehyde group integrated into a bicyclic heterostructure displayed more potent antileishmanial effects and were relatively safe for host cells. Docking studies of podophyllotoxin and podophyllic aldehyde derivatives showed that these compounds share a similar pattern of interaction at the colchicine site of Leishmania tubulin, thus pointing to a common mechanism of action. However, the results obtained suggested that despite tubulin is a remarkable target against leishmaniasis, there is a poor correlation between inhibition of tubulin polymerization and antileishmanial effect of many of the compounds tested, fact that points to alternative pathways to kill the parasites. Keywords: Leishmania, Tubulin, DNA

  16. Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

    Czech Academy of Sciences Publication Activity Database

    Grekov, Igor; Svobodová, M.; Nohýnková, E.; Lipoldová, Marie

    2011-01-01

    Roč. 87, č. 3 (2011), s. 273-277 ISSN 0167-7012 R&D Projects: GA MŠk(CZ) LC06009; GA ČR GA310/08/1697 Institutional research plan: CEZ:AV0Z50520514 Keywords : Leishmania * SNB-9 * biphasic culture medium * parasite infectivity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.086, year: 2011

  17. Isolation of Leishmania braziliensis from Lutzomyia ovallesi (Diptera:Psychodidae) in Guatemala.

    Science.gov (United States)

    Rowton, E D; de Mata, M; Rizzo, N; Porter, C H; Navin, T R

    1992-04-01

    Leishmania braziliensis is endemic in Guatemala and Belize in Central America. To help identify the vector(s) of this parasite in Guatemala, phlebotomine sand flies that were aspirated from the clothing of collectors at Tikal National Park in the Department of the Peten were examined for flagellates. Lutzomyia ovallesi was found infected with flagellates that were identified as L. braziliensis by isoenzyme electrophoresis. The isoenzyme profile of this isolate matched those from humans from the same area.

  18. Whatman Paper (FTA Cards) for Storing and Transferring Leishmania DNA for PCR Examination

    OpenAIRE

    A Amin-Mohammadi; E Eskandari; AA Akhavan; M Ganjbakhsh; Z Hosseininejad; M Afzalaghaei; F Berenji; M Mohajery; A Khamesipour; A Fata

    2009-01-01

    "nBackground: Diagnosis of cutaneous leishmaniasis (CL) is often made based on clinical manifesta­tion. Correct diagnosis and identification of the parasite are crucial for choosing the effective treat­ment and for epidemiological studies. On the other hand, determination of Leishmania species is nec­essary for designing appropriate control programs. Diagnosis by PCR is becoming a 'gold standard'. For PCR preparation, storage and shipments of specimens are necessary. In this study, ...

  19. Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

    Directory of Open Access Journals (Sweden)

    Daniel P Depledge

    2010-09-01

    Full Text Available A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes.We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are

  20. An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

    Science.gov (United States)

    Costa Duarte, Mariana; dos Reis Lage, Letícia Martins; Lage, Daniela Pagliara; Mesquita, Juliana Tonini; Salles, Beatriz Cristina Silveira; Lavorato, Stefânia Neiva; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Alves, Ricardo José; Tavares, Carlos Alberto Pereira; Tempone, André Gustavo; Coelho, Eduardo Antonio Ferraz

    2016-02-15

    The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

    Science.gov (United States)

    de Almeida, Marcos E; Koru, Ozgur; Steurer, Francis; Herwaldt, Barbara L; da Silva, Alexandre J

    2017-01-01

    Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate. Copyright © 2016 American Society for Microbiology.

  2. HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Lívia O Santos

    Full Text Available BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania

  3. Differential Midgut Attachment of Leishmania (Viannia braziliensis in the Sand Flies Lutzomyia (Nyssomyia whitmani and Lutzomyia (Nyssomyia intermedia

    Directory of Open Access Journals (Sweden)

    Rodrigo P. Soares

    2010-01-01

    Full Text Available The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG. An upregulation of β-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V. braziliensis with its natural vectors Lutzomyia (Nyssomyia intermedia and Lutzomyia (Nyssomyia whitmani. Using two in vitro binding techniques, phosphoglycans (PGs derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was ∼11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL in Brazil is discussed.

  4. Characterization of isotypes of antibody response against leishmania parasite

    Energy Technology Data Exchange (ETDEWEB)

    Elassad, Asma M.S.; Ghalib, Hashim W [Medical Parasitology Project NIH/Sudan, Khartoum (Sudan); Younis, Saddia A [Department of Zoology, Faculty of Science, University of Khartoum, Khartoum (Sudan)

    1994-12-01

    In this study an enzyme linked immunosorbent assay (ELIZA) was developed to detect IgG,IgM and IgA response in visceral leishmaniasis patients (VL) against L.donovain and L. major antigens compared to control groups; cutaneous leishmaniasis patients (CL), mucosal leishmaniasis patients (ML), patients with other tropical diseases and healthy controls.Highly specific IgG were found in VL patients with test specificity (93.7%) and sensitivity(93.4%). A moderate IgG were found in VL patients but non-specific while no IgA were detected in all studied groups. Also VL patients showed high specificity and sensitivity (95.2 and 96.6% respectively) against L.major antigen.The distribution of IgG subclasses (IgG1,IgG2,IgG3 and IgG4) antibodies in VL patients were assayed.IgG3 showed the highest specificity and sensitivity and titers followed by IgG1.Also the diagnostic value of ELIZA test for different leishmaniasis forms were discussed. (Author). 18 refs., 1 fig., 3 tabs.

  5. Characterization of isotypes of antibody response against leishmania parasite

    International Nuclear Information System (INIS)

    Elassad, Asma M.S.; Ghalib, Hashim W.; Younis, Saddia A.

    1994-01-01

    In this study an enzyme linked immunosorbent assay (ELIZA) was developed to detect IgG,IgM and IgA response in visceral leishmaniasis patients (VL) against L.donovain and L. major antigens compared to control groups; cutaneous leishmaniasis patients (CL), mucosal leishmaniasis patients (ML), patients with other tropical diseases and healthy controls.Highly specific IgG were found in VL patients with test specificity (93.7%) and sensitivity(93.4%). A moderate IgG were found in VL patients but non-specific while no IgA were detected in all studied groups. Also VL patients showed high specificity and sensitivity (95.2 and 96.6% respectively) against L.major antigen.The distribution of IgG subclasses (IgG1,IgG2,IgG3 and IgG4) antibodies in VL patients were assayed.IgG3 showed the highest specificity and sensitivity and titers followed by IgG1.Also the diagnostic value of ELIZA test for different leishmaniasis forms were discussed. (Author)

  6. Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

    OpenAIRE

    Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

    2010-01-01

    Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gen...

  7. Long- and short-term selective forces on malaria parasite genomes

    DEFF Research Database (Denmark)

    Nygaard, Sanne; Braunstein, Alexander; Malsen, Gareth

    2010-01-01

    Plasmodium parasites, the causal agents of malaria, result in more than 1 million deaths annually. Plasmodium are unicellular eukaryotes with small ~23 Mb genomes encoding ~5200 protein-coding genes. The protein-coding genes comprise about half of these genomes. Although evolutionary processes ha...

  8. Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

    Directory of Open Access Journals (Sweden)

    Venkateswara Reddy Gogulamudi

    2015-10-01

    Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

  9. Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

    Science.gov (United States)

    Diaz, E; Zacarias, A K; Pérez, S; Vanegas, O; Köhidai, L; Padrón-Nieves, M; Ponte-Sucre, A

    2015-11-01

    In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

  10. Expression of hsa Let-7a MicroRNA of Macrophages Infected by Leishmania Major

    Directory of Open Access Journals (Sweden)

    Nooshin Hashemi

    2016-10-01

    Full Text Available Leishmaniasis is a vector-born disease caused by species of the genus Leishmania and is transmitted from host to host through the bite of an infected sandfly. MicroRNAs (miRNAs are non-coding small RNAs with 22-nucleotide length. They are involved in some biological and cellular processes. We aimed to evaluate the expression of let-7a in human macrophages miRNA when are infected by Leishmania major. We also evaluated the impact of Leishmania major infection on the expression of let-7a at two different times, 24 and 48 hours, after infection. Blood samples were collected from ten healthy volunteers with no history of leishmaniasis. Development of macrophages from peripheral monocytes and infection with stationary phase of Leishmania major promastigotes were done through serial cultures under 5% CO2 environment and 37C. To measure the expression levels of let-7a real-time PCR was performed with specific related primers using the SYBR® Green master mix Kit™. The real-time PCR showed let-7a was expressed in cells infected with parasites after 24 and 48h post-infection. Comparison of let-7a miRNA expression after 24 and 48 h revealed that let-7a miRNAs were down-regulated at 48 h post-infection more than 24h after infection. The results of this study suggest that according to the main function of miRNA in repression of mRNA translation it could be possible to manipulate host cells in order to alter miRNA levels and regulate macrophage functions after establishment of intracellular parasites such as Leishmania.

  11. Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

    Science.gov (United States)

    Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

    2013-01-11

    Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

  12. Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission.

    Directory of Open Access Journals (Sweden)

    Michaela Vlkova

    2011-10-01

    Full Text Available BACKGROUND: Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins. CONCLUSIONS: Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.

  13. Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

    Science.gov (United States)

    Sardar, Abul Hasan; Jardim, Armando; Ghosh, Ayan Kumar; Mandal, Abhishek; Das, Sushmita; Saini, Savita; Abhishek, Kumar; Singh, Ruby; Verma, Sudha; Kumar, Ajay; Das, Pradeep

    2016-01-01

    Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments

  14. Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management.

    Directory of Open Access Journals (Sweden)

    Abul Hasan Sardar

    2016-03-01

    Full Text Available Reactive oxygen and nitrogen species (ROS and RNS produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL. There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed

  15. Centrins in unicellular organisms: functional diversity and specialization.

    Science.gov (United States)

    Zhang, Yu; He, Cynthia Y

    2012-07-01

    Centrins (also known as caltractins) are conserved, EF hand-containing proteins ubiquitously found in eukaryotes. Similar to calmodulins, the calcium-binding EF hands in centrins fold into two structurally similar domains separated by an alpha-helical linker region, shaping like a dumbbell. The small size (15-22 kDa) and domain organization of centrins and their functional diversity/specialization make them an ideal system to study protein structure-function relationship. Here, we review the work on centrins with a focus on their structures and functions characterized in unicellular organisms.

  16. Winter-summer succession of unicellular eukaryotes in a meso-eutrophic coastal system.

    Science.gov (United States)

    Christaki, Urania; Kormas, Konstantinos A; Genitsaris, Savvas; Georges, Clément; Sime-Ngando, Télesphore; Viscogliosi, Eric; Monchy, Sébastien

    2014-01-01

    The objective of this study was to explore the succession of planktonic unicellular eukaryotes by means of 18S rRNA gene tag pyrosequencing in the eastern English Channel (EEC) during the winter to summer transition. The 59 most representative (>0.1%, representing altogether 95% of total reads), unique operational taxonomic units (OTUs) from all samples belonged to 18 known high-level taxonomic groups and 1 unaffiliated clade. The five most abundant OTUs (69.2% of total reads) belonged to Dinophyceae, Cercozoa, Haptophyceae, marine alveolate group I, and Fungi. Cluster and network analysis between samples distinguished the winter, the pre-bloom, the Phaeocystis globosa bloom and the post-bloom early summer conditions. The OTUs-based network revealed that P. globosa showed a relatively low number of connections-most of them negative-with all other OTUs. Fungi were linked to all major taxonomic groups, except Dinophyceae. Cercozoa mostly co-occurred with the Fungi, the Bacillariophyceae and several of the miscellaneous OTUs. This study provided a more detailed exploration into the planktonic succession pattern of the EEC due to its increased depth of taxonomic sampling over previous efforts based on classical monitoring observations. Data analysis implied that the food web concept in a coastal system based on predator-prey (e.g. grazer-phytoplankton) relationships is just a part of the ecological picture; and those organisms exploiting a variety of strategies, such as saprotrophy and parasitism, are persistent and abundant members of the community.

  17. Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

    Science.gov (United States)

    MacLean, Lorna; Price, Helen; O'Toole, Peter

    2016-01-01

    Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

  18. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru

    Science.gov (United States)

    Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area. PMID:26735142

  19. Mechanisms of cellular invasion by intracellular parasites.

    Science.gov (United States)

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  20. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after...... the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL...... stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from...

  1. Fish parasites

    DEFF Research Database (Denmark)

    This book contains 22 chapters on some of the most important parasitic diseases in wild and farmed fish. International experts give updated reviews and provide solutions to the problems......This book contains 22 chapters on some of the most important parasitic diseases in wild and farmed fish. International experts give updated reviews and provide solutions to the problems...

  2. Parasitic diseases

    International Nuclear Information System (INIS)

    Rozenshtraukh, L.S.

    1983-01-01

    Foundations of roentgenological semiotics of parasitic diseases of lungs, w hich are of the greatest practical value, are presented. Roentgenological pictu res of the following parasitic diseases: hydatid and alveolar echinococcosis, pa ragonimiasis, toxoplasmosis, ascariasis, amebiasis, bilharziasis (Schistosomias is) of lungs, are considered

  3. Seroepidemiology of Leishmania spp. in dogs residing in Telêmaco Borba, Paraná, Brazil

    Directory of Open Access Journals (Sweden)

    Caroline Constantino

    2014-12-01

    Full Text Available Leishmaniasis is an important metazoonosis caused by protozoa of the genus Leishmania and has a heteroxenic life cycle involving invertebrate and vertebrate hosts. Transmission occurs during the blood meal of infected phlebotomine sand flies in wild species, domestic animals, and humans. The dog is a reservoir for the parasite causing visceral leishmaniasis (VL, whereas in American tegumentary leishmaniasis (ATL, dogs are erratic hosts that are accidentally infected, as in humans. Dogs are considered an important indicator of the parasite and its vectors in the environment, thus highlighting the importance of diagnosis in these animals. This study aimed to assess the seroepidemiology of Leishmania spp. in dogs in the municipality of Telêmaco Borba that were part of a castration campaign. Blood samples from 191 dogs were collected, and their owners were surveyed on various epidemiological variables. Serological analysis was performed using indirect immunofluorescence (IIF and rapid immunochromatography (DPP®. Screening by IIF identified 13 (6.81% positive animals, none of which were positive for the DPP® test, which is specific for VL. Statistical analysis of the questionnaire responses indicated a significant association between seropositivity and the presence of stacked or composting leaves in the backyard (p = 0.0498, forest areas (squares, woods, parks near the residence (p = 0.0015, and poorly healing ulcerated or nodular epidermal lesions in the dog (p = 0.0138. This study revealed the presence of anti-Leishmania spp. IgG antibodies in dogs residing in Telêmaco Borba, suggesting the presence of the parasite and vector in the environment. In addition, the existence of stacked or composting leaves in the backyard, forest areas near the residence, and epidermal lesions in dogs are factors associated with Leishmania spp. infection in pet dogs.

  4. Gentamicin-attenuated Leishmania infantum: a clinicopathological study in dogs.

    Science.gov (United States)

    Daneshvar, Hamid; Molaei, Mohammad M; Afshar, Reza Malekpour; Kamiabi, Hosein; Burchmore, Richard; Hagan, Paul; Phillips, R Stephen

    2009-05-15

    The clinicopathological changes following infection with an attenuated line of Leishmania infantum (L. infantum H-line) were evaluated in mixed breed dogs. Two groups of dogs were infected intravenously (i.v.) or intradermally (i.d.) with L. infantum H-line and two control groups were infected i.v. or i.d. with L. infantum wild-type (L. infantum WT). None of the dogs, which were infected i.v. or i.d. with L. infantum H-line, showed any abnormalities during the observation period. In contrast, two out of three dogs, which were infected i.v. with L. infantum WT, developed clinical signs of disease. In addition, no histopathological changes were seen in the liver and spleen of the dogs infected with the attenuated line of parasite, whereas the histopathological changes in the two dogs infected i.v. with L. infantum WT were severe in form and manifested by infiltration of high numbers of inflammatory cells. No promastigotes were found in cultures set up from spleens and livers of dogs infected with L. infantum H-line at 12 months post-infection, whereas promastigotes were seen in the spleen and liver cultures from 2 dogs infected i.v. with L. infantum WT. Serum levels of total IgG anti-Leishmania antibody were raised in all dogs. The antibody level in the serum of dogs infected i.v. with L. infantum WT was higher than that in dogs infected with L. infantum H-line. These results show no clinicopathological abnormalities in the dogs infected with gentamicin-attenuated L. infantum H-line. Moreover, L. infantum H-line induced IgG anti-Leishmania antibody in the dogs.

  5. Comparison of Protein Extracts from Various Unicellular Green Sources.

    Science.gov (United States)

    Teuling, Emma; Wierenga, Peter A; Schrama, Johan W; Gruppen, Harry

    2017-09-13

    Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation techniques, proteins were extracted and isolated from four different unicellular sources (Arthrospira (spirulina) maxima, Nannochloropsis gaditana, Tetraselmis impellucida, and Scenedesmus dimorphus). Despite differences in protein contents of the sources (27-62% w/w) and in protein extractability (17-74% w/w), final protein isolates were obtained that had similar protein contents (62-77% w/w) and protein yields (3-9% w/w). Protein solubility as a function of pH was different between the sources and in ionic strength dependency, especially at pH < 4.0. Overall, the characterization and extraction protocol used allows a relatively fast and well-described isolation of purified proteins from novel protein sources.

  6. Experimental mixed infection of Leishmania (Leishmania) amazonensis and Leishmania (L.) infantum in hamsters (Mesocricetus auratus).

    Science.gov (United States)

    DE Lima Celeste, Jordanna Luíza; Venuto Moura, Ana Paula; França-Silva, João Carlos; Matos DE Sousa, Gabriela; Oliveira Silva, Soraia; Norma Melo, Maria; Luiz Tafuri, Wagner; Carvalho Souza, Carolina; Monteiro DE Andrade, Hélida

    2017-08-01

    In South America, visceral leishmaniasis is frequently caused by Leishmania infantum and, at an unknown frequency, by Leishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections with L. amazonensis and L. infantum in a hamster model. We show that mixed infections are associated with more severe clinical disease than infection with L. amazonensis or L. infantum alone. In spleens with mixed infections, L. infantum outcompeted L. amazonensis in the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected with L. infantum. Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected with L. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected with L. infantum. This finding has important implications regarding the biology of Leishmania and humoral immune responses to infections with these organisms.

  7. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

    Science.gov (United States)

    Zhang, Wen-Wei; Matlashewski, Greg

    2015-07-21

    The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this

  8. Leishmania species identification using FTA card sampling directly from patients' cutaneous lesions in the state of Lara, Venezuela.

    Science.gov (United States)

    Kato, Hirotomo; Watanabe, Junko; Mendoza Nieto, Iraida; Korenaga, Masataka; Hashiguchi, Yoshihisa

    2011-10-01

    A molecular epidemiological study was performed using FTA card materials directly sampled from lesions of patients with cutaneous leishmaniasis (CL) in the state of Lara, Venezuela, where causative agents have been identified as Leishmania (Viannia) braziliensis and L. (Leishmania) venezuelensis in previous studies. Of the 17 patients diagnosed with CL, Leishmania spp. were successfully identified in 16 patients based on analysis of the cytochrome b gene and rRNA internal transcribed spacer sequences. Consistent with previous findings, seven of the patients were infected with L. (V.) braziliensis. However, parasites from the other nine patients were genetically identified as L. (L.) mexicana, which differed from results of previous enzymatic and antigenic analyses. These results strongly suggest that L. (L.) venezuelensis is a variant of L. (L.) mexicana and that the classification of L. (L.) venezuelensis should be reconsidered. Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

  9. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    International Nuclear Information System (INIS)

    Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

    2007-01-01

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

  10. Effect of Kelussia odoratissima Mozaff essential oil on promastigot form of Leishmania major (in vitro

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    Pirali Kheirabadi Khodadad

    2015-01-01

    Full Text Available Introduction: Leishmaniasis is a zoonotic disease caused by a protozoan of the genus Leishmania. In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were studied. Methods: In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were assessed by calculating the average number of surviving promastigots after exposure to different concentrations of essential oil, relative to the control Glucantime, at different time intervals. To achieve this, various essential oil concentrations (7.5 μl, 15 μl, 25 μl, 35.25 μl, 50 μl were added to parasites. Different groups in this study were kept in a 26°C incubator under identical conditions. 24, 48 and 72 hours after incubation, living promastigots were counted. Results: The effect of the essential oil of Kelussia odoratissima Mozaff differed from the negative and positive controls and depended on the concentration: higher concentrations (35.25 μl, 50 μl had a stronger effect on promastigots, causing total mortality. Conclusion: This study showed that Kelussia odoratissima Mozaff essential oil had effects on promastigot form of Leishmania major. So it might be possible to use the essential oil of Kelussia odoratissima instead of chemical drugs.

  11. Occurrence of Leishmania (Leishmania chagasi in a domestic cat (Felis catus in Andradina, São Paulo, Brazil: case report Ocorrência de Leishmania (Leishmania chagasi em gato doméstico (Felis catus em Andradina, São Paulo, Brasil: relato de caso

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    Willian Marinho Dourado Coelho

    2010-12-01

    Full Text Available This work describes natural infection by Leishmania in a domestic cat where amastigote forms of the parasite were observed in the popliteal lymph node imprint. Positive and negative serological reactions were observed by enzyme-linked immunosorbent assay (ELISA and indirect immunofluorescence assay (IFA, respectively. Polymerase chain reaction (PCR revealed that the nucleotide sequence of the sample was identical to Leishmania (L. chagasi. This is the first report of the disease in felines of the city of Andradina, SP, an area considered endemic for canine and human visceral leishmaniasis.Neste trabalho, é relatada a infecção natural por Leishmania em um gato doméstico no qual, formas amastigotas do parasito foram observadas em imprint de linfonodo poplíteo. Reações sorológicas positivas e negativas foram observadas pelo teste de imunoadsorção enzimática (ELISA e reação de imunofluorescência indireta (RIFI, respectivamente. A reação em cadeia da polimerase (PCR revelou que a sequência de nucleotídeos foi idêntica à Leishmania (L. chagasi. Este é o primeiro relato da doença em felino da cidade de Andradina, Estado de São Paulo, Brasil, área considerada endêmica para leishmaniose visceral canina e humana.

  12. Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis.

    Science.gov (United States)

    Ito, Marcos Massayuki; Catanhêde, Lilian Motta; Katsuragawa, Tony Hiroshi; Silva Junior, Cipriano Ferreira da; Camargo, Luis Marcelo Aranha; Mattos, Ricardo de Godoi; Vilallobos-Salcedo, Juan Miguel

    2015-01-01

    Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  13. Histochemical and molecular evaluation of the prevalence of Leishmania spp. in hematophagous insects

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    Willian Marinho Dourado Coelho

    2016-06-01

    Full Text Available The prevalence study of Leishmania spp. in hematophagous insects captured from the environment in bat roosts and pigeon nests, or feeding their hosts (cattle, pigs, horses, dogs and humans in urban, peri-urban and rural areas, between 2012 and 2014. For this study, the amastigotes present in these insects were detected by histochemical and PCR techniques. Positive gene amplification for Leishmania was found in two horseflies of the species Tabanus importunus collected in the environment, and amastigote forms of Leishmania spp., as well as erythrocytes and leukocytes, were histochemically detected in one of that insect. The other analyzed insects were not positive by PCR our by direct parasitological examination. Only horseflies captured in urban and peri-urban areas were positive. During the collection, no phlebotomine sand flies were captured in rural areas far from the city limits. It can be concluded that the discovery of horseflies positive for Leishmania spp. in urban and peri-urban areas indicates the likelihood that urban areas and their surroundings provide vector parasites with an environment suitable for the spread and consequent perpetuation of the biological cycle of this protozoan.

  14. The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

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    Sergio Sifontes-Rodríguez

    2015-04-01

    Full Text Available Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC and 50% inhibitory concentration (IC50 values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl-furan (furvina and 2-bromo-5-(2-methyl-2-nitrovinyl-furan (UC245 also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r (a furvina-containing antifungal ointment for the treatment of CL.

  15. In-silico Leishmania Target Selectivity of Antiparasitic Terpenoids

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    Ifedayo Victor Ogungbe

    2013-07-01

    Full Text Available Neglected Tropical Diseases (NTDs, like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

  16. Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

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    R Salehi

    2009-07-01

    Full Text Available "nBackground: Leishmania is an obligatory intracellular protozoan parasite, which infects human be­ings when infected sand fly vector takes a blood meal.  Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has spe­cial im­portant. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. "nMethods: Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was de­signed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. "nResults: Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltrans­ferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and de­posited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank "nConclusion: We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully.

  17. Contribution of arsenic species in unicellular algae to the cycling of arsenic in marine ecosystems.

    Science.gov (United States)

    Duncan, Elliott G; Maher, William A; Foster, Simon D

    2015-01-06

    This review investigates the arsenic species produced by and found in marine unicellular algae to determine if unicellular algae contribute to the formation of arsenobetaine (AB) in higher marine organisms. A wide variety of arsenic species have been found in marine unicellular algae including inorganic species (mainly arsenate--As(V)), methylated species (mainly dimethylarsenate (DMA)), arsenoribosides (glycerol, phosphate, and sulfate) and metabolites (dimethylarsenoethanol (DMAE)). Subtle differences in arsenic species distributions exist between chlorophyte and heterokontophyte species with As(V) commonly found in water-soluble cell fractions of chlorophyte species, while DMA is more common in heterokontophyte species. Additionally, different arsenoriboside species are found in each phyla with glycerol and phosphate arsenoribosides produced by chlorophytes, whereas glycerol, phosphate, and sulfate arsenoribosides are produced by heterokontophytes, which is similar to existing data for marine macro-algae. Although arsenoribosides are the major arsenic species in many marine unicellular algal species, AB has not been detected in unicellular algae which supports the hypothesis that AB is formed in marine animals via the ingestion and further metabolism of arsenoribosides. The observation of significant DMAE concentrations in some unicellular algal cultures suggests that unicellular algae-based detritus contains arsenic species that can be further metabolized to form AB in higher marine organisms. Future research establishing how environmental variability influences the production of arsenic species by marine unicellular algae and what effect this has on arsenic cycling within marine food webs is essential to clarify the role of these organisms in marine arsenic cycling.

  18. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests

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    Chen Hai-Tang

    2011-05-01

    Full Text Available Abstract Background Canine leishmaniasis (CanL is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China, which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp fragment of the conserved region in the minicircle kinetoplast DNA (kDNA and the results of phylogenetic analyses based on the sequence of the amplified fragment. Results The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease. Conclusions More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of

  19. Riesgo de transmisión de Leishmania (Kinetoplastida: Trypanosomatidae en Mérida Venezuela

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    Elsa Nieves

    2014-09-01

    Full Text Available La leishmaniasis es una enfermedad causada por la infección de un parásito protozoario del género Leishmania, transmitido por la picada de insectos hematófagos conocidos como flebotominos. El estudio tiene como objetivo determinar la presencia de flebotominos en los Distritos Sanitarios del estado Mérida y diseñar un mapa de riesgo de transmisión entomológico. Se utilizaron cuatro métodos de captura de flebotominos, los ejemplares se identificaron y se les determinó la infección natural por Leishmania. Se estimó la riqueza de especies, y se realizó un proceso analítico Jerárquico. Los resultados muestran la presencia de diversas especies de flebotominos en los Distritos Sanitarios del estado Mérida, siendo las especies de mayor frecuencia L. youngi, L. gomezi, L. ovallesi y L. walkeri. Se detectó 2,1% de infección natural con Leishmania, la cual se encontró en las 4 especies más frecuentes. Se presenta un mapa de riesgo de transmisión entomológico para el estado Mérida. El conocimiento de la situación actual de los vectores de Leishmania en el estado Mérida y el riesgo de transmisión son relevantes a la hora de considerar la prevención y posible surgimiento de nuevos brotes de leishmaniasis. Abstract (english The leishmaniasis is a disease caused by infection with a protozoan parasite of the genus Leishmania, transmitted by the bite of blood-sucking insects known as sandflies. The study aims to determine the presence of sandflies in Merida state health districts and design a map of entomological risk of transmission. Four methods capture sandflies were used, the specimens were identified and natural Leishmania infection was determined. The richness species was estimated and analityc Hierarchie procesess was performed. The results show the presence of various species of sandflies in Merida state health districts, L. youngi, L. gomezi, L. ovallesi and L. walkeri were most abundant species. The 2.1% of natural infection

  20. A non-commercial approach for the generation of transgenic Leishmania tarentolae and its application in antileishmanial drug discovery.

    Science.gov (United States)

    Pineda, Tatiana; Valencia, Yesenia; Flórez, María F; Pulido, Sergio A; Vélez, Iván D; Robledo, Sara M

    2016-08-01

    Leishmaniasis is a parasitic infection caused by several species of the genus Leishmania that is considered as a neglected disease. Drug development process requires a robust and updated high-throughput technology to the evaluation of candidate compounds that imply the manipulation of the pathogenic species of the parasite in the laboratory. Therefore, it is restricted to trained personal and level II biosafety environments. However, it has been established the utility of Leishmania tarentolae as a model for in vitro screening of antileishmanial agents without the necessity of level II biosafety setups. In parallel the transfection of Leishmania parasites with reporter genes as the eGFP using non-commercial integration vectors like the pIRmcs3(-) has proved to be a powerful tool for the implementation of semi automatized high-throughput platforms for the evaluation of antileishmanial compounds. Here we report the generation of a new L. tarentolae strain overexpressing the eGFP gene harboured by the non-commercial vector pIR3(-). We also demonstrate its utility for the semi-automatized screening of antileshmanial compounds in intracellular forms of the L. tarentolae parasite.

  1. Sequence analysis of the 3’-untranslated region of HSP70 (type I genes in the genus Leishmania: its usefulness as a molecular marker for species identification

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    Requena Jose M

    2012-04-01

    Full Text Available Abstract Background The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods In the present study, we analyzed the 3’-untranslated region (UTR of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3´-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.

  2. Leishmania spp. Epidemiology of Canine Leishmaniasis in the Yucatan Peninsula

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    A. López-Céspedes

    2012-01-01

    Full Text Available Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico. A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

  3. Leishmania spp. epidemiology of canine leishmaniasis in the Yucatan Peninsula.

    Science.gov (United States)

    López-Céspedes, A; Longoni, S S; Sauri-Arceo, C H; Sánchez-Moreno, M; Rodríguez-Vivas, R I; Escobedo-Ortegón, F J; Barrera-Pérez, M A; Bolio-González, M E; Marín, C

    2012-01-01

    Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico). A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

  4. Antimony Resistance in Leishmania, Focusing on Experimental Research

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    Fakhri Jeddi

    2011-01-01

    Full Text Available Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases. There are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions. However, resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules. In this paper, we reviewed the studies carried out on antimonial resistance in Leishmania. Several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related. Afterwards, phenotypic determination of resistance is described, then confronted to clinical outcome. Finally, we detail molecular mechanisms and targets involved in resistance and already identified in vitro within selected mutant strains or in clinical isolates.

  5. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

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    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  6. Studies on Using Cattle and Sheep Hydatid Cyst Fluid Instead of the Fetal Calf Serum in Leishmania Culture

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    Hossein Rezvan

    2013-12-01

    Full Text Available Background: Leishmania is a single cell parasite causing leishmaniasis, which is a common disease between humans and animals. Due to the importance of in-vitro culture of the parasite in leishmania research, developing new methods for in-vitro cultivation of the parasite has always been a goal for leishmania researchers. The main objective of7T 5T7Tthis study was to use sheep and bovine hydatid cyst fluids as alternatives for fetal calf serum (FCS in leishmania in-vitro5T culture5T. Materials and Methods: 7TA total of 5T7T1 million leishmania promastigotes were added to 4 flasks as follow5T7T. A f5T7Tlask containing DMEM medium with 105T7T% 5T7Tfetal bovine serum5T7T, a f5T7Tlask containing DMEM and 10% sheep hydatid cyst fluid5T7T, a f5T7Tlask containing DMEM medium with 105T7T% 5T7Tbovine hydatid cyst fluid and a5T7T f5T7Tlask containing DMEM medium alone. After 2, 45T7T, 5T7T7, 95T7T, 11, 5T7T21 and 24 days, the number of parasites were counted and compared5T7T. Results: The result of this study showed that, DMEM medium enriched with 10% sheep hydatid cyst fluid in 168 hours and medium enriched with 10% bovine hydatid cyst fluid in 96 hours can act as a good alternative for fetal bovine serum in the culture Leishmania major. Conclusion: 5TThe results showed that sheep and bovine hydatid cyst fluid can be used as alternatives to FCS for dense cultivation of leishmania. The results also showed that5T, 5Tthe growth of promastigotes in medium enriched with bovine cyst fluid is more rapid than the medium enriched with sheep5T c5Tyst fluid5T in5T the beginning of cultivation.

  7. Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

    Science.gov (United States)

    Barrera, Maria Claudia; Rojas, Laura Jimena; Weiss, Austin; Fernandez, Olga; McMahon-Pratt, Diane; Saravia, Nancy G; Gomez, Maria Adelaida

    2017-12-01

    The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results

  8. Detection and characterization of Leishmania in tissues of patients with post kala-azar dermal leishmaniasis using a specific monoclonal antibody

    DEFF Research Database (Denmark)

    Ismail, A; Kharazmi, A; Permin, H

    1997-01-01

    Sections from skin lesions and draining lymph nodes of patients with post kala-azar dermal leishmaniasis were examined using an immunoperoxidase method and a monoclonal antibody directed against Leishmania donovani. Parasites were detected in 22 of 25 biopsies (88%). In parallel sections stained...

  9. Study of ionizing radiation as a tool for select promastigotes forms of Leishmania Amazonensis, and the megalomaniac response in experimental models

    International Nuclear Information System (INIS)

    Bonetti, Franco Claudio

    2006-01-01

    Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a cutaneous disease to a visceral one. The cutaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a 60 Co font, had being successes used to promote physical-chemical transformations on different protozoan, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of meta cyclic forms of the parasite in axenic culture, for a possible efficient irradiated immuno gene production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metacyclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didn't produce immunoglobulin nor on the immunization or on the challenge. (author)

  10. Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications.

    Science.gov (United States)

    Chaara, Dhekra; Ravel, Christophe; Bañuls, Anne- Laure; Haouas, Najoua; Lami, Patrick; Talignani, Loïc; El Baidouri, Fouad; Jaouadi, Kaouther; Harrat, Zoubir; Dedet, Jean-Pierre; Babba, Hamouda; Pratlong, Francine

    2015-04-01

    The taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica. Thirty-five L. killicki and 25 L. tropica strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches. The results of the genetic and phylogenetic analyses strongly support the hypothesis that L. killicki belongs to the L. tropica complex. Our data suggest that L. killicki emerged from a single founder event and that it evolved independently from L. tropica. However, they do not validate the hypothesis that L. killicki is a distinct complex. Therefore, we suggest naming this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination. This study provides taxonomic and phylogenetic information on L. killicki and improves our knowledge on the evolutionary history of this taxon.

  11. Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

    Science.gov (United States)

    Bichaud, Laurence; Souris, Marc; Mary, Charles; Ninove, Laëtitia; Thirion, Laurence; Piarroux, Raphaël P; Piarroux, Renaud; De Lamballerie, Xavier; Charrel, Rémi N

    2011-09-01

    Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between

  12. Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

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    Laurence Bichaud

    2011-09-01

    Full Text Available Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus, an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i in individuals and (ii at a spatial level in the city of Marseille (south-eastern France. Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of

  13. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

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    Jose M. Requena

    2015-01-01

    Full Text Available Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges for drug discovery and improving of current treatments against leishmaniasis.

  14. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    Science.gov (United States)

    Requena, Jose M.; Montalvo, Ana M.; Fraga, Jorge

    2015-01-01

    Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis. PMID:26167482

  15. Can equids be a reservoir of Leishmania braziliensis in endemic areas?

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    Jessé Henrique Truppel

    Full Text Available In this study, we detected Leishmania (Viannia braziliensis infection in equids living in endemic regions of cutaneous leishmaniasis. To determine the role of these animals in the Leishmania cycle, we used two approaches: serological and molecular methods. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay (ELISA. Blood samples were collected and tested by polymerase chain reaction (PCR, and the positive products were sequenced. The results showed that 11.0% (25/227 of the equids were seropositive for Leishmania sp, and 16.3% (37/227 were PCR positive. Antibodies were detected in 20 horses, 3 donkeys, and 2 mules, and the parasite DNA was detected in 30 horses, 5 donkeys, and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. braziliensis. Our results show that equids are infected with L. braziliensis, which could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the cutaneous leishmaniasis cycle.

  16. Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media.

    Science.gov (United States)

    Castelli, Germano; Galante, Antonella; Lo Verde, Vincenza; Migliazzo, Antonella; Reale, Stefano; Lupo, Tiziana; Piazza, Maria; Vitale, Fabrizio; Bruno, Federica

    2014-04-01

    The aim of this study is to improve the cultivation of Leishmania promastigotes without the use of common, semisolid culture media such as Evans' modified Tobie's medium (EMTM), liquid RPMI 1640, and Peptone-yeast extract medium (P-Y). Although EMTM medium permits the growth of a high number of parasites, it is technically difficult to prepare as it requires the use of fresh rabbit blood from animals bred on farms, while RPMI 1640 and P-Y show lower growth rates than the EMTM. There is, therefore, a need to develop new blood-free and time-saving culture systems. The aim of this paper is to propose new modified microbiological media, named RPMI-PY and Tobie-PY, to isolate Leishmania and cultivate parasites for research and diagnostic purposes. This study compares classic culture media to the new media, RPMI-PY and Tobie-PY, and demonstrates that the new media have superior performance in terms of time and parasitic load. The growth rate of the parasite was significantly higher at 24, 48, and 72 hr cultivation, based on counts using Bürker's chambers, when compared to classic media. This study was carried out at the National References Centre for Leishmaniasis (C.Re.Na.L.) where the isolation procedures are conducted daily from a number of different biological matrices.

  17. Experimental Infection of Lutzomyia (Nyssomyia) whitmani (Diptera: Psychodidae: Phlebotominae) With Leishmania (Viannia) braziliensis and Leishmania (L.) amazonensis, Etiological Agents of American Tugumentary Leishmaniasis.

    Science.gov (United States)

    Fonteles, Raquel S; Pereira Filho, Adalberto A; Moraes, Jorge L P; Kuppinger, Oliver; Rebêlo, José M M

    2016-01-01

    Leishmania (L.) amazonensis (Lainson & Shaw, 1972) and Leishmania (Viannia) braziliensis (Vianna, 1911) are the principal causative agents of American tegumentary leishmaniasis (ATL) in Brazil. L. amazonensis also causes diffuse cutaneous leishmaniasis (DCL) vectored principally by Lutzomyia flaviscutellata and secondarily by Lutzomyia whitmani (Antunes & Coutinho, 1939). The latter is the most common phlebotomine in the state of Maranhão, and it is the focal species for potential ATL transmission. For this reason, we tested the ability of L. whitmani to become infected with Lutzomyia parasites. Phlebotomines were derived from a colony maintained in the laboratorial conditions. The first generation, uninfected females were offered a bloodmeal with mice infected with the strains of both parasites. We found that L. whitmani can become infected with both parasite species, with infection rates of 65.2% (L. braziliensis) and 47.4% (L. amazonensis). We conclude that in Maranhão, L. whitmani is likely an important vector in the transmission of ATL and may function as a vector of DCL. This possibility should be further investigated. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

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    Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

    2014-02-27

    The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

  19. Histopathological and parasitological investigations of ear healthy skin of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

    Science.gov (United States)

    Figueiredo, Maria Marta; Moura, Eliane Perlatto; Costa, Miriam Maria; Ribeiro, Vitor Marcio; Michalick, Marilene Suzan; Tafuri, Washington Luiz; Tafuri, Wagner Luiz

    2010-07-01

    Although 90% of clinical cases of American visceral leishmaniasis (AVL) occur in the northeastern region of Brazil, the incidence of cases in recent years has increased in southeastern states such as Minas Gerais (MG), where the disease has been reported in several cities, including Belo Horizonte, the state capital. Some studies have shown a strong correlation between the incidence of AVL and canine visceral leishmaniasis (CVL) in Belo Horizonte. A study of 108 dogs with parasite Leishmania chagasi detected by immuno-histochemistry in healthy ear skin was obtained from two distinct geographical areas: 55 from a metropolitan area of the municipality (Santa Luzia, MG) and 53 dogs from a central area of Belo Horizonte. In parallel, a group of 10 beagles were experimentally infected with L. chagasi. Considering the clinical aspects of all naturally infected dogs, symptomatic dogs were more frequent than asymptomatic ones, especially animals from the metropolitan area compared with the central area (79.6% and 20.3%, respectively). A chronic exudate was observed in the ear of 51 out of 55 dogs naturally infected from the metropolitan area (92.7%) and 45 out of 53 dogs naturally infected from the central area (84.9%). Importantly, asymptomatic dogs from the central area harbor more parasites in the skin than the asymptomatic ones from the metropolitan area. In addition, a profound difference was noted in the intensity of the inflammatory reaction and parasite load in the skin of experimental infected dogs.

  20. Different host complement systems and their interactions with saliva from Lutzomyia longipalpis (Diptera, Psychodidae and Leishmania infantum promastigotes.

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    Antonio Ferreira Mendes-Sousa

    Full Text Available BACKGROUND: Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH and 8.15 (the midgut pH immediately after a blood meal. We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. RESULTS: The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%, and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C had no effect on Leishmania viability during our assays. CONCLUSION: Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite. Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

  1. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

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    Felix Hugentobler

    Full Text Available BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H1 CD4(+ and CD8(+ T cells and a systemic LACK-specific T(H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

  2. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    Science.gov (United States)

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  3. Different host complement systems and their interactions with saliva from Lutzomyia longipalpis (Diptera, Psychodidae) and Leishmania infantum promastigotes.

    Science.gov (United States)

    Mendes-Sousa, Antonio Ferreira; Nascimento, Alexandre Alves Sousa; Queiroz, Daniel Costa; Vale, Vladimir Fazito; Fujiwara, Ricardo Toshio; Araújo, Ricardo Nascimento; Pereira, Marcos Horácio; Gontijo, Nelder Figueiredo

    2013-01-01

    Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays. Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

  4. Leishmania major glycosylation mutants require phosphoglycans (lpg2- but not lipophosphoglycan (lpg1- for survival in permissive sand fly vectors.

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    Anna Svárovská

    2010-01-01

    Full Text Available Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG and other phosphoglycans (PGs in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1(-, and the phosphoglycan (PG-deficient mutant lpg2(-. The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.The lpg2(- mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2(- parasite killing. The lpg1(- mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT parasites. In contrast, in P. duboscqi the lpg1(- mutants developed significantly worse than the WT parasites.In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.

  5. Natural hybrid of Leishmania infantum/L. donovani: development in Phlebotomus tobbi, P. perniciosus and Lutzomyia longipalpis and comparison with non-hybrid strains differing in tissue tropism.

    Science.gov (United States)

    Seblova, Veronika; Myskova, Jitka; Hlavacova, Jana; Votypka, Jan; Antoniou, Maria; Volf, Petr

    2015-11-25

    Infection caused by parasites from L. donovani complex can manifest as a serious visceral disease or a self-healing milder cutaneous form. The different tropism and pathology in humans is caused by the interaction between parasites, host and vector determinants but the mechanisms are not well understood. In Cukurova region in Turkey we previously identified a major focus of cutaneous leishmaniasis caused by L. donovani/infantum hybrids (CUK strain) and isolated this parasite from the locally abundant sand fly, Phlebotomus tobbi. Here, we present the first experimental study with P. tobbi. We tested the susceptibility of this species to various Leishmania under laboratory conditions, characterized glycoproteins in the P. tobbi midgut putatively involved in parasite-vector interaction and compared the development of the CUK strain in the sand fly with one other dermotropic and three viscerotropic strains belonging to the L. donovani complex. Females of laboratory reared P. tobbi, P. perniciosus and Lutzomyia longipalpis were infected using membrane feeding on rabbit blood containing promastigotes of various Leishmania species with different tropisms. The individual guts were checked microscopically for presence and localization of Leishmania parasites; the number of parasites was assessed more precisely by qPCR. In addition, glycosylation of midgut proteins of P. tobbi was studied by lectin blotting of midgut lysate with lectins specific for terminal sugars of N-type and O-type glycans. High infection rates, heavy parasite loads and late-stage infection with colonization of the stomodeal valve were observed in P. tobbi infected by Leishmania major or L. infantum CUK hybrid. In parallel, lectin blotting revealed the presence of O-glycosylated proteins in the P. tobbi midgut. In P. perniciosus and L. longipalpis all five Leishmania strains tested developed well. In both vectors, significantly higher parasite numbers were detected by qPCR for dermotropic L. donovani

  6. Imaging of the host/parasite interplay in cutaneous leishmaniasis.

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    Millington, Owain R; Myburgh, Elmarie; Mottram, Jeremy C; Alexander, James

    2010-11-01

    An understanding of host-parasite interplay is essential for the development of therapeutics and vaccines. Immunoparasitologists have learned a great deal from 'conventional'in vitro and in vivo approaches, but recent developments in imaging technologies have provided us (immunologists and parasitologists) with the ability to ask new and exciting questions about the dynamic nature of the parasite-immune system interface. These studies are providing us with new insights into the mechanisms involved in the initiation of a Leishmania infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations can be further developed to understand the immunology of cutaneous Leishmania infection in vivo. (c) 2010 Elsevier Inc. All rights reserved.

  7. Massive expansion of the calpain gene family in unicellular eukaryotes

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    Zhao Sen

    2012-09-01

    Full Text Available Abstract Background Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists. Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Results Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. Conclusions The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

  8. Massive expansion of the calpain gene family in unicellular eukaryotes.

    Science.gov (United States)

    Zhao, Sen; Liang, Zhe; Demko, Viktor; Wilson, Robert; Johansen, Wenche; Olsen, Odd-Arne; Shalchian-Tabrizi, Kamran

    2012-09-29

    Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

  9. Using the marine unicellular algae in biological monitoring

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    Kapkov V. I.

    2017-06-01

    Full Text Available The possibility of using marine unicellular algae from natural plankton community in biomonitoring of pollution by heavy metals has been investigated. Algae of different taxa from the Mediterranean Sea have been allocated to culture. In the laboratory the culture conditions – i. e. growth medium, temperature, photoperiod, level of artificial light and initial density – have been selected for every species. The impact of heavy metals (Hg, Cd, Cu, Pb in the form of chloride salts on the growth of axenic algae culture has been studied in the modelling experiments. The unicellular marine algae have a very short life cycle, therefore it is possible to use them in the experiments of studying the effect of anthropogenic factors at cellular and population levels on the test-object. With biomonitoring pollution of marine environment by heavy metals and others dangerous toxicants, the major indicators of algae community condition are the cellular cycle and the condition of the photosynthetic apparatus of the cell. The subsequent lysis of cells under the influence of heavy metals leads to the excretion of secondary metabolites which can essentially affect the metal toxicity. The established scales of threshold and lethal concentration of heavy metals for algae of different taxon make it possible to use the ratio of sensitive and resistant species to heavy metals as biological markers when forecasting ecological consequences of pollution of the marine environment by heavy metals. Distinctions in the resistance of different taxon to heavy metals can result in implementing the strategy of selection of test-objects depending on the purposes of the research.

  10. An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections.

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    Omar A Saldarriaga

    2016-04-01

    Full Text Available Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V. braziliensis, L. (V. panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V. braziliensis (n = 33, L. (V. guyanensis (n = 17, L. (V. panamensis (n = 9. The less common L. (V. lainsoni, L. (V. shawi, and L. (V. naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania subgenus. In a small number of clinical samples (n = 13 we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.

  11. Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

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    Andrade, Juvana M; Baba, Elio H; Machado-de-Avila, Ricardo A; Chavez-Olortegui, Carlos; Demicheli, Cynthia P; Frézard, Frédéric; Monte-Neto, Rubens L; Murta, Silvane M F

    2016-08-01

    Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani

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    Aditya Verma

    2017-12-01

    Full Text Available Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL in the Indian subcontinent. Paromomycin (PMM, an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R and the corresponding PMM sensitive (PMM-S parasites revealed modulated expression of 500 genes (1.5 fold cut off in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a reduced oxidative phosphorylation; b increased glycosomal succinate fermentation and substrate level phosphorylation; c dependency on lipids and amino acids for energy generation; d reduced DNA synthesis and increased DNA damage repair and e decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO levels while reactive oxygen species (ROS level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania. Keywords: Leishmania donovani, Drug resistance, Paromomycin, Transcriptome, ABC transporters, Nitric oxide, Visceral

  13. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

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    Raymond Wilson

    2010-09-01

    Full Text Available The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s that mediate binding is not fully understood.To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti. The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms, but is absent in the early blood meal and final stages (procyclic

  14. Survey of wild mammal hosts of cutaneous leishmaniasis parasites in panamá and costa rica.

    Science.gov (United States)

    González, Kadir; Calzada, José E; Saldaña, Azael; Rigg, Chystrie A; Alvarado, Gilbert; Rodríguez-Herrera, Bernal; Kitron, Uriel D; Adler, Gregory H; Gottdenker, Nicole L; Chaves, Luis Fernando; Baldi, Mario

    2015-03-01

    The eco-epidemiology of American cutaneous leishmaniasis (ACL) is driven by animal reservoir species that are a source of infection for sand flies that serve as vectors infecting humans with Leishmania spp parasites. The emergence and re-emergence of this disease across Latin America calls for further studies to identify reservoir species associated with enzootic transmission. Here, we present results from a survey of 52 individuals from 13 wild mammal species at endemic sites in Costa Rica and Panama where ACL mammal hosts have not been previously studied. For Leishmania spp. diagnostics we employed a novel PCR technique using blood samples collected on filter paper. We only found Leishmania spp parasites in one host, the two-toed sloth, Choloepus hoffmanni. Our findings add further support to the role of two-toed sloths as an important ACL reservoir in Central America.

  15. Immunity to Visceral Leishmaniasis Using Genetically Defined Live-Attenuated Parasites

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    Angamuthu Selvapandiyan

    2012-01-01

    Full Text Available Leishmaniasis is a protozoan parasitic disease endemic to the tropical and subtropical regions of the world, with three major clinical forms, self-healing cutaneous leishmaniasis (CL, mucocutaneous leishmaniasis (MCL, and visceral leishmaniasis (VL. Drug treatments are expensive and often result in the development of drug resistance. No vaccine is available against leishmaniasis. Subunit Leishmania vaccine immunization in animal models has shown some efficacy but little or none in humans. However, individuals who recover from natural infection are protected from reinfection and develop life-long protection, suggesting that infection may be a prerequisite for immunological memory. Thus, genetically altered live-attenuated parasites with controlled infectivity could achieve such memory. In this paper, we discuss development and characteristics of genetically altered, live-attenuated Leishmania donovani parasites and their possible use as vaccine candidates against VL. In addition, we discuss the challenges and other considerations in the use of live-attenuated parasites.

  16. Apoptotic markers in protozoan parasites

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    Fasel Nicolas

    2010-11-01

    Full Text Available Abstract The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms. In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

  17. Comparative study of structural models of Leishmania donovani and human GDP-mannose pyrophosphorylases.

    Science.gov (United States)

    Daligaux, Pierre; Bernadat, Guillaume; Tran, Linh; Cavé, Christian; Loiseau, Philippe M; Pomel, Sébastien; Ha-Duong, Tâp

    2016-01-01

    Leishmania is the parasite responsible for the neglected disease leishmaniasis. Its virulence and survival require biosynthesis of glycoconjugates, whose guanosine diphospho-d-mannose pyrophosphorylase (GDP-MP) is a key player. However, experimentally resolved structures of this enzyme are still lacking. We herein propose structural models of the GDP-MP from human and Leishmania donovani. Based on a multiple sequences alignment, the models were built with MODELLER and then carefully refined with all atom molecular dynamics simulations in explicit solvent. Their quality was evaluated against several standard criteria, including their ability to bind GDP-mannose assessed by redocking calculations. Special attention was given in this study to interactions of the catalytic site residues with the enzyme substrate and competitive inhibitors, opening the perspective of medicinal chemistry developments. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Concurrent cutaneous, visceral and ocular leishmaniasis caused by Leishmania (Viannia braziliensis in a kidney transplant patient

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    Gontijo Célia MF

    2002-01-01

    Full Text Available Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V. braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.

  19. Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector.

    Science.gov (United States)

    Akopyants, Natalia S; Kimblin, Nicola; Secundino, Nagila; Patrick, Rachel; Peters, Nathan; Lawyer, Phillip; Dobson, Deborah E; Beverley, Stephen M; Sacks, David L

    2009-04-10

    Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore full genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fly vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

  20. Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis.

    Science.gov (United States)

    Sevilha-Santos, L; Dos Santos Júnior, A C M; Medeiros-Silva, V; Bergmann, J O; da Silva, E F; Segato, L F; Arabi, A Y M; de Paula, N A; Sampaio, R N R; Lima, B D; Gomes, C M

    2018-05-03

    Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 10 6 , range 4.0-781.7, and 75.4 × 10 6 , range 8.0-244.5, mean Leishmania parasite equivalents per μg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 10 6 , range 8.0-24.1, mean Leishmania parasite equivalents per μg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  1. Parasitism as the main factor shaping peptide vocabularies in current organisms.

    Science.gov (United States)

    Zemková, Michaela; Zahradník, Daniel; Mokrejš, Martin; Flegr, Jaroslav

    2017-06-01

    Self/non-self-discrimination by vertebrate immune systems is based on the recognition of the presence of peptides in proteins of a parasite that are not contained in the proteins of a host. Therefore, a reduction of the number of 'words' in its own peptide vocabulary could be an efficient evolutionary strategy of parasites for escaping recognition. Here, we compared peptide vocabularies of 30 endoparasitic and 17 free-living unicellular organisms and also eight multicellular parasitic and 16 multicellular free-living organisms. We found that both unicellular and multicellular parasites used a significantly lower number of different pentapeptides than free-living controls. Impoverished pentapeptide vocabularies in parasites were observed across all five clades that contain both the parasitic and free-living species. The effect of parasitism on a number of peptides used in an organism's proteins is larger than effects of all other studied factors, including the size of a proteome, the number of encoded proteins, etc. This decrease of pentapeptide diversity was partly compensated for by an increased number of hexapeptides. Our results support the hypothesis of parasitism-associated reduction of peptide vocabulary and suggest that T-cell receptors mostly recognize the five amino acids-long part of peptides that are presented in the groove of major histocompatibility complex molecules.

  2. Rapid Sequestration of Leishmania mexicana by Neutrophils Contributes to the Development of Chronic Lesion.

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    Benjamin P Hurrell

    2015-05-01

    Full Text Available The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing.

  3. Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp.

    Science.gov (United States)

    Betzler de Oliveira de Siqueira, Luciana; da Silva Cardoso, Verônica; Almeida Rodrigues, Igor; Lúcia Vazquez-Villa, Ana; Pereira dos Santos, Elisabete; da Costa Leal Ribeiro Guimarães, Bruno; dos Santos Cerqueira Coutinho, Cristal; Vermelho, Alane Beatriz; Ricci Junior, Eduardo

    2017-02-01

    Photodynamic therapy (PDT) combines light with photosensitizers (PS) for production of reactive oxygen species (ROS) that can kill infectious microorganisms such as bacteria, fungi and protozoa. The application of nanotechnology has enabled the advancement of PDT because many PS are insoluble in water, necessitating a nanocarrier as a physiologically acceptable carrier. Nanoemulsions are efficient nanocarriers for solubilizing liposoluble drugs, like the PS, in water. Cutaneous (CL) and mucocutaneous leishmaniasis (ML) are caused by different species of the genus Leishmania, transmitted to humans by sandfly bites. Parasites are hosted in skin macrophages producing ulcerative lesions. Thus, a topical treatment, effective and inexpensive, for CL and ML is preferable to systemic interventions. There are topical treatments like paromomycin and amphotericin B, but they have many local side effects or a very high cost, limiting their use. This work aimed to develop a zinc phthalocyanine (photosensitizer) oil-in-water nanoemulsion, essential clove oil and polymeric surfactant (Pluronic® F127) for the formulation of a topical delivery system for use in PDT against Leishmania amazonensis and Leishmania infantum. The nanoemulsion was produced by a high-energy method and characterized by size, polydispersity, morphology, pH, content and stability studies. The toxicity in the dark and the photobiological activity of the formulations were evaluated in vitro for Leishmania and macrophages. The formulation presented was pH compatible with topical use, approximately 30 nm in size, with a polydispersity index ≤0.1 and remained stable at room and refrigerator temperature during the stability study (60 days). The zinc phthalocyanine nanoemulsion is effective in PDT against Leishmania spp.; use against skin infections can be a future application of this topical formulation, avoiding the use of oral or injectable medications, decreasing systemic adverse effects.

  4. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

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    Marie Samanovic

    2009-01-01

    Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

  5. Computational prediction of protein-protein interactions in Leishmania predicted proteomes.

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    Antonio M Rezende

    Full Text Available The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks

  6. L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

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    Jasdeep Singh

    2017-12-01

    Full Text Available Emergence of Amphotericin B (AmB resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania. Keywords: Leishmania donovani, L-asparaginase, Amphotericin B resistance, Metabolic target

  7. Comparative Metabolism of Free-living Bodo saltans and Parasitic Trypanosomatids

    Czech Academy of Sciences Publication Activity Database

    Opperdoes, F. R.; Butenko, A.; Flegontov, P.; Yurchenko, V.; Lukeš, Julius

    2016-01-01

    Roč. 63, č. 5 (2016), s. 657-678 ISSN 1066-5234 R&D Projects: GA ČR(CZ) GA14-23986S Grant - others:EU COST Action CM1307 Institutional support: RVO:60077344 Keywords : adaptation * Leishmania * Leptomonas * lateral gene transfer * parasitism * Phytomonas * Trypanosoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2016

  8. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    Science.gov (United States)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  9. Comparison of codon usage bias across Leishmania and Trypanosomatids to understand mRNA secondary structure, relative protein abundance and pathway functions.

    Science.gov (United States)

    Subramanian, Abhishek; Sarkar, Ram Rup

    2015-10-01

    Understanding the variations in gene organization and its effect on the phenotype across different Leishmania species, and to study differential clinical manifestations of parasite within the host, we performed large scale analysis of codon usage patterns between Leishmania and other known Trypanosomatid species. We present the causes and consequences of codon usage bias in Leishmania genomes with respect to mutational pressure, translational selection and amino acid composition bias. We establish GC bias at wobble position that governs codon usage bias across Leishmania species, rather than amino acid composition bias. We found that, within Leishmania, homogenous codon context coding for less frequent amino acid pairs and codons avoiding formation of folding structures in mRNA are essentially chosen. We predicted putative differences in global expression between genes belonging to specific pathways across Leishmania. This explains the role of evolution in shaping the otherwise conserved genome to demonstrate species-specific function-level differences for efficient survival. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

    International Nuclear Information System (INIS)

    Hashim, Amna Osman Yousif

    1997-06-01

    Leishmaniasis, manifesting on it's different clinical forms is endemic in different regions of the Sudan. diagnosis of the disease in the Sudan is usually done using simple methods such as microscopical examination of slit smirs, Histological sections and cultures. Serological diagnosis using enzymes linked immunosorbent assay (ELISA)- and direct agglutination test (DAT) are sometimes used as more sensitive tool has thrown light on the epidemiology of the disease in the Sudan. This study was conducted on 126 subjects to identify the parasites-causing the different clinical manifestations, to determine the genetic diversity of different isolates of Lieshmania- and to detect parasites in the peripheral blood of subjects from highly endemic foci. The study population consisted of 7 with suspected VL, 12 with suspected ML, 14 with suspected PKDL, 2 with sporotrichoid CL and 89 healthy games wardens and army soldiers from highly endemic foci. Parasites were cultured in biphasic medium and subcultured in liquid medium until mass production was stabilized. Extraction of DNA was done using three methods which were phenol/ chloroform/ isoamylalcohol, K buffer and proteinase K as well as lysing of the parasite with distilled water. The KDNA was amplified using species namely AJSI and DeB8. The products were analysed on 1.5% agarose gel-and were visualized and photographed with U.V. transilluminator and camera. Characteristic bands of 700 and 800 b.p corresponding to the full length of mini circle of L.major and L.donovani respectively were obtained on amplification of KDNA from patients with VL and CL. In some cases lower bands of 400 and 500 b.p PKDL and multiple bands for sporotrichoid CL.Leishmania DNA was detected from the conjucativa of the eye of a patient with PKDL. The genetic diversity of Leishmania parasite was determined by digesting PCR products from PKDL, sporotrichoids CL and VL patients. Different patterns were produced for each digesting product. This result

  11. The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

    Energy Technology Data Exchange (ETDEWEB)

    Hashim, Amna Osman Yousif [Department of Zoology, Faculty of Science, University of Khartoum, Khartoum (Sudan)

    1997-06-01

    Leishmaniasis, manifesting on it`s different clinical forms is endemic in different regions of the Sudan. diagnosis of the disease in the Sudan is usually done using simple methods such as microscopical examination of slit smirs, Histological sections and cultures. Serological diagnosis using enzymes linked immunosorbent assay (ELISA)- and direct agglutination test (DAT) are sometimes used as more sensitive tool has thrown light on the epidemiology of the disease in the Sudan. This study was conducted on 126 subjects to identify the parasites-causing the different clinical manifestations, to determine the genetic diversity of different isolates of Lieshmania- and to detect parasites in the peripheral blood of subjects from highly endemic foci. The study population consisted of 7 with suspected VL, 12 with suspected ML, 14 with suspected PKDL, 2 with sporotrichoid CL and 89 healthy games wardens and army soldiers from highly endemic foci. Parasites were cultured in biphasic medium and subcultured in liquid medium until mass production was stabilized. Extraction of DNA was done using three methods which were phenol/ chloroform/ isoamylalcohol, K buffer and proteinase K as well as lysing of the parasite with distilled water. The KDNA was amplified using species namely AJSI and DeB8. The products were analysed on 1.5% agarose gel-and were visualized and photographed with U.V. transilluminator and camera. Characteristic bands of 700 and 800 b.p corresponding to the full length of mini circle of L.major and L.donovani respectively were obtained on amplification of KDNA from patients with VL and CL. In some cases lower bands of 400 and 500 b.p PKDL and multiple bands for sporotrichoid CL.Leishmania DNA was detected from the conjucativa of the eye of a patient with PKDL. The genetic diversity of Leishmania parasite was determined by digesting PCR products from PKDL, sporotrichoids CL and VL patients. Different patterns were produced for each digesting product. This result

  12. Leishmania in sand flies: comparison of quantitative polymerase chain reaction with other techniques to determine the intensity of infection

    Czech Academy of Sciences Publication Activity Database

    Myšková, J.; Votýpka, Jan; Volf, P.

    2008-01-01

    Roč. 45, č. 1 (2008), s. 133-138 ISSN 0022-2585 R&D Projects: GA MŠk(CZ) LC06009 Grant - others:Univerzita Karlova v Praze(CZ) 195/2005/B-BIO/PrF Institutional research plan: CEZ:AV0Z60220518 Keywords : sand fly * Leishmania * Phlebotomus * parasite-vector interaction * RT-PCR Subject RIV: EG - Zoology Impact factor: 1.967, year: 2008

  13. Surface reaction of Leishmania. III. Ulex europaeus II lectin affinity for excreted factor (EF) serotype A strains.

    Science.gov (United States)

    Greenblatt, C L; Meline, D; Slutzky, G M; Schnur, L F; Levene, C

    1984-04-01

    Eukaryotic parasites, including species of Leishmania, acquire or synthesize carbohydrate moieties similar to human blood group antigens. Leishmanial strains separate into three serotypes: A, B and AB. All strains containing the A component are agglutinated by Ulex europaeus lectin. Inhibition by haptene sugar suggests that a Ulex II-like receptor is involved. Organic solvents, but not protease treatment, remove its reactivity, suggesting that the receptor is a glycolipid.

  14. Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae

    Directory of Open Access Journals (Sweden)

    Alejandra Vivenes

    2001-04-01

    Full Text Available Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.

  15. Study of ionizing radiation as a tool for select promastigotes forms of Leishmania Amazonensis, and the megalomaniac response in experimental models; Estudo do uso da radiacao ionizante como ferramenta de selecao de formas promastigotas metaciclicas de Leishmania amazonensis, e a inducao de resposta imunologica em modelos experimentais

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco Claudio

    2006-07-01

    Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a cutaneous disease to a visceral one. The cutaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a {sup 60}Co font, had being successes used to promote physical-chemical transformations on different protozoan, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of meta cyclic forms of the parasite in axenic culture, for a possible efficient irradiated immuno gene production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metacyclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didn't produce immunoglobulin nor on the immunization or on the challenge. (author)

  16. Study of ionizing radiation as a tool for select promastigotes forms of Leishmania Amazonensis, and the megalomaniac response in experimental models; Estudo do uso da radiacao ionizante como ferramenta de selecao de formas promastigotas metaciclicas de Leishmania amazonensis, e a inducao de resposta imunologica em modelos experimentais

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco Claudio

    2006-07-01

    Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a cutaneous disease to a visceral one. The cutaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a {sup 60}Co font, had being successes used to promote physical-chemical transformations on different protozoan, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of meta cyclic forms of the parasite in axenic culture, for a possible efficient irradiated immuno gene production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metacyclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didn't produce immunoglobulin nor on the immunization or on the challenge. (author)

  17. [MiRNA system in unicellular eukaryotes and its evolutionary implications].

    Science.gov (United States)

    Zhang, Yan-Qiong; Wen, Jian-Fan

    2010-02-01

    microRNAs (miRNAs) in higher multicellular eukaryotes have been extensively studied in recent years. Great progresses have also been achieved for miRNAs in unicellular eukaryotes. All these studies not only enrich our knowledge about the complex expression regulation system in diverse organisms, but also have evolutionary significance for understanding the origin of this system. In this review, Authors summarize the recent advance in the studies of miRNA in unicellular eukaryotes, including that on the most primitive unicellular eukaryote--Giardia. The origin and evolution of miRNA system is also discussed.

  18. Signaling mechanisms of apoptosis-like programmed cell death in unicellular eukaryotes.

    Science.gov (United States)

    Shemarova, Irina V

    2010-04-01

    In unicellular eukaryotes, apoptosis-like cell death occurs during development, aging and reproduction, and can be induced by environmental stresses and exposure to toxic agents. The essence of the apoptotic machinery in unicellular organisms is similar to that in mammals, but the apoptotic signal network is less complex and of more ancient origin. The review summarizes current data about key apoptotic proteins and mechanisms of the transduction of apoptotic signals by caspase-like proteases and mitochondrial apoptogenic proteins in unicellular eukaryotes. The roles of receptor-dependent and receptor-independent caspase cascades are reviewed. 2010 Elsevier Inc. All rights reserved.

  19. Protection mediated by chemokine CXCL10 in BALB/c mice infected by Leishmania infantum

    Science.gov (United States)

    Figueiredo, Webertty Mayk Eufrásio; Viana, Sayonara de Melo; Alves, Dorotheia Teixeira; Guerra, Priscila Valera; Coêlho, Zirlane Castelo Branco; Barbosa, Helene Santos; Teixeira, Maria Jania

    2017-01-01

    BACKGROUND Visceral leishmaniasis (VL) caused by Leishmania infantum is characterised by the loss of the ability of the host to generate an effective immune response. Chemokines have a direct involvement in the pathogenesis of leishmaniasis, causing a rapid change in the expression of these molecules during infection by Leishmania. OBJECTIVES Herein, it was investigated the role of CXCL10 in controlling infection by L. infantum. METHODS RAW 264.7 macrophages were infected with L. infantum in vitro and treated or not with CXCL10 (25, 50 and 100 ng/mL). Parasite load, as well as nitric oxide (NO), IL-4 and IL-10 production were assessed at 24 and 48 h after infection. In vivo, BALB/c mice were infected and treated or not with CXCL10 (5 μg/kg) at one, three and seven days of infection. Parasite load, IFN-g, IL-4, TGF-β and IL-10 were evaluated one, seven and 23 days post treatment. FINDINGS In vitro, CXCL10 reduced parasitic load, not dependent on NO, and inhibited IL-10 and IL-4 secretion. In vivo, CXCL10 was able to reduce the parasite load in both liver and spleen, four weeks after infection, representing a higher decrease in the number of parasites in these organs, also induced IFN-γ at day 23 after treatment, correlating with the decrease in parasite load, and reduced IL-10 and TGF-β. MAIN CONCLUSIONS This study suggests a partial protective role of CXCL10 against L. infantum, mediated by IFN-g, not dependent on NO, and with suppression of IL-10 and TGF-β. These data may provide information for the development of new approaches for future therapeutic interventions for VL. PMID:28767981

  20. The development of Leishmania turanica in sand flies and competition with L. major.

    Science.gov (United States)

    Chajbullinova, Alsu; Votypka, Jan; Sadlova, Jovana; Kvapilova, Katerina; Seblova, Veronika; Kreisinger, Jakub; Jirku, Milan; Sanjoba, Chizu; Gantuya, Sambuu; Matsumoto, Yoshitsugu; Volf, Petr

    2012-10-02

    In Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear. In this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy. Late-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi. Our data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late

  1. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

    Science.gov (United States)

    Abbasi, Ibrahim; Aramin, Samar; Hailu, Asrat; Shiferaw, Welelta; Kassahun, Aysheshm; Belay, Shewaye; Jaffe, Charles; Warburg, Alon

    2013-03-27

    Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

  2. Photodynamic Vaccination of BALB/c Mice for Prophylaxis of Cutaneous Leishmaniasis Caused by Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Sayonara M. Viana

    2018-02-01

    Full Text Available Background: Photosensitizers (PS, like porphyrins and phthalocyanines (PC are excitable by light to generate cytotoxic singlet oxygen and other reactive oxygen species in the presence of atmospheric O2. Photodynamic inactivation of Leishmania by this means renders them non-viable, but preserves their effective use as vaccines. Leishmania can be photo-inactivated after PS-sensitization by loading via their endocytic uptake of PC or endogenous induction of transgenic mutants with delta-aminolevulinate (ALA to accumulate cytosolic uroporphyrin I (URO. Here, PS-sensitization and photo-inactivation of Leishmaniaamazonensis was further examined in vitro and in vivo for vaccination against cutaneous leishmaniasis (CL.Methods and Results:Leishmania amazonensis promastigotes were photodynamically inactivated in vitro by PC-loading followed by exposure to red light (1–2 J/cm2 or ALA-induction of uroporphyrinogenic transfectants to accumulate cytosolic URO followed by longwave UV exposure. When applied individually, both strategies of photodynamic inactivation were found to significantly, albeit incompletely abolish the MTT reduction activities of the promastigotes, their uptake by mouse bone marrow-derived macrophages in vitro and their infectivity to mouse ear dermis in vivo. Inactivation of Leishmania to completion by using a combination of both strategies was thus used for the sake of safety as whole-cell vaccines for immunization of BALB/c mice. Different cutaneous sites were assessed for the efficacy of such photodynamic vaccination in vivo. Each site was inoculated first with in vitro doubly PS-sensitized promastigotes and then spot-illuminated with white light (50 J/cm2 for their photo-inactivation in situ. Only in ear dermis parasites were photo-inactivated beyond detection. Mice were thus immunized once in the ear and challenged 3 weeks later at the tail base with virulent L. amazonensis. Prophylaxis was noted in mice photodynamically

  3. First molecular detection of Leishmania major within naturally infected Phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern Iran.

    Science.gov (United States)

    Azizi, K; Fakoorziba, M R; Jalali, M; Moemenbellah-Fard, M D

    2012-03-01

    Human cutaneous leishmaniasis (CL) is a major notifiable public health problem in many parts of Iran. It is often caused by the zooflagellate parasite Leishmania major which is mainly transmitted by the bites of female phlebotomine sandflies belonging to the genus Phlebotomus (Diptera: Psychodidae). The annual incidence of CL in Fars province, southern Iran, was about 108-144 in 2007. The leishmanial infections of wild sandflies that may act as vectors were thus investigated at an endemic focus in this province. In all 330 female Phlebotomus sandflies were screened for the detection of Leishmania-specific kinetoplast DNA (kDNA) by polymerase chain reaction (PCR) methods. A two stage nested PCR protocol was used to establish the identity of Leishmania major species in naturally infected sandflies. The L. major kDNA was detected in 18 (5.5%) individual sandflies which belonged to four different Phlebotomus species (Phlebotomus papatasi, Phlebotomus salehi, Phlebotomus sergenti and P. major group). For the first time, one naturally infected P. salehi specimen contained the kDNA of the protozoan parasite, L. major, with a main band of 560 base pairs identified using the nested PCR method. It seems most likely therefore that P. salehi is potentially a rare sylvatic vector of L. major parasites in parts of this province. This is the first combined morphological and molecular studies of P. salehi in Iran.

  4. Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

    International Nuclear Information System (INIS)

    Petrillo-Peixoto, M.L.; Beverley, S.M.

    1988-01-01

    We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis

  5. Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

    Science.gov (United States)

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L