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Sample records for da leishmania amazonensis

  1. The activity of azithromycin against Leishmania (Viannia braziliensis and Leishmania (Leishmania amazonensis in the golden hamster model A atividade da azitromicina contra a Leishmania (Viannia braziliensis e a Leishmania (Leishmania amazonensis no modelo golden hamster

    Ángel Sinagra

    2007-12-01

    Full Text Available 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 untreated controls and animals treated with meglumine antimoniate. Footpad thickness, lesion cultures and dissemination sites were analyzed. Treatment of golden hamsters with oral azithromycin at 450mg/kg had no activity against infections with Leishmania (Leishmania amazonensis. For infections due to Leishmania (Viannia braziliensis, azithromycin demonstrated significant activity relative to untreated controls, but inferior to meglumine antimoniate, for controlling lesion size. Neither drug was able to totally eliminate parasites from the lesions. It was concluded that azithromycin has activity against Leishmania (Viannia braziliensis but not against Leishmania (Leishmania amazonensis in this model.Novas alternativas terapêuticas contra a leishmaniose são ainda uma prioridade. A atividade da azitromicina contra a Leishmania (Leishmania major foi anteriormente demonstrada. Diferentes respostas entre as espécies de Leishmania fazem com que um screening de drogas específicas para espécies seja necessário. A atividade da azitromicina contra a Leishmania (Viannia braziliensis e a Leishmania (Leishmania amazonensis foi avaliada em Golden hamsters infectados a través de injeções de promastigotas metacíclicas e comparando com controles sem tratamento e animais tratados com antimoniato de N-metil-glucamina. Foram analisadas a espessura da pata, a cultura das lesões e disseminação para órgãos internos. A azitromicina oral em dose de 450mg/kg não teve

  2. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    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.

  3. Effects of Brazilian propolis on Leishmania amazonensis

    Diana Copi Ayres

    2007-03-01

    Full Text Available Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 µg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

  4. 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

    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)

  5. Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine.

    Coelho, Adriano C; Gentil, Luciana G; da Silveira, José Franco; Cotrim, Paulo C

    2008-09-01

    Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene.

  6. Leishmania in synanthropic rodents (Rattus rattus): new evidence for the urbanization of Leishmania (Leishmania) amazonensis.

    Caldart, Eloiza Teles; Freire, Roberta Lemos; Ferreira, Fernanda Pinto; Ruffolo, Bruno Bergamo; Sbeghen, Mônica Raquel; Mareze, Marcelle; Garcia, João Luis; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico

    2017-02-06

    This study aimed to detect parasites from Leishmania genus, to determine the prevalence of anti-Leishmania spp. antibodies, to identify circulating species of the parasite, and to determine epidemiological variables associated with infection in rats caught in urban area of Londrina, Paraná, Brazil. Animal capture was carried out from May to December 2006, serological and molecular methods were performed. DNA was extracted from total blood, and nested-PCR, targeting SSu rRNA from Leishmania genus, was performed in triplicate. The positive samples were sequenced twice by Sanger method to species determination. In total, 181 rodents were captured, all were identified as Rattus rattus and none showed clinical alterations. Forty-one of the 176 (23.3%) animals were positive for Leishmania by ELISA and 6/181 (3.3%) were positive by IFAT. Nine of 127 tested animals (7.1%) were positive by PCR; seven were identified as L. (L.) amazonensis, one as L. (L.) infantum. Four rats were positive using more than one test. This was the first description of synanthropic rodents naturally infected by L. (L.) amazonensis (in the world) and by L. (L.) infantum (in South Brazil). Regarding L. (L.) amazonensis, this finding provides new evidence of the urbanization of this etiological agent.

  7. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

    Kendi Okuda

    2016-06-01

    Full Text Available Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection.

  8. LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

    De Cicco, Nuccia N T; Pereira, Miria G; Corrêa, José R; Andrade-Neto, Valter V; Saraiva, Felipe B; Chagas-Lima, Alessandra C; Gondim, Katia C; Torres-Santos, Eduardo C; Folly, Evelize; Saraiva, Elvira M; Cunha-E-Silva, Narcisa L; Soares, Maurilio J; Atella, Georgia C

    2012-04-01

    Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

  9. Interleukin- 2 production during murine infection by Leishmania mexicana amazonensis

    Manoel Barral-Netto

    1986-03-01

    Full Text Available Highly susceptible BALB/c mice, resistant C57B1/6 and their F1 progeny (BDF1 were infected subcutaneously in the foot pad with Leishmania mexicana amazonenesis. At various times after infection, spleen or draining popliteal lymph node cells were assayed for their capacity to generate Interleukin-2 (I1-2 by Concanavalin A (ConA stimulation. In both BALB/c and C57B1/6 strains there was a transient increase in their capacity to produce I1-2, from the 3rd to the 10th week post-infection. Return to pre-infection levels ocurred between 13th to 16th week post-infection in all three strains. BALB/c mice always produced higher titers of 11-2 than C57B1/6, but such differences were statistically significant only at 3 and 10 weeks post-infection. BDF1 mice had titers similar to those observed in BALB/c mice. I1-2 production by ConA-stimulated lymph node cells was lower as compared to the spleen, but with a similar pattern among the three mice strains. Our data show that susceptibility to infection by l. mexicana amazonenesis is not associated with deficient ConA-stimulated I1-2 production.Camundongos BALB/c (susceptíveis, C57B1/6 (resistentes ou sua geração F1 (BDF foram infectados subcutaneamente na pata traseira com Leishmania mexicana amazonensis. Avaliamos, em diferentes períodos de infecção, a capacidade de células do baço ou de linfonodo poplíteo, de produzir Interleucina-2 (I1-2 em resposta à estimulação por Conconavalina A (ConA. Nos camundongos BALB/c e C57B1/6 observamos, da 3ª à 10ª semana pós-infecção transitória da capacidade de produzir I1-2. Da 13ª à 16ª semana pós-infecção houve um retorno dos níveis de produção pré-infecção. Camundongos BALB/c produziram títulos mais elevados de I2- que os C57B1/6, mas tais diferenças só foram estatisticamente significantes na 3ª e 10ª semanas pós-infecção. Camundongos BDF1 apresentaram títulos semelhantes aos dos BALB/c. Os níveis de I1-2 (estimulada por Con

  10. Metacyclic promastigotes of Leishmania amazonensis selection using gamma irradiation

    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. Efficacy of a diarylheptanoid derivative against Leishmania amazonensis

    Alves Luciana Vignólio

    2003-01-01

    Full Text Available The activity of several diarylheptanoid derivatives (curcuminoids was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was the [1-(4-methoxy-phenyl-7-(3,4-methoxy-4-hydroxy-phenyl-1,6-heptadien-3, 5-dione]. This derivative was chosen to be assayed in vivo in a treatment trial. For these experiments, the curcuminoid compound was used in a concentration equivalent to the IC50/24 h, obtained from the previous study. Balb/c mice were inoculated subcutaneously in the footpad with L. amazonensis infective promastigotes and 4 weeks after the inoculation, the animals were treated with different schemes, varying from 1 to 3 doses. In all the experiments, Pentamidine Isethionate was used as reference drug under the same experimental conditions. The results showed that one dose was not enough to heal the lesion, however, with 2 and 3 doses the efficiency of the assayed compound was clear. On the other hand, treatment with Pentamidine Isethionate using the three different schemes was not satisfactory when compared to the curcuminoid derivative.

  12. 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

    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)

  13. Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control

    Letícia da Cruz Sanches

    Full Text Available Abstract Leishmaniasis is a major public health problem worldwide. Because Leishmania can adapt to new hosts or vectors, knowledge concerning the current etiological agent in dogs is important in endemic areas. This study aimed to identify the Leishmania species detected in 103 samples of peripheral blood from dogs that were naturally infected with these protozoa. The diagnosis of leishmaniasis was determined through parasitological examination, the indirect enzyme-linked immunosorbent assay (ELISA and the polymerase chain reaction (PCR. The Leishmania species were identified by means of PCR-restriction fragment length polymorphism (PCR-RFLP. The samples were subjected to PCR using oligonucleotide primers that amplify the intergenic region ITS1 of the rRNA gene in order to identify the species. The amplified DNA was digested using the restriction enzyme HaeIII. A restriction profile identical to L. amazonensis was shown in 77/103 samples and the profile was similar to L. infantum in 17/103. However, a mixed profile was shown in 9/103 samples, which impeded species identification. In conclusion, the infection in these dogs was predominantly due to L. amazonensis, thus indicating that diagnosing of cases of canine leishmaniasis needs to be reexamined, since the causative agent identified is not restricted to L. infantum.

  14. Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania amazonensis

    MORA Ana Mariela

    1999-01-01

    Full Text Available In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania amazonensis (LLa with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin. The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60 and 10.77UI/ml of IFN-g production. When crude extract of L. (L. amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that

  15. Leishmania (Viannia braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania amazonensis

    Andrea Niño

    2005-05-01

    Full Text Available We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V. braziliensis replication rates and decreased the time of maximum replication to 48 h.

  16. 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

    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

  17. 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.

    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.

  18. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda; Real, Fernando

    2016-05-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

  19. Bioactivity of flavonoids isolated from Lychnophora markgravii against Leishmania amazonensis amastigotes.

    Salvador, Marcos José; Sartori, Fabiana Terezinha; Sacilotto, Ana Claudia B C; Pral, Elizabeth M F; Alfieri, Silvia Celina; Vichnewski, Walter

    2009-01-01

    The bioactivity of the flavonoids pinostrobin (1), pinocembrin (2), tectochrysin (3), galangin 3-methyl ether (4), and tiliroside (5) isolated from Lychnophora markgravii aerial parts was investigated in vitro against amastigote stages of Leishmania amazonensis. The compounds were isolated by several chromatographic techniques and their chemical structures were established by ESI-MS and NMR spectroscopic data. The flavonoids 1 and 3 were the most active compounds; they markedly reduced the viability of Leishmania amastigotes.

  20. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; Reis, Ana Beatriz de Bragança Dos; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-05-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

  1. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  2. Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis Efeito combinado do óleo de essência de Chenopodium ambrosioides e drogas anti-leishmaniose nos promastigotas de Leishmania amazonensis

    Lianet Monzote

    2007-08-01

    Full Text Available To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.Até hoje não temos vacina contra a Leishmania e a quimioterapia é a indicação para o controle desta doença. Os remédios que hoje utilizamos são tóxicos e muito caros e além disso o resultado não é sempre o desejado. Por isso, uma terapia de combinação é a melhor opção. Este trabalho mostra que o óleo de essência de C. ambrosioides tem atividade sinérgica junto com a pentamidina sobre os promastigotas de L. amazonensis, diferente do resultado da combinação de antimônio de meglumine e anfotericina B e o óleo de essência.

  3. Therapeutic trial in experimental tegumentary leishmaniasis caused by Leishmania (Leishmania amazonensis. A comparative study between mefloquine and aminosidine Ensaio terapêutico na leishmaniose tegumentar experimental causada por Leishmania (Leishmania amazonensis. Um estudo comparativo entre mefloquina e aminosidine

    Letícia Oba Galvão

    2000-08-01

    Full Text Available One hundred and eighty-two male inbred C57/BL/6 mice were infected with 3 x 106 Leishmania (Leishmania amazonensis promastigotes of the MHOM/BR/PH8 strain by means of a subcutaneous injection in the right ear. The animals were separated in three groups: 1 oral mefloquine hydrochloride treatment (16mg/kg/day/10 days, 2 intramuscular aminosidine (Paromomycin® treatment (20mg/kg/20 days and 3 control. Twenty six mice of each treated group were sacrificed, one at the end of treatment (nine weeks after inoculation, and one six weeks later (fifteen weeks after inoculation. Control Group animals were sacrificed at weeks six, nine and fifteen after inoculation. There was no significant difference between Group 1 (mefloquine and Group 3 (control subjects. Group 2 animals (aminosidine presented the smallest differences of all, both at the end of the treatment and six weeks later. The histopato-logical parameters have shown the following findings: a there was no significant difference between the mefloquine treated group and the control group; the group treated with aminosidine showed fewer of vacuolated macrophages than the control group, at week 9 (end of treatment. b both at the end of treatment and six weeks later, evaluation of tissue necrosis and tissue fibrosis revealed no differences between the treated groups. It was found that six weeks after the end of treatment, mice in the control group presented significantly more severe degrees of fibrosis than mice in the other groups. It can be concluded that mefloquine showed limited therapeutic effect in this experimental model, whereas aminosidine had a significant effect. Nevertheless, neither of them resulted in cure of the lesions.Foram utilizados 182 camundongos machos, isogênicos, da linhagem C57BL/6 inoculados na orelha direita com 3,0 x 10(6 formas promastigotas da cepa MHOM/BR/PH8 de Leishmania (Leishmania amazonensis. Os animais foram separados em três grupos: 1 52 animais tratados com

  4. Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species.

    Da Silva, B J M; Da Silva, R R P; Rodrigues, A P D; Farias, L H S; Do Nascimento, J L M; Silva, E O

    2016-03-01

    Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2(-)) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2(-) generation to induce Leishmania death.

  5. Immune responses induced by a Leishmania (Leishmania amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

    Ana Paula FERNANDES

    1997-03-01

    Full Text Available In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania amazonensis recombinant protein of 33 kD (Larp33 which is recognized by antibodies and peripheral blood leukocytes (PBL from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L. amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L. amazonensis and L. (Viannia braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10 against L. (L. amazonensis after vaccination using Bacille Calmette-Guerin (BCG as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasisA resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania amazonensis de 33 kD (Larp33 foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L. amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao ant

  6. Propolis reduces Leishmania amazonensis-induced inflammation in the liver of BALB/c mice.

    da Silva, Suelen S; Mizokami, Sandra S; Fanti, Jacqueline R; Miranda, Milena M; Kawakami, Natalia Y; Teixeira, Fernanda Humel; Araújo, Eduardo J A; Panis, Carolina; Watanabe, Maria A E; Sforcin, José M; Pavanelli, Wander R; Verri, Waldiceu A; Felipe, Ionice; Conchon-Costa, Ivete

    2016-04-01

    Experimental models of mouse paw infection with L. amazonensis show an induction of a strong inflammatory response in the skin, and parasitic migration may occur to secondary organs with consequent tissue injury. There are few studies focusing on the resolution of damage in secondary organs caused by Leishmania species-related cutaneous leishmaniasis. We investigated the propolis treatment effect on liver inflammation induced by Leishmania amazonensis infection in the mouse paw. BALB/c mice were infected in the hind paw with L. amazonensis (10(7)) promastigote forms. After 15 days, animals were treated daily with propolis (5 mg/kg), Glucantime (10 mg/kg), or with propolis plus Glucantime combined. After 60 days, mice were euthanized and livers were collected for inflammatory process analysis. Liver microscopic analysis showed that propolis reduced the inflammatory process compared to untreated infected control. There was a decrease of liver myeloperoxidase and N-acetyl-β-glucosaminidase activity levels, collagen fiber deposition, pro-inflammatory cytokine production, and plasma aspartate transaminase and alanine transaminase levels. Furthermore, propolis treatment enhanced anti-inflammatory cytokine levels and reversed hepatosplenomegaly. Our data demonstrated that daily low doses of Brazilian propolis reduced the secondary chronic inflammatory process in the liver caused by L. amazonensis subcutaneous infection in a susceptible mice strain.

  7. Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

    2004-01-01

    Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates ...

  8. Identificação de espécies de Leishmania isoladas de casos humanos em Mato Grosso do Sul por meio da reação em cadeia da polimerase Identification of Leishmania species isolated in human cases in Mato Grosso do Sul, by means of the polymerase chain reaction

    Manoel Sebastião da Costa Lima Junior

    2009-06-01

    Full Text Available As leishmanioses são zoonoses endêmicas em Mato Grosso do Sul e têm por agentes etiológicos nessa região Leishmania (Leishmania chagasi, Leishmania (Leishmania amazonensis e Leishmania (Viannia braziliensis. Como método para identificação de espécies de Leishmania, a reação em cadeia da polimerase é uma ferramenta com elevada especificidade e sensibilidade. Analisaram-se 39 isolados de Leishmania criopreservados, obtidos por meio de aspirado medular e/ou biópsia de lesão, conforme a suspeita clínica. Os isolados foram submetidos à extração de DNA e à reação em cadeia da polimerase com os iniciadores: RV1/RV2 para Leishmania (Leishmania chagasi, a1/a2 para a identificação de Leishmania (Leishmania amazonensis e b1/b2 para Leishmania (Viannia braziliensis. Leishmania (Leishmania chagasi foi a única espécie identificada em 37 casos de leishmaniose visceral. Leishmania (Leishmania amazonensis foi identificada em dois isolados de pacientes com diagnóstico de leishmaniose tegumentar. Os resultados obtidos confirmam a possibilidade do uso dos três pares de iniciadores como uma ferramenta na caracterização de isolados de Leishmania.Leishmaniases are endemic zoonoses in the State of Mato Grosso do Sul. Their etiological agents in this region of Brazil are Leishmania (Leishmania chagasi, Leishmania (Leishmania amazonensis and Leishmania (Viannia braziliensis. The polymerase chain reaction (PCR is a tool with high specificity and sensitivity for identifying Leishmania species. This study examined 39 cryopreserved isolates of Leishmania that had been collected by bone marrow aspiration and/or lesion biopsy, depending on the clinical suspicion. The isolates were subjected to DNA extraction and PCR using the following primers: RV1/RV2 for identifying Leishmania (Leishmania chagasi, a1/a2 for Leishmania (Leishmania amazonensis and b1/b2 for Leishmania (Viannia braziliensis.Leishmania (Leishmania chagasi was the only species

  9. Leishmania donovani Nucleoside Hydrolase Terminal Domains in Cross-Protective Immunotherapy Against Leishmania amazonensis Murine Infection

    Nico, Dirlei; Gomes, Daniele Crespo; Palatnik-de-Sousa, Iam; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa Beatriz

    2014-01-01

    Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani nucleoside hydrolase (NH36) induced a main CD4+ T cell driven protective response against L. chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1–103), central domain (F2 aminoacids 104–198), and C-terminal domain (F3 amino acids 199–314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48 and 64%, and the parasite load in footpads to 82.6 and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated immune responses

  10. Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.

    Chau Huynh

    Full Text Available Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

  11. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  12. Eupomatenoid-5 Isolated from Leaves of Piper regnellii Induces Apoptosis in Leishmania amazonensis

    Francielle Pelegrin Garcia

    2013-01-01

    Full Text Available Leishmania spp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves of Piper regnellii var. pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 against L. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death in L. amazonensis promastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest.

  13. Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

    da Silva, Marcelo Santos; Monteiro, Jomar Patrício; Nunes, Vinícius Santana; Vasconcelos, Elton José; Perez, Arina Marina; Freitas-Júnior, Lúcio de Holanda; Elias, Maria Carolina; Cano, Maria Isabel Nogueira

    2013-01-01

    Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus. PMID:24278433

  14. Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

    Hervé Lecoeur

    Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

  15. HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

    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

  16. The T-cell anergy induced by Leishmania amazonensis antigens is related with defective antigen presentation and apoptosis

    Roberta O. Pinheiro

    2004-09-01

    Full Text Available Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg but not L. braziliensis promastigotes (LbAg contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100ºC/1h and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.A Leishmania amazonensis é o principal agente etiológico da leishmaniose cutânea difusa, uma doença associada a respostas imunes anérgicas. Neste estudo nós mostramos que o extrato bruto de promastigotas de Leishmania amazonensis (LaAg, mas não de L. braziliensis (LbAg, contém substâncias que suprimem respostas proliferativas, espontâneas e mitogênicas, de células T. As substâncias supressoras no LaAg são termo-resistentes (100°C/1h e parcialmente dependentes da atividade de proteases. A anergia de células T não foi devida à diminuição na produção de fatores de crescimento, uma vez que não foi revertida pela adição de: IL-2, IL-4, IFN-gama ou IL-12. O LaAg não inibiu a ativação de células T induzida por anti-CD3, sugerindo que a anergia

  17. Synthesis and biological evaluation against Mycobacterium tuberculosis and Leishmania amazonensis of a series of diaminated terpenoids.

    Dos Reis, Dijovani Batista; Souza, Thalita Cristina Alves; Lourenço, Maria Cristina S; de Almeida, Mauro Vieira; Barbosa, Adriano; Eger, Iriane; Saraiva, Mauricio Frota

    2016-12-01

    We report the synthesis of a series of diaminated terpenoids containing, as side-chain of the diamine core, the "head-to-tail" prenyl derivatives, with amino amino spacers of variable length. In vitro biological activity of these compounds was evaluated against Mycobacterium tuberculosis and Leishmania amazonensis, and the structure-activity relationships are discussed. Different biological results were observed depending on the terpenic side-chain length. The best results were obtained for trans,trans-farnesol derivatives. Moreover, these results demonstrated that the stereochemistry of the double bond could play an important role in determining antitubercular and antileishmanial activities of these compounds.

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

    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.

  19. Activity of the Lupane isolated from Combretum leprosum against Leishmania amazonensis promastigotes

    Teles, Carolina B.G.; Silva-Jardim, Izaltina; Silva, Alexandre de A.E.; Zuliani, Juliana P.; Stabeli, Rodrigo G., E-mail: izaltina.jardim@pq.cnpq.b [Instituto de Pesquisas em Patologias Tropicais de Rondonia (IPEPATRO), Porto Velho, RO (Brazil); Moreira, Leandro S.; Facundo, Valdir A. [Universidade Federal de Rondonia, Porto Velho, RO (Brazil)

    2011-07-01

    This paper describes the activity of the ethanolic extract (EE), obtained from the fruits of Combretum leprosum, the triterpene 3{beta}, 6{beta}, 16{beta}-trihydroxylup-20(29)-ene (1) and its synthetic derivatives 1a-1d on Leishmania Amazonensis promastigotes. The EE displayed leishmanicidal activity and the IC{sub 50} was 24.8 {mu}g mL{sup -1}. However, the triterpene 3{beta}, 6{beta}, 16{beta}-trihydroxylup-20(29)-ene (1), at a concentration of 5.0 {mu}g mL{sup -1}, showed a potent inhibitory activity on promastigotes proliferation (IC{sub 50} = 3.3 {mu}g mL{sup -1}). Among the synthetic derivatives, only (1b) and (1d) were active against promastigotes (IC{sub 50} = 3.48 {mu}g mL{sup -1} and 5.8 {mu}g mL{sup -1}, respectively). Moreover, the synthetic derivative 1a showed no activity on promastigotes of L. Amazonensis. EE, (1) and the synthetic derivatives 1a-1d showed no cytotoxic effect on mice peritoneal macrophages. These results provide evidence that the ethanolic extract and the lupane isolated from C. leprosum was active against promastigotes of L. amazonensis, and may be used as a tool in the studies of new antileishmanial drugs. (author)

  20. Antiproliferative, Ultrastructural, and Physiological Effects of Amiodarone on Promastigote and Amastigote Forms of Leishmania amazonensis

    Sara Teixeira de Macedo-Silva

    2011-01-01

    Full Text Available Amiodarone (AMIO, the most frequently antiarrhythmic drug used for the symptomatic treatment of chronic Chagas' disease patients with cardiac compromise, has recently been shown to have also specific activity against fungi, Trypanosoma cruzi and Leishmania. In this work, we characterized the effects of AMIO on proliferation, mitochondrial physiology, and ultrastructure of Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values were 4.21 and 0.46 μM against promastigotes and intracellular amastigotes, respectively, indicating high selectivity for the clinically relevant stage. We also found that treatment with AMIO leads to a collapse of the mitochondrial membrane potential (ΔΨm and to an increase in the production of reactive oxygen species, in a dose-dependent manner. Fluorescence microscopy of cells labeled with JC-1, a marker for mitochondrial energization, and transmission electron microscopy confirmed severe alterations of the mitochondrion, including intense swelling and modification of its membranes. Other ultrastructural alterations included (1 presence of numerous lipid-storage bodies, (2 presence of large autophagosomes containing part of the cytoplasm and membrane profiles, sometimes in close association with the mitochondrion and endoplasmic reticulum, and (3 alterations in the chromatin condensation and plasma membrane integrity. Taken together, our results indicate that AMIO is a potent inhibitor of L. amazonensis growth, acting through irreversible alterations in the mitochondrial structure and function, which lead to cell death by necrosis, apoptosis and/or autophagy.

  1. Benzaldehyde thiosemicarbazone derived from limonene complexed with copper induced mitochondrial dysfunction in Leishmania amazonensis.

    Elizandra Aparecida Britta

    Full Text Available BACKGROUND: Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC(50 concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC(50 of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis. CONCLUSION/SIGNIFICANCE: Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

  2. The action of ionizing radiation on the morphology, physiology and growth of Leishmania Amazonensis, with evaluation of their immunogenic power in experimental models; Acao da radiacao ionizante sobre a morfologia, fisiologia e crescimento da Leishmania amazonensis, com avaliacao de seu poder imunogenico em modelos experimentais

    Bonetti, Franco Claudio

    2002-07-01

    Leishmaniasis is a disease which affects thousands of people in the Tropical regions around the world, is caused by a protozoan of the genus Leishmania spp., with urban and wild mammals acting as reservoirs. In the mammal host, the amastigote form of the parasite infects and multiplies into macrophages. Treatments for leishmaniasis have a high cost and are long lasting, frequently resulting in therapy interruption. This procedure culminates with a selection of resistant parasite strains, inducing tolerance to the therapy. Either the control of vectors or the mammal host are difficult due the social and economic implications. Thus, the search for alternatives treatments against these protozoans have been stimulated. The gamma radiation ({sup 60}CO) shown to be an efficient toll to kill these parasites maintaining their immunogenicity. Cellular viability, Electronically microscopy and Multiplex-PCR techniques showed that, after irradiation, the parasites had their growth inhibited by cytoplasmatic and nucleic material disorganisation, appointing the gamma radiation as important in terms of immunogens improvement. (author)

  3. Oral effectiveness of PMIC4, a novel hydroxyethylpiperazine analogue, in Leishmania amazonensis

    Mariela Ferreira de Vasconcelos

    2014-12-01

    Full Text Available Pentavalent antimonials have saved the lives of thousands of Leishmania-infected patients more than seventy years but, unfortunately, they are highly toxic and require parenteral delivery. Therefore, the search for safer and orally delivered alternative is a need. This paper describes the antileishmanial properties of PMIC4, a novel hydroxyethylpiperazine analogue. PMIC4 showed potent activity against intracellular amastigotes of Leishmania amazonensis, with IC50 of 1.8 μM and selectivity index higher than 100-fold, calculated in relation to the toxicity on the host cell. Following laboratory animal welfare policies, we analyzed the absorption, distribution, metabolism, excretion and toxicity (ADMET properties and calculated the Lipinski’s rule of five of PMIC4 before proceeding to in vivo tests. PMIC4 satisfied Lipinski’s rule of five and presented high probability of human intestinal absorption, suggesting a good chance of druglikeness and oral bioavailability. For in vivo studies, PMIC4 was administered via intralesional injection (3.4 mg/kg/day, three times a week or orally (34.0 mg/kg/day, five times a week to L. amazonensis-infected BALB/c mice throughout the 98 day experiments. At the end of the treatment period, serum markers of toxicity were measured. When administered orally, PMIC4 controlled the lesions in L. amazonensis-infected BALB/c mice without altering serological markers of toxicity. These results demonstrate that PMIC4 is a promising molecular scaffold, orally effective against experimental leishmaniasis.

  4. Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L. amazonensis Promastigote

    Marcos Hikari Toyama

    2013-03-01

    Full Text Available The sulfated polysaccharides from Solieria filiformis (Sf, Botryocladia occidentalis (Bo, Caulerpa racemosa (Cr and Gracilaria caudata (Gc were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L. amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL. The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL. In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L. amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.

  5. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    Bidyottam Mittra

    2016-01-01

    Full Text Available Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast and mitoferrin-1 (human that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1 showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

  6. Impact of tumor necrosis factor receptor p55 deficiency in susceptibility of C57BL/6 mice to infection with Leishmania (Leishmania) amazonensis.

    Cargnelutti, Diego Esteban; Salomón, María Cristina; Celedon, Verónica; Cuello-Carrión, Fernando Darío; Gea, Susana; Di Genaro, María Silvia; Scodeller, Eduardo Alberto

    2016-04-01

    Tumor necrosis factor (TNF) is involved in host resistance to several intracellular pathogens. Although the critical role of TNF receptor (TNFR)p55 in Leishmania (Leishmania) major infection has been demonstrated, the impact of TNFRp55 deficiency on L. (L.) amazonensis infection has not been explored. L. (L.) amazonensis-infected TNFRp55(-/-) mice failed to resolve lesions, whereas C57BL/6 wild-type mice completely healed. The susceptibility of the TNFRp55(-/-) mice was characterized by higher lesion size and histopathological damage in comparison with the wild-type mice. A marked increased of the splenic index was observed in the TNFRp55(-/-) mice after 15 weeks infection. These results show that in the absence of TNFRp55, L. (L.) amazonensis-infected knockout mice fail to resolve lesions, whereas wild-type mice completely heal.

  7. In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis.

    de Macedo-Silva, Sara Teixeira; Urbina, Julio A; de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2013-01-01

    Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent

  8. In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis.

    Sara Teixeira de Macedo-Silva

    Full Text Available Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ and posaconazole (POSA, two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51. Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm, which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and

  9. In Vitro Activity of the Antifungal Azoles Itraconazole and Posaconazole against Leishmania amazonensis

    de Macedo-Silva, Sara Teixeira; Urbina, Julio A.; de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2013-01-01

    Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent

  10. In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

    Rizk, Yasmin Silva; Fischer, Alice; Cunha, Marillin de Castro; Rodrigues, Patrik Oening; Marques, Maria Carolina Silva; Matos, Maria de Fátima Cepa; Kadri, Mônica Cristina Toffoli; Carollo, Carlos Alexandre; de Arruda, Carla Cardozo Pinto

    2014-01-01

    This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis. PMID:25591109

  11. Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis

    Kumari, Shraddha; Samant, Mukesh; Khare, Prashant; Misra, Pragya; Dutta, Sujoy; Kolli, Bala Krishna; Sharma, Sharad; Chang, Kwang Poo; Dube, Anuradha

    2016-01-01

    Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme-biosynthesis renders them inducible with δ-aminolevulinate to develop porphyria for selective photolysis, leaving infected host-cells unscathed. Delivery of released “vaccines” to antigen-presenting cells is thus expected to enhance immune response, while their self-destruction presents added advantages of safety. Such suicidal-L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat-killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal-mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania-specific delayed-type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN-γ and IL-12 expressions and of Leishmania-specific IgG2 in the serum. Moreover, a single intravenous administration of T-cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis. PMID:19053149

  12. Opsonization modulates Rac-1 activation during cell entry by Leishmania amazonensis.

    Morehead, J; Coppens, I; Andrews, N W

    2002-08-01

    Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.

  13. Isolation of an enriched plasma membrame subpellicular microtubule fraction of Leishmania mexicana amazonensis

    Solange L. Timm

    1980-01-01

    Full Text Available A cell fractionation procedure previously developed for Trypanosoma cruzi was applied to isolated the plasma membrane of promastigotes of Leishania mexicana amazonensis. The cell, swollen in an hypotonic mediun, were disrupted in the presence of a nonionic detergent and the membrane fraction isolated by differencial centrifugation. Electron microscopy showed that the fraction consisted of pieces of the plasma membrane associated with subpellicular microtubules. It was also shown that this fraction is able to induce cell-mediated immune response in mice.Um método de fracionamento subcelular, previamente desenvolvido para Trypanosoma cruzi, foi aplicado para isolar a membrana plasmática de promastigotas de Leishmania mexicana amazonensis. As células, após turgimento em meio hipotônico, foram rompidas na presença de um detergente não iônico e a fração de membrana isolada por centrifugação diferencial. A microscopia eletrônica mostrou consistir a fração de fragmentos de membrana plasmática associados com microtúbulos subpeliculares. Foi também mostrado que esta fração era capaz de induzir resposta celular em camundongos.

  14. Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes

    Lang Thierry

    2009-03-01

    Full Text Available Abstract Background Mammal macrophages (MΦ display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L. Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MΦ. Results Using BALB/c mouse bone marrow-derived MΦ loaded or not with amastigotes, we analyzed the transcriptional signatures of MΦ 24 h later, when the amastigote population was growing. Total RNA from MΦ cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips®, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR. A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software® pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02 involving several genes (1.95 to 4.30 fold change values, and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. Conclusion Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MΦ lipid and polyamine pathways. Moreover, these MΦ hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

  15. Tamoxifen is effective in the treatment of Leishmania amazonensis infections in mice.

    Danilo C Miguel

    Full Text Available BACKGROUND: Chemotherapy is still a critical issue in the management of leishmaniasis. Until recently, pentavalent antimonials, amphotericin B or pentamidine compounded the classical arsenal of treatment. All these drugs are toxic and have to be administered by the parenteral route. Tamoxifen has been used as an antiestrogen in the treatment and prevention of breast cancer for many years. Its safety and pharmacological profiles are well established in humans. We have shown that tamoxifen is active as an antileishmanial compound in vitro, and in this paper we analyzed the efficacy of tamoxifen for the treatment of mice infected with Leishmania amazonensis, an etiological agent of localized cutaneous leishmaniasis and the main cause of diffuse cutaneous leishmaniasis in South America. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were infected with L. amazonensis promastigotes. Five weeks post-infection, treatment with 15 daily intraperitoneal injections of 20 mg/kg tamoxifen was administered. Lesion and ulcer sizes were recorded and parasite burden quantified by limiting dilution. A significant decrease in lesion size and ulcer development was noted in mice treated with tamoxifen as compared to control untreated animals. Parasite burden in the inoculation site at the end of treatment was reduced from 10(8.5+/-0.7 in control untreated animals to 10(5.0+/-0.0 in tamoxifen-treated mice. Parasite load was also reduced in the draining lymph nodes. The reduction in parasite number was sustained: 6 weeks after the end of treatment, 10(15.5+/-0.5 parasites were quantified from untreated animals, as opposed to 10(5.1+/-0.1 parasites detected in treated mice. CONCLUSIONS/SIGNIFICANCE: Treatment of BALB/c mice infected with L. amazonensis for 15 days with tamoxifen resulted in significant decrease in lesion size and parasite burden. BALB/c mice infected with L. amazonensis represents a model of extreme susceptibility, and the striking and sustained reduction

  16. 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

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

    2010-01-01

    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 capt...

  17. Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

    Tavares, Natália; Afonso, Lilian; Suarez, Martha; Ampuero, Mariana; Prates, Deboraci Brito; Araújo-Santos, Théo; Barral-Netto, Manoel; DosReis, George A; Borges, Valéria Matos; Brodskyn, Cláudia

    2016-02-15

    Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

  18. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae) against the infection and infectivity of macrophages by Leishmania amazonensis

    BERNARDO MELO NETO; JOSEANA M.S.R. LEITÃO; OLIVEIRA,LUCIANO G.C.; SANTOS,SÉRGIO E.M.; SABRINA M.P. CARNEIRO; Rodrigues, Klinger A. F.; Chaves, Mariana H.; DANIEL D.R. ARCANJO; CARVALHO,FERNANDO A.A.

    2016-01-01

    ABSTRACT Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated ...

  19. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis.

    Camila Marques-da-Silva

    Full Text Available Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L. amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L. amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L. amazonensis escape mechanism is discussed.

  20. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis.

    Marques-da-Silva, Camila; Chaves, Mariana Martins; Rodrigues, Juliany Cola; Corte-Real, Suzana; Coutinho-Silva, Robson; Persechini, Pedro Muanis

    2011-01-01

    Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L. amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L. amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L. amazonensis escape mechanism is discussed.

  1. Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

    Souza, Géssika Silva; do Nascimento, Viviane Veiga; de Carvalho, Laís Pessanha; de Melo, Edésio José Tenório; Fernandes, Keysson Vieira; Machado, Olga Lima Tavares; Retamal, Claudio Andres; Gomes, Valdirene Moreira; Carvalho, André de Oliveira

    2013-09-01

    Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

  2. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein✩

    Rocha, Marcele N.; Corrêa, Célia M.; Melo, Maria N.; Beverley, Stephen M.; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P.

    2013-01-01

    Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

  3. Effects of medicinal plant extracts on growth of Leishmania (L. amazonensis and Trypanosoma cruzi Efeito de extratos de plantas medicinais no crescimento de Leishmania (L. amazonensis e Trypanosoma cruzi

    Patrícia Shima Luize

    2005-03-01

    Full Text Available This study describes the screening of extracts obtained from 19 species of plants used in Brazilian traditional medicine for treatment of a variety of diseases. The extracts were tested against axenic amastigote and promastigote forms of Leishmania (L. amazonensis, and epimastigote forms of Trypanosoma cruzi in vitro at a concentration of 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, and Tanacetum vulgare showed significant effects against one or both parasites, with a percentage of growth inhibition between 49.5 and 99%. The extracts showed no cytotoxic effect on sheep erythrocytes. These medicinal plants may be sources of new compounds that are clinically active against L. amazonensis and T. cruzi.Este estudo descreve a triagem de extratos obtidos de 19 espécies de plantas usadas na medicina tradicional brasileira para o tratamento de várias doenças. Os extratos foram testados contra formas amastigota axênica e promastigota de Leishmania (L. amazonensis, e formas epimastigota de Trypanosoma cruzi in vitro na concentração de 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, e Tanacetum vulgare apresentaram efeito significante contra um ou ambos parasitas, com a porcentagem de inibição de crescimento entre 49,5 e 99%. Os extratos não mostraram efeito citotóxico em hemácias de carneiro. Essas plantas medicinais podem ser fontes alternativas de novos compostos clinicamente ativos contra L. amazonensis e T. cruzi.

  4. In Vitro Assessment of Plants Growing in Cuba Belonging to Solanaceae Family Against Leishmania amazonensis.

    Monzote, Lianet; Jiménez, Jenny; Cuesta-Rubio, Osmany; Márquez, Ingrid; Gutiérrez, Yamile; da Rocha, Cláudia Quintino; Marchi, Mary; Setzer, William N; Vilegas, Wagner

    2016-11-01

    In this study, an in vitro antileishmanial assessment of plant extracts from 12 genera and 46 species growing in Cuba belonging to Solanaceae family was performed. A total of 226 extracts were screened against promastigotes of Leishmania amazonensis, and cytotoxicity of active extracts [median inhibitory concentration (IC50 ) promastigotes 5 were then assayed against intracellular amastigote. Metabolomics analysis of promissory extracts was performed using chemical profile obtained by ultra performance liquid chromatography. Only 11 extracts (4.9%) from nine plants were selected as potentially actives: Brunfelsia cestroides A. Rich, Capsicum annuum L., Capsicum chinense Jacq., Cestrum nocturnum L., Nicotiana plumbaginifolia Viv., Solanum havanense Jacq., Solanum myriacanthum Dunal, Solanum nudum Dunal and Solanum seaforthianum And., with IC50  5. Metabolomics analysis demonstrated significant differences in the chemical profiles with an average of 42.8 (range 31-88) compounds from m/z 104 to 1477, which demonstrated the complex mixture of compounds. In addition, no common markers among active extracts were identified. The results demonstrate the importance of the Solanaceae family to search new antileishmanial agents, particularly in unexplored species of this family. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Determination of femto Newton forces and fluid viscosity using optical tweezers: application to Leishmania amazonensis

    Fontes, Adriana; Giorgio, Selma; de Castro, Archimedes B., Jr.; Neto, Vivaldo M.; Pozzo, Liliana d. Y.; Marques, Gustavo P.; Barbosa, Luiz C.; Cesar, Carlos L.

    2005-03-01

    The objective of this research is to use the displacements of a polystyrene microsphere trapped by an optical tweezers (OT) as a force transducer in mechanical measurements in life sciences. To do this we compared the theoretical optical and hydrodynamic models with experimental data under a broad variation of parameters such as fluid viscosity, refractive index, drag velocity and wall proximities. The laser power was measured after the objective with an integration sphere because normal power meters do not provide an accurate measurement for beam with high numerical apertures. With this careful laser power determination the plot of the optical force (calculated by the particle displacement) versus hydrodynamic force (calculated by the drag velocity) under very different conditions shows an almost 45 degrees straight line. This means that hydrodynamic models can be used to calibrate optical forces and vice-versa. With this calibration we observed the forces of polystyrene bead attached to the protozoa Leishmania amazonensis, responsible for a serious tropical disease. The force range is from 200 femto Newtons to 4 pico Newtons and these experiments shows that OT can be used for infection mechanism and chemotaxis studies in parasites. The other application was to use the optical force to measure viscosities of few microliters sample. Our result shows 5% accuracy measurements.

  6. The in Vitro Biological Activity of the Brazilian Brown Seaweed Dictyota mertensii against Leishmania amazonensis

    Amanda Silva dos Santos Aliança

    2014-09-01

    Full Text Available Seaweeds present a wide variety of interesting bioactive molecules. In the present work we evaluated the biological activity of the dichloromethane/methanol (2:1 extract (DME from the brown seaweed Dictyota mertensii against Leishmania amazonensis and its cytotoxic potential on mammalian cells. The extract showed significant inhibitory effect on the growth of promastigote forms (IC50 = 71.60 μg/mL and low toxicity against mammalian cells (CC50 = 233.10 μg/mL. The DME was also efficient in inhibiting the infection in macrophages, with CC50 of 81.4 μg/mL and significantly decreased the survival of amastigote forms within these cells. The selectivity index showed that DME was more toxic to both promastigote (SI = 3.25 and amastigote (SI = 2.86 forms than to macrophages. Increased NO production was observed in treated macrophages suggesting that besides acting directly on the parasites, the DME also shows an immunomodulatory effect on macrophages. Drastic ultrastructural alterations consistent with loss of viability and cell death were observed in treated parasites. Confocal microscopy and cytometry analyzes showed no significant impairment of plasma membrane integrity, whereas an intense depolarization of mitochondrial membrane could be observed by using propidium iodide and rhodamine 123 staining, respectively. The low toxicity to mammalian cells and the effective activity against promastigotes and amastigotes, point to the use of DME as a promising agent for the treatment of cutaneous leishmaniasis.

  7. The in vitro biological activity of the Brazilian brown seaweed Dictyota mertensii against Leishmania amazonensis.

    dos Santos Aliança, Amanda Silva; dos Anjos, Keicyanne Fernanda Lessa; de Vasconcelos Reis, Thiago Nogueira; Higino, Taciana Mirely Maciel; Brelaz-de-Castro, Maria Carolina Accioly; Bianco, Éverson Miguel; de Figueiredo, Regina Celia Bressan Queiroz

    2014-09-09

    Seaweeds present a wide variety of interesting bioactive molecules. In the present work we evaluated the biological activity of the dichloromethane/methanol (2:1) extract (DME) from the brown seaweed Dictyota mertensii against Leishmania amazonensis and its cytotoxic potential on mammalian cells. The extract showed significant inhibitory effect on the growth of promastigote forms (IC50=71.60 μg/mL) and low toxicity against mammalian cells (CC50=233.10 μg/mL). The DME was also efficient in inhibiting the infection in macrophages, with CC50 of 81.4 μg/mL and significantly decreased the survival of amastigote forms within these cells. The selectivity index showed that DME was more toxic to both promastigote (SI=3.25) and amastigote (SI=2.86) forms than to macrophages. Increased NO production was observed in treated macrophages suggesting that besides acting directly on the parasites, the DME also shows an immunomodulatory effect on macrophages. Drastic ultrastructural alterations consistent with loss of viability and cell death were observed in treated parasites. Confocal microscopy and cytometry analyzes showed no significant impairment of plasma membrane integrity, whereas an intense depolarization of mitochondrial membrane could be observed by using propidium iodide and rhodamine 123 staining, respectively. The low toxicity to mammalian cells and the effective activity against promastigotes and amastigotes, point to the use of DME as a promising agent for the treatment of cutaneous leishmaniasis.

  8. Treatment of Leishmania (Leishmania) Amazonensis-Infected Mice with a Combination of a Palladacycle Complex and Heat-Killed Propionibacterium acnes Triggers Protective Cellular Immune Responses

    Paladi, Carolina S.; da Silva, Danielle A. M.; Motta, Priscila D.; Garcia, Daniel M.; Teixeira, Daniela; Longo-Maugéri, Ieda M.; Katz, Simone; Barbiéri, Clara L.

    2017-01-01

    Palladacycle complex DPPE 1.2 was previously reported to inhibit the in vitro and in vivo infection by Leishmania (Leishmania) amazonensis. The aim of the present study was to compare the effect of DPPE 1.2, in association with heat-killed Propionibacterium acnes, on L. (L.) amazonensis infection in two mouse strains, BALB/c and C57BL/6, and to evaluate the immune responses of the treated animals. Foot lesions of L. (L.) amazonensis-infected mice were injected with DPPE 1.2 alone, or associated with P. acnes as an adjuvant. Analysis of T-cell populations in the treated mice and in untreated controls was performed by FACS. Detection of IFN-γ-secreting lymphocytes was carried out by an ELISPOT assay and active TGF-β was measured by means of a double-sandwich ELISA test. The treatment with DPPE 1.2 resulted in a significant reduction of foot lesion sizes and parasite burdens in both mouse strains, and the lowest parasite burden was found in mice treated with DPPE 1.2 plus P. acnes. Mice treated with DPPE 1.2 alone displayed a significant increase of TCD4+ and TCD8+ lymphocytes and IFN-γ secretion which were significantly higher in animals treated with DPPE 1.2 plus P. acnes. A significant reduction of active TGF-β was observed in mice treated with DPPE 1.2 alone or associated with P. acnes. Moreover, DPPE 1.2 associated to P. acnes was non-toxic to treated animals. The destruction of L. (L.) amazonensis by DPPE 1.2 was followed by host inflammatory responses which were exacerbated when the palladacycle complex was associated with P. acnes. PMID:28321209

  9. Impacto de Leishmania amazonensis y la Sangre de Ave en el Potencial Biológico y Fecundidad de Lutzomyia migonei y Lutzomyia ovallesi (Diptera: Psychodidae

    Elsa Nieves

    2011-03-01

    Resumo. Nos flebotomíneos (Diptera: Psychodidae o hábito pela hematofagia é responsável pela indução de vários processos fisiológicos também na transmissão de Leishmania Ross. O presente estudo compara o sangue de ave, de mamífero e com infecção por Leishmania amazonensis Lainson & Shaw sobre o potencial biológico de Lutzomyia migonei (França e de Lutzomyia ovallesi Ortiz. Foram utilizadas fêmeas das duas espécies alimentadas artificialmente com sangue de hamster (Mesocricetus auratus Waterhouse e frango (Gallus gallus Linnaeus, infectados com L. amazonensis. Os grupos controle foram alimentados somente com sangue, sem parasitas. Foram determinados o grau de repasto sanguíneo, o tempo de digestão, o padrão de diurese, o tempo de oviposição, a sobrevivencia a oviposição e a fecundidade. A espécie L. migonei quando alimentada com sangue de hamster e frango apresentaram maior fecundidade do que as fêmeas de L. ovallesi, a maior fecundidade foi com sangue de frango. A presença de Leishmania no sangue de frango ou sangue de hamster diminuiu significativamente o seu consumo, o que resultou na diminuição da sobrevida das fêmeas após a oviposição em L. migonei alimentados com sangue de frango e não com sangue de hamsters. Entretanto, não afetar a quantidade de sangue e a sobrevivência de oviposição de L. ovallesi. A infecção com L. amazonensis causo um aumento no número de ovos retidos e diminuiu o número de ovos postos por L. migonei e L. ovallesi, especialmente com sangue de frango e também reduz o tempo de digestão do sangue em ambas as espécies com sangue de frango, mas não com sangue de hamster. Embora o sangue de frango foi menos eficaz do que o sangue de hamster sobre o potencial biológico de L. migonei e L. ovallesi, não exclui o sangue de frango como uma fonte de sangue para a manutenção das populações de ambas as espécies nas casas.

  10. Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation.

    Borghi, Sergio M; Fattori, Victor; Ruiz-Miyazawa, Kenji W; Miranda-Sapla, Milena M; Casagrande, Rúbia; Pinge-Filho, Phileno; Pavanelli, Wander R; Verri, Waldiceu A

    2017-02-17

    Cutaneous leishmaniasis (CL) is the most common form of the leishmaniasis in humans. Ulcerative painless skin lesions are predominant clinical features of CL. Wider data indicate pain accompanies human leishmaniasis, out with areas of painless ulcerative lesions per se. In rodents, Leishmania (L.) major infection induces nociceptive behaviors that correlate with peripheral cytokine levels. However, the role of the spinal cord in pain processing after Leishmania infection has not been investigated. Balb/c mice received intraplantar (i.pl.) injection of Leishmania (L). amazonensis and hyperalgesia, edema, parasitism, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression, and NFκB activation were evaluated. The effects of intrathecal (i.t.) injection of morphine, TNFα, TNFα inhibitors (etanercept and adalimumab) and NFκB inhibitor (PDTC) were investigated. The present study demonstrates that Leishmania (L.) amazonensis infection in balb/c mice induces chronic mechanical and thermal hyperalgesia in an opioid-sensitive manner. Spinal cord TNFα mRNA expression increased in a time-dependent manner, peaking between 30 and 40 days after infection. At the peak of TNFα mRNA expression (day 30), there was a concomitant increase in TNFR1 and TNFR2 mRNA expression. TNFα i.t. injection enhanced L. (L.) amazonensis-induced hyperalgesia. Corroborating a role for TNFα in L. (L.) amazonensis-induced hyperalgesia, i.t. treatment with the TNFα inhibitors, etanercept and adalimumab inhibited the hyperalgesia. L. (L.) amazonensis also induced spinal cord activation of NFκB, and PDTC (given i.t.), also inhibited L. (L.) amazonensis-induced hyperalgesia, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression. Moreover, L. (L.) amazonensis-induced spinal cord activation of NFκB was also inhibited by etanercept and adalimumab as well as PDTC i.t.

  11. The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis

    Yamamoto, Eduardo S.; Campos, Bruno L. S.; Jesus, Jéssica A.; Laurenti, Márcia D.; Ribeiro, Susan P.; Kallás, Esper G.; Rafael-Fernandes, Mariana; Santos-Gomes, Gabriela; Silva, Marcelo S.; Sessa, Deborah P.; Lago, João H. G.; Levy, Débora; Passero, Luiz F. D.

    2015-01-01

    Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 μg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 μg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment

  12. 17-AAG Kills Intracellular Leishmania amazonensis while Reducing Inflammatory Responses in Infected Macrophages

    Petersen, Antonio Luis de Oliveira Almeida; Guedes, Carlos Eduardo Sampaio; Versoza, Carolina Leite; Lima, José Geraldo Bomfim; de Freitas, Luiz Antônio Rodrigues; Borges, Valéria Matos; Veras, Patrícia Sampaio Tavares

    2012-01-01

    Background Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory. Methodology/Principal Findings We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25–500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O2−) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment. Conclusions/Significance The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor’s potential in the development of new generations of anti-leishmanials. PMID:23152914

  13. Determination of fluid viscosity and femto Newton forces of Leishmania amazonensis using optical tweezers

    Fontes, Adriana; Giorgio, Selma; de Castro, Archimedes, Jr.; Neto, Vivaldo M.; de Y. Pozzo, Liliana; de Thomaz, Andre A.; Barbosa, Luiz C.; Cesar, Carlos L.

    2005-08-01

    The displacements of a polystyrene microsphere trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences such as the measurement of forces of living microorganisms or the viscosity of local fluids. The technique we used allowed us to measure forces on the 200 femto Newtons to 4 pico Newtons range of the protozoa Leishmania amazonensis, responsible for a serious tropical disease. These observations can be used to understand the infection mechanism and chemotaxis of these parasites. The same technique was used to measure viscosities of few microliters sample with agreement with known samples better than 5%. To calibrate the force as a function of the microsphere displacement we first dragged the microsphere in a fluid at known velocity for a broad range of different optical and hydrodynamical parameters. The hydrodynamical model took into account the presence of two walls and the force depends on drag velocity, fluid viscosity and walls proximities, while the optical model in the geometric optics regime depends on the particle and fluid refractive indexes and laser power. To measure the high numerical (NA) aperture laser beam power after the objective we used an integration sphere to avoid the systematic errors of usual power meters for high NA beams. After this careful laser power measurement we obtained an almost 45 degrees straight line for the plot of the optical force (calculated by the particle horizontal displacement) versus hydrodynamic force (calculated by the drag velocity) under variation of all the parameters described below. This means that hydrodynamic models can be used to calibrate optical forces, as we have done for the parasite force measurement, or vice-versa, as we did for the viscosity measurements.

  14. In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis.

    Kaplum, Vanessa; Cogo, Juliana; Sangi, Diego Pereira; Ueda-Nakamura, Tânia; Corrêa, Arlene Gonçalves; Nakamura, Celso Vataru

    2016-06-01

    Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (ΔΨm), mitochondrial superoxide anion (O2·(-)) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ΔΨm, an increase in the O2·(-) concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of

  15. N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

    Gasparotto, Juciano; Kunzler, Alice; Senger, Mario Roberto; de Souza, Celeste da Silva Freitas; de Simone, Salvatore Giovanni; Bortolin, Rafael Calixto; Somensi, Nauana; Dal-Pizzol, Felipe; Moreira, José Claudio Fonseca; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva; Silva, Floriano Paes; Gelain, Daniel Pens

    2017-01-01

    BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function. PMID:28177049

  16. Glycosphingolipid antigens from Leishmania (L. amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

    A.H. Straus

    1997-03-01

    Full Text Available Specific glycosphingolipid antigens of Leishmania (L. amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC. Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L. amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

  17. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    Debora B. Petropolis

    2014-04-01

    Full Text Available Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D environment mainly composed of Collagen I (COL I. This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

  18. Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

    Nogueira, Paula M.; Assis, Rafael R.; Torrecilhas, Ana C.; Saraiva, Elvira M.; Pessoa, Natália L.; Campos, Marco A.; Marialva, Eric F.; Ríos-Velasquez, Cláudia M.; Pessoa, Felipe A.; Secundino, Nágila F.; Rugani, Jerônimo N.; Nieves, Elsa; Turco, Salvatore J.; Melo, Maria N.

    2016-01-01

    The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly. PMID:27508930

  19. Exploring the Association of Surface Plasmon Resonance with Recombinant MHC:Ig Hybrid Protein as a Tool for Detecting T Lymphocytes in Mice Infected with Leishmania (Leishmania amazonensis

    Lenilton Silva da Silveira-Júnior

    2017-01-01

    Full Text Available A surface plasmon resonance- (SPR- based recognition method applying H-2 Ld:Ig/peptides complexes for ex vivo monitoring cellular immune responses during murine infection with Leishmania (Leishmania amazonensis is described. Lymphocytes from lesion-draining popliteal lymph nodes were captured on a carboxylated sensor chip surface previously functionalized with H-2 Ld:Ig (DimerX protein bound to synthetic peptides derived from the COOH-terminal region of cysteine proteinase B of L. (L. amazonensis. In computational analysis, these peptides presented values of kinetic constants favorable to form complexes with H-2 Ld at neutral pH, with a Gibbs free energy ΔG°<0. The assayed DimerX:peptide complexes presented the property of attaching to distinct T lymphocytes subsets, obtained from experimentally infected BALB/c mice, in each week of infection, thus indicating a temporal variation in specific T lymphocytes populations, each directed to a different COOH-terminal region-derived peptide. The experimental design proposed herein is an innovative approach for cellular immunology studies of a neglected disease, providing a useful tool for the analysis of specific T lymphocytes subsets.

  20. Leishmaniose cutânea na Amazônia: registro do primeiro caso humano de infecção mista, determinado por duas espécies distintas de Leishmnias: Leishmania brasiliensis e Leishmania mexicana amazonensis Cutaneous leishmaniasis in Amazonia: the first record of a case of cutaneous leishmaniasis caused by two different parasites: Leishmania braziliensis braziliensis and Leishmania mexicana amazonensis

    F. T. Silveira

    1984-10-01

    Full Text Available Fez-se o registro, na Amazônia, do primeiro caso humano de infecção cutânea mista determinada por duas espécies distintas de Leishmania: a Leishmania braziliensis braziliensis e a Leishmania mexicana amazonensis. As duas amostras, em questão, foram isoladas de lesões distintas de um mesmo paciente, e a caracterização das espécies foi feita com base em observações de infecção experimental em hamsters, comportamento em meios artificiais de cultura, desenvolvimento de infecção experimental em Lutzomyia longipalpis, e eletroforese de isoenzimas em gel de amido. Conclui-se ser de interesse o achado que, combinado com o fato já conhecido de ausência de imunidade cruzada entre a maioria das leishmânias, sugere a necessidade do emprego de uma vacina polivalente para a região.For the first time, in the Amazon region, a mixed infection of two Leishmania was found in a patient suffering from dermal leishmaniasis. L. mexicana amazonensis was isolated from one lesion and L. braziliensis braziliensis from another. Both parasites were characterized in sandflies, hamsters, in vitro cultures, by their morphology and by isoenzyme studies in starch gel. The Authors conclude that the occurrence of this case combined with the known lack of cross immunity between most leishmanial parasites means that a vaccine for this region must be polyvalent.

  1. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

    Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-01

    The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

  2. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

    Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro dos Santos; Behrens, Maria Dutra

    2016-01-01

    The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment. PMID:27579922

  3. Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

    Brandt, Artur M L; Batista, Paulo Ricardo; Souza-Silva, Franklin; Alves, Carlos Roberto; Caffarena, Ernesto Raul

    2016-04-01

    New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

  4. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change

    Carvalho, Bruno M.; Ready, Paul D.

    2015-01-01

    Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia) flaviscutellata and the parasite it transmits, Leishmania (Leishmania) amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector’s climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest). Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: “stabilization” and “high increase”. Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador, Colombia and

  5. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia flaviscutellata (Diptera: Psychodidae: Phlebotominae, Vector of Leishmania (Leishmania amazonensis in South America, under Climate Change.

    Bruno M Carvalho

    Full Text Available Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia flaviscutellata and the parasite it transmits, Leishmania (Leishmania amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector's climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest. Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: "stabilization" and "high increase". Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador

  6. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change.

    Carvalho, Bruno M; Rangel, Elizabeth F; Ready, Paul D; Vale, Mariana M

    2015-01-01

    Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia) flaviscutellata and the parasite it transmits, Leishmania (Leishmania) amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector's climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest). Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: "stabilization" and "high increase". Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador, Colombia and Venezuela

  7. In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

    Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

    2016-11-01

    The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

  8. Impacto de Leishmania amazonensis y la Sangre de Ave en el Potencial Biológico y Fecundidad de Lutzomyia migonei y Lutzomyia ovallesi (Diptera: Psychodidae)

    2011-01-01

    En los flebótomos (Diptera: Psychodidae), la ingesta sanguínea es responsable de la inducción de varios procesos fisiológicos y es determinante en la transmisión de Leishmania Ross. El presente trabajo estudia la sangre de ave, de mamífero y mezclada con Leishmania amazonensis Lainson & Shaw sobre el potencial biológico de Lutzomyia migonei França y Lutzomyia ovallesi Ortiz. Se utilizaron hembras de ambas especies alimentadas arti...

  9. Inhibition of Leishmania (Leishmania amazonensis and rat arginases by green tea EGCG, (+-catechin and (--epicatechin: a comparative structural analysis of enzyme-inhibitor interactions.

    Matheus Balduíno Goncalves dos Reis

    Full Text Available Epigallocatechin-3-gallate (EGCG, a dietary polyphenol (flavanol from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+-catechin and (--epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L and rat liver arginase (ARG-1. The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+-catechin and (--epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+-catechin (IC50 = 0.8 µM followed by (--epicatechin (IC50 = 1.8 µM, gallic acid (IC50 = 2.2 µM and EGCG (IC50 = 3.8 µM. Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC50 values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.

  10. Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP

    Silva-Jr FP

    2002-01-01

    Full Text Available The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

  11. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

    Celia Maria Vieira Vendrame

    2014-01-01

    Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

  12. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae) against the infection and infectivity of macrophages by Leishmania amazonensis.

    Melo, Bernardo; Leitão, Joseana M S R; Oliveira, Luciano G C; Santos, Sérgio E M; Carneiro, Sabrina M P; Rodrigues, Klinger A F; Chaves, Mariana H; Arcanjo, Daniel D R; Carvalho, Fernando A A

    2016-01-01

    Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow cytometry were also evaluated. The infection rate and the number of internalized amastigote forms were markedly decreased after pretreatment of macrophages or promastigotes when compared with non-treated cells. The increase in phagocytic capability and nitrite content was also observed. Furthermore, the decrease of DNA content, size and number of promastigotes was also observed. In conclusion, ZR-EEtOH and ZR-FHEX promoted a markedly significant antileishmanial effect and reduction of infection of macrophages, probably underlying defense mechanisms activation in macrophages. These findings reinforce the potential application of Z. rhoifolium in the treatment of leishmaniasis.

  13. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae against the infection and infectivity of macrophages by Leishmania amazonensis

    BERNARDO MELO NETO

    2016-01-01

    Full Text Available ABSTRACT Zanthoxylum rhoifolium Lam. (Rutaceae has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH and its n-hexane fraction (ZR-FHEX on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow cytometry were also evaluated. The infection rate and the number of internalized amastigote forms were markedly decreased after pretreatment of macrophages or promastigotes when compared with non-treated cells. The increase in phagocytic capability and nitrite content was also observed. Furthermore, the decrease of DNA content, size and number of promastigotes was also observed. In conclusion, ZR-EEtOH and ZR-FHEX promoted a markedly significant antileishmanial effect and reduction of infection of macrophages, probably underlying defense mechanisms activation in macrophages. These findings reinforce the potential application of Z. rhoifolium in the treatment of leishmaniasis.

  14. DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

    Lira, C B B; Gui, K E; Perez, A M; da Silveira, R C V; Gava, L M; Ramos, C H I; Cano, M I N

    2009-02-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

  15. Enhancement of Leishmania amazonensis infection in BCG non-responder mice by BCG-antigen specific vaccine

    Kátia da Silva Calabrese

    1992-01-01

    Full Text Available Different patterns of cutaneous leishmaniasis can be induced when a challenge of alike dose of Leishmania amazonensis amastigotes in various inbred strains was applied. Two strains of mice, the Balb/c and C57 BL/10J, showed exceptional suscepbility, and 10(elevado a sexta potência amastigotes infective dose lead, to ulcerative progressive lesions with cutaneous metastasis and loss by necrosis of leg on wich the footpad primary lesion occured. Lesions were also progressive but in a lower degree when C3H/HeN and C57BL/6 were infected. Lesions progress slowly in DBA/2 mice presenting lesions wich reach a discreet peack after 12 weeks, do not heal but do not uncerate. DBA/2 mice is, therefore, a good model for immunomodualtion. In attempt to determine the influence of BCG in vaccination schedule using microsomal fraction, DBA/2 became an excellent model, since it is also a non-responder to BCG. Vaccination of DBA/2 mice, receiving the same 10(elevado a sexta potência BCG viable dose and 10 *g or 50 *g of protein content of microsomal fraction, lead to a progressive disease with time course similar to those observed in susceptible non-vaccinated C57BL/10J mice after 6 months of observation. An enhancement of infection in BCG non-responder mice suggests that use of BCG as immunostimulant in humans could be critical for both vaccination and immunoprophylactic strategies.

  16. In Vitro Activity of the Antifungal Azoles Itraconazole and Posaconazole against Leishmania amazonensis

    Sara Teixeira de Macedo-Silva; Urbina, Julio A; Wanderley de Souza; Juliany Cola Fernandes Rodrigues

    2013-01-01

    Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is a...

  17. Nanobiotechnologic approach to a promising vaccine prototype for immunisation against leishmaniasis: a fast and effective method to incorporate GPI-anchored proteins of Leishmania amazonensis into liposomes.

    Colhone, Marcelle Carolina; Silva-Jardim, Izaltina; Stabeli, Rodrigo Guerino; Ciancaglini, Pietro

    2015-01-01

    Liposomes are known to be a potent adjuvant for a wide range of antigens, as well as appropriate antigen carriers for antibody generation response in vivo. In addition, liposomes are effective vehicles for peptides and proteins, thus enhancing their immunogenicity. Considering these properties of liposomes and the antigenicity of the Leishmania membrane proteins, we evaluated if liposomes carrying glycosylphosphatidylinositol (GPI)-anchored proteins of Leishmania amazonensis promastigotes could induce protective immunity in BALB/c mice. To assay protective immunity, BALB/c mice were intraperitoneally injected with liposomes, GPI-protein extract (EPSGPI) as well as with the proteoliposomes carrying GPI-proteins. Mice inoculated with EPSGPI and total protein present in constitutive proteoliposomes displayed a post-infection protection of about 70% and 90%, respectively. The liposomes are able to work as adjuvant in the EPSGPI protection. These systems seem to be a promising vaccine prototype for immunisation against leishmaniasis.

  18. In Vitro Activities of ER-119884 and E5700, Two Potent Squalene Synthase Inhibitors, against Leishmania amazonensis: Antiproliferative, Biochemical, and Ultrastructural Effects▿

    Fernandes Rodrigues, Juliany Cola; Concepcion, Juan Luis; Rodrigues, Carlos; Caldera, Aura; Urbina, Julio A.; de Souza, Wanderley

    2008-01-01

    ER-119884 and E5700, novel arylquinuclidine derivatives developed as cholesterol-lowering agents, were potent in vitro growth inhibitors of both proliferative stages of Leishmania amazonensis, the main causative agent of cutaneous leishmaniasis in South America, with the 50% inhibitory concentrations (IC50s) being in the low-nanomolar to subnanomolar range. The compounds were very potent noncompetitive inhibitors of native L. amazonensis squalene synthase (SQS), with inhibition constants also being in the nanomolar to subnanomolar range. Growth inhibition was strictly associated with the depletion of the parasite's main endogenous sterols and the concomitant accumulation of exogenous cholesterol. Using electron microscopy, we identified the intracellular structures affected by the compounds. A large number of lipid inclusions displaying different shapes and electron densities were observed after treatment with both SQS inhibitors, and these inclusions were associated with an intense disorganization of the membrane that surrounds the cell body and flagellum, as well as the endoplasmic reticulum and the Golgi complex. Cells treated with ER-119884 but not those treated with E5700 had an altered cytoskeleton organization due to an abnormal distribution of tubulin, and many were arrested at cytokinesis. A prominent contractile vacuole and a phenotype typical of programmed cell death were frequently found in drug-treated cells. The selectivity of the drugs was demonstrated with the JC-1 mitochondrial fluorescent label and by trypan blue exclusion tests with macrophages, which showed that the IC50s against the host cells were 4 to 5 orders of magnitude greater that those against the intracellular parasites. Taken together, our results show that ER-119884 and E5700 are unusually potent and selective inhibitors of the growth of Leishmania amazonensis, probably because of their inhibitory effects on de novo sterol biosynthesis at the level of SQS, but some of our

  19. Chemotaxis study using optical tweezers to observe the strength and directionality of forces of Leishmania amazonensis

    Pozzo, Liliana d. Y.; Fontes, Adriana; de Thomaz, André A.; Barbosa, Luiz C.; Ayres, Diana C.; Giorgio, Selma; Cesar, Carlos L.

    2006-08-01

    The displacements of a dielectric microspheres trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences. This system can measure forces on the 50 femto Newtons to 200 pico Newtons range, of the same order of magnitude of a typical forces induced by flagellar motion. The process in which living microorganisms search for food and run away from poison chemicals is known is chemotaxy. Optical tweezers can be used to obtain a better understanding of chemotaxy by observing the force response of the microorganism when placed in a gradient of attractors and or repelling chemicals. This report shows such observations for the protozoa Leishmania amazomenzis, responsible for the leishmaniasis, a serious tropical disease. We used a quadrant detector to monitor the movement of the protozoa for different chemicals gradient. This way we have been able to observe both the force strength and its directionality. The characterization of the chemotaxis of these parasites can help to understand the infection mechanics and improve the diagnosis and the treatments employed for this disease.

  20. Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

    Almeida-Souza, Fernando; de Souza, Celeste da Silva Freitas; Taniwaki, Noemi Nosomi; Silva, João José Mendes; de Oliveira, Renata Mondêgo; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-31

    Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 μg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 μg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes.

  1. Leishmaniose cutânea na Amazônia: registro do primeiro caso humano de infecção mista, determinado por duas espécies distintas de Leishmnias: Leishmania brasiliensis e Leishmania mexicana amazonensis

    F. T. Silveira

    1984-10-01

    Full Text Available Fez-se o registro, na Amazônia, do primeiro caso humano de infecção cutânea mista determinada por duas espécies distintas de Leishmania: a Leishmania braziliensis braziliensis e a Leishmania mexicana amazonensis. As duas amostras, em questão, foram isoladas de lesões distintas de um mesmo paciente, e a caracterização das espécies foi feita com base em observações de infecção experimental em hamsters, comportamento em meios artificiais de cultura, desenvolvimento de infecção experimental em Lutzomyia longipalpis, e eletroforese de isoenzimas em gel de amido. Conclui-se ser de interesse o achado que, combinado com o fato já conhecido de ausência de imunidade cruzada entre a maioria das leishmânias, sugere a necessidade do emprego de uma vacina polivalente para a região.

  2. TROMBOCITOPENIA IMMUNOMEDIATA SECONDARIA IN CANI NATURALMENTE INFETTI DA LEISHMANIA INFANTUM

    2009-01-01

    Lo studio si prefigge di indagare, mediante immunofluorescenza indiretta e citometria a flusso, se la presenza di anticorpi anti-piastrine può essere associata all’occorrenza di trombocitopenia immunomediata in cani naturalmente infetti da Leishmania infantum.

  3. Lutzomyia reducta Feliciangeli et al., 1988, a host of Leishmania amazonensis, sympatric with two other members of the Flaviscutellata complex in southern Amazonas and Rondônia, Brazil (Diptera: Psychodidae Lutzomyia reducta Feliciangeli et al., 1988 um hospedeiro de Leishmania amazonensis, simpátrico com duas outras espécies do complexo flaviscutellata no sul do Amazonas e Rondônica, Brasil (Diptera: Psychodidae

    R. A. Freitas

    1989-09-01

    Full Text Available A member of the Lutzomyia flaviscutellata complex from Rondônia and southern Amazonas States, Brazil, is so close to the Venezuelan Lutzomyia olmeca recuta Feliciangeli et al., 1988, that it is regarded as belonging to the same species. Since this phlebotomine co-extis with L. olmeca nociva in Brazil, the subspecific status of the former is untenable and is rased to specific rank, as Lutzomyia reducta. The Brazilian material is described and illustrated, and compared with specimens of L. o. nociva and L. flaviscutellata from the same area. Keys to the known taxa of the flaviscutellata complex are presented. Leishmania amazonensis was isolated from one heavily infected specimen of L. reducta, making this the third species of the flaviscutellata complex to be implicated as a vector of this parasite in Brazil. The relative abundance of the three sympatric flaviscutellata complex species varies locally and appears to be related to soil drainage. L. reducta constituted about 25% if all phlebotomines captured in Disney traps at poorly drained and well drained site, but appears not to coloniza areas subject to periodic flooding. L. olmeca nociva was restricted to poorly drained areas not subject to flooding, whereas L. flaviscutellata was ubiquitous L. reducta has never been detected north of the Amazon river in Brazil, but absence of recosrds from western and northwestern Amazonas State may reflect lack of collecting in these areas.Um flebotomíneo do complexo Lutzomyia flaviscutellata, de Rondônia e sul do Amazonas, Brasil é tão parecido com Lutzomyia olmeca reducta, que é considerado como sendo da mesma espécie. Este flebotomíneo ocorre junto com L. olmeca nociva, portanto o nome é emendado para o nível de espécie, como Lutzomyia reducta. O material do Brasil é descrito e ilustrado, e comparado com exemplares de L. o. nociva e L. flaviscutellata da mesma área. Chaves para as espécies e subespécies do complexo flaviscutellata são inclu

  4. El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis

    José F. Gil

    2011-10-01

    Full Text Available Es importante conocer si la variabilidad de especies de Leishmania circulantes en una región afecta la performance de las pruebas de ELISA estandarizadas para el diagnostico de la leishmaniasis. El objetivo de este trabajo fue analizar la reactividad de la prueba de ELISA utilizando homogenados de promastigotes de Leishmania (V. braziliensis (ELISAb, L (L amazonensis (ELISAa y L (V. guyanensis (ELISAg frente a distintos grupos de sueros. Se estudiaron muestras de personas con leishmaniasis cutánea (n = 37, leishmaniasis mucocutánea (n = 8, no infectados (n = 52, infectadas por Trypanosoma cruzi (n = 11 e infecciones mixtas (n = 14. Se calcularon las sensibilidades, especificidades, cut off, valores predictivos, y se compararon las tres pruebas usando ANOVA, índice de concordancia kappa, comparación de curvas ROC e intervalos de confianza construidos por el método de bootstrap. Se encontraron diferencias significativas al comparar los niveles de DO de los sueros de pacientes con leishmaniasis cutánea respecto a los controles negativos, pero no se encontraron diferencias entre pruebas. Las sensibilidades calculadas fueron de 84.6% para ELISAb y ELISAa y de 88.5 para ELISAg, mientras que el valor de especificidad para las tres pruebas fue de 96.2. El índice de concordancia kappa y la comparación de curvas ROC mostraron performances similares para las tres pruebas (p = 0.225. La elevada reactividad obtenida para estas ELISAs frente a sueros de pacientes con leishmaniasis mucocutánea indica un importante potencial de esta técnica como complemento en el diagnóstico de la enfermedad.It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V. braziliensis (ELISAb, Leishmania (L amazonensis

  5. Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

    Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen

    2015-02-01

    Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

  6. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA Induce Protective Immune Responses in Dogs.

    Elodie Petitdidier

    2016-05-01

    Full Text Available Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA, from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA or its carboxy terminal part LaPSA-12S (Cter-rPSA, combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

  7. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  8. Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania Vacinação de camundongos C57BL/10 contra leishmaniose com promastigotas mortas de diferentes cepas e espécies de Leishmania

    Wilson Mayrink

    2002-04-01

    Full Text Available Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L. amazonensis infection.Estudos anteriores revelaram que uma vacina preparada com promastigotas mortas de cinco cepas de Leishmania pode induzir uma imunidade protetora para a leishmaniose tegumentar americana no homem e em modelos experimentais. Um dos problemas do uso desta vacina é a complexidade de sua composição e a necessidade de se incorporar diferentes cepas de Leishmania. Por esta razão, extratos antigênicos de cada uma das cinco cepas constituintes da vacina foram preparados e usados individualmente em estudos imunológicos com camundongos C57BL/10. A Leishvacin® e a vacina monovalente PH8 induziram os maiores níveis de Interferon-g (IFN-gama detectado no sobrenadante de células esplênicas dos animais vacinados. Cada grupo imunizado com vacinas monovalentes desenvolveram uma imunidade protetora seis meses após a infecção desafio com promastigotas de Leishmania (Leishmania amazonensis e nenhuma diferença estatística foi observada entre os grupos vacinados. Pode-se concluir que vacinas compostas por cepas isoladas de Leishmania protegem camundongos C57BL/10 contra, pelo menos, da infecção por L. (L. amazonensis.

  9. Sobre a sensibilidade da cultura de leucócitos circulantes na detecção de Leishmania no sangue periférico de pacientes com leishmaniose tegumentar

    Fernando T. Silveira

    1989-09-01

    Full Text Available Foi investigada a presença de Leishmania, através da cultura de leucócitos circulantes, no sangue periférico de 60 pacientes portadores de leishmaniose tegumentar americana, nas suas diferentes formas clínicas, assim como nas principais fases evolutivas da doença. Biópsias de lesões cutâneas e/ou de mucosa desses pacientes foram obtidas com a finalidade de isolar e caracterizar os parasitas, através da técnica de anticorpos monoclonais. Dos 60 pacientes examinados, foram isoladas 40 amostras de Leishmania das lesões biopsiadas, sendo 5 de Leishmania (V. brasiliensis, 3 de L. (V. guyanensis, 1 de L. (V. lainsoni, 13 de L. (L. amazonensis e 18 não puderam ser caracterizados a nível específico, porém, reagiram com anticorpos monoclonais do grupo braziliensis. Quanto àpesquisa através das culturas de leucócitos circulantes, esta revelou resultados completamente negativos. Com base nesses achados, os autores concluíram ser pouco consistente atribuir valor à cultura de leucócitos para o diagnóstico da leishmaniose tegumentar.The possible presence of Leishmania in the peripheral blood of 60 patients with American cutaneous leishmaniasis was investigated by the culture of circulating leucocytes. Patients were selected with a variety ofclinical forms ofthe disease and in different evolutionary stages of infection. Biopsies of skin and/or mucosal lesions were made in order to isolate the parasites, which were identified using monoclonal antibodies. 40 isolations were obtained, including 5 of Leishmania (Viannia braziliensis, 3 L. (V. guyanensis, 1 L. (V. lainsoni, 13 L. (Leishmania amazonensis and 18 which could only be identified as parasites of the braziliensis complex. Cultures of circulanting leucocytes were consistently negative, and the authors conclude that this method is of little use in diagnosis of cutaneous or mucocutaneous leishmaniasis.

  10. El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria

    José F. Gil

    2011-10-01

    Full Text Available Es importante conocer si la variabilidad de especies de Leishmania circulantes en una región afecta la performance de las pruebas de ELISA estandarizadas para el diagnostico de la leishmaniasis. El objetivo de este trabajo fue analizar la reactividad de la prueba de ELISA utilizando homogenados de promastigotes de Leishmania (V. braziliensis (ELISAb, L (L amazonensis (ELISAa y L (V. guyanensis (ELISAg frente a distintos grupos de sueros. Se estudiaron muestras de personas con leishmaniasis cutánea (n = 37, leishmaniasis mucocutánea (n = 8, no infectados (n = 52, infectadas por Trypanosoma cruzi (n = 11 e infecciones mixtas (n = 14. Se calcularon las sensibilidades, especificidades, cut off, valores predictivos, y se compararon las tres pruebas usando ANOVA, índice de concordancia kappa, comparación de curvas ROC e intervalos de confianza construidos por el método de bootstrap. Se encontraron diferencias significativas al comparar los niveles de DO de los sueros de pacientes con leishmaniasis cutánea respecto a los controles negativos, pero no se encontraron diferencias entre pruebas. Las sensibilidades calculadas fueron de 84.6% para ELISAb y ELISAa y de 88.5 para ELISAg, mientras que el valor de especificidad para las tres pruebas fue de 96.2. El índice de concordancia kappa y la comparación de curvas ROC mostraron performances similares para las tres pruebas (p = 0.225. La elevada reactividad obtenida para estas ELISAs frente a sueros de pacientes con leishmaniasis mucocutánea indica un importante potencial de esta técnica como complemento en el diagnóstico de la enfermedad.

  11. Determinación de la producción de Öxido Nítrico en Macrófagos Activados J774.1 como respuesta al tamaño de la carga fagocítica de partículas de látex y Leishmania amazonensis Determination of Nitric Oxide Production in Activate Macrophages J774.1 as effect of Phagocitic Load Volume of Latex Beads and Leishmania amazonensis

    Camargo Jiménez Maria Helena

    2000-06-01

    Full Text Available La leishmaniosis es una enfermedad parasitaria causada por el protozoario Leishmania. Cercade 12 millones de personas padecen esta enfermedad y 350 millones de personas están enriesgo de contraerla. Existe evidencia de que la infección por Leishmania amazonensisdisminuyela producción de óxido nítrico (NO de macrófagos; se ha interpretado que este deterioroes inducido por el parásito. Esta investigación corrobora esta idea, pero contradice que seaexclusivo de la infección. En este trabajo, cultivos celulares de macrófagos fueron expuestosa partículas de látex en diferentes proporciones buscando cargas fagocíticas y volúmenes defagosoma semejantes a los de la infección. Las concentraciones de nitrito y parámetros mor-fológicos se midieron a las 48 horas post fagocitosis e infección con 24 horas de activaciónmediante IFN-γy LPS. Se determinó que volúmenes similares de fagosomas generados porpartículas de látex o amastigotes de L. amazonensis, deterioran en la misma proporción laproducción de NO. Esto sugiere que este deterioro no depende de la naturaleza de la partículafagocitada. El incremento en el volumen de fagosoma se correlaciona con la disminución en laproducción de nitrito, por tanto, la expansión del fagosoma puede ser uno de los mecanismosimplicados en la disminución de la producción de NO. Los resultados apoyan la evidencia deque Leishmaniadisminuye la producción de NO pero contradicen la interpretación,comúnmente aceptada, de que este fenómeno es específico de la infección.Leishmaniasis is a parasitic disease caused by a protozoa of the genera Leishmania. Around 12million people are infected and 350 millions of people are risk to contract it. There is evidencethat infection by Leishmania amazonensisdecreases nitric oxide production; it has beeninterpreted that this impairment is induced by the parasite. This study confirmed this idea, butcontradicts that this impairment is exclusive of infection. In

  12. Busca de genes de Leishmania spp que possam codificar a leishporina e expressão dos produtos gênicos de hemolisinas do tipo III putativas de L.major

    Natalia Tomich Paiva Miranda

    2010-01-01

    A leishporina é uma citolisina formadora de poros descrita pelo nosso grupo, inicialmente em Leishmania amazonensis, mas cuja atividade citolítica foi também detectada em L. major, L. panamensis e L. guyanensis (Noronha,1996; Noronha et al., 1996; Noronha et al., 2000; Almeida-Campos & Horta, 2000). A atividade citolítica da leishporina de L. amazonensis foi detectada em extratos de promastigotas e de amastigotas, mas toda a caracterização desta atividade foi feita em promastigotas. Diversas ...

  13. Acercamiento al estudio de la interacción y salida de Leishmania amazonensis en un modelo in vitro con macrófagos murinos de la línea celular J774a.1

    León Cabrera Sonia Andrea

    2006-06-01

    Full Text Available Los miembros del género Leishmania son parásitos intracelulares obligados, responsables de numerosas enfermedades humanas. Cumplen una parte muy importante de su ciclo de vida dentro del mamífero hospedero, en donde después de la fagocitosis por los macrófagos, los parásitos son confinados dentro de un compartimiento endolisosomal denominado vacuola parasitófora (VP, en el cual se replican siendo finalmente liberados infectando otros macrófagos y de esta forma ampliando la infección. Poco es el conocimiento que se tiene acerca de cómo ocurre el proceso de liberación de amastigotes de Leishmania que infectan macrófagos. Se sospecha que este mecanismo puede estar ocurriendo por un proceso de fusión de membranas. Mediciones de capacitancia
    de la membrana del macrófago y el uso de inhibidores de fusión de membranas soportan esta idea. El objetivo de este trabajo fue realizar seguimientos del ciclo infectivo de Leishmania amazonensis, para confirmar los hallazgos
    previos en cuanto a los tiempos en que probablemente puede estar ocurriendo la salida del amastigote. Además, se buscó determinar la viabilidad del parásito a lo largo del ciclo infectivo con el fin de comprender mejor la interacción hospedero-patógeno en el modelo in vitro; para ello se midió: viabilidad del parásito con tinción de diacetato de fluoresceína (DAF y ioduro de propidio (IP, porcentaje de infección y número de parásitos por célula (p/c. Los resultados sugieren que la salida de los parásitos puede presentarse entre las 72 y 78 horas post infección (hpi y entre las 96 y 120 hpi. Con los resultados de trabajos previos, y los datos presentados en este estudio, se ha propuesto que L. amazonensis puede presentar dos ciclos infectivos que se desarrollan durante cinco días en nuestras condiciones de cultivo in vitro. En las primeras 36-48 hpi el parásito se diferencia a amastigote. Después de su diferenciación comienza su división celular

  14. Diagnostic Antigens of Leishmania.

    1994-01-31

    L. major (LTM p-2), L. major (Friedlander), and Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) were used. Leishmania promastigotes and T. cruzi ...some weak hybridization was observed with L. amazonensis, but none was seen with L. braziliensis, L. guyanensis, or T cruzi . A similar, overlapping... cruzi (8) have been previously isolated by us. To address this possibility in rLt-1, a portion of the repeat was expressed separately as rLt-lr. The

  15. Leishmaniose cutânea disseminada produzida por Leishmania viannia braziliensis no Estado do Maranhão - Brasil

    Cloves Eduardo S. Galvão

    1993-06-01

    Full Text Available Os autores relatam o caso de um paciente procedente do Município de Barreirinhas, MA, portador de leishmaniose cutânea disseminada, que apresentava 58 lesões distribuídas pelo corpo, sob os mais variados aspectos com predominância da lesão ulcerada. Discutem a dificuldade do diagnóstico laboratorial na fase inicial da investigação e suas implicações com a terapêutica. O parasita isolado e caracterizado pela técnica de anticorpos monoclonais foi a Leishmania viannia braziliensis. Esta forma da doença é diferente da leishmaniose cutânea difusa, encontrada no Maranhão, cujo agente etiológico é a Leishmania (Leishmania amazonensis, responsável pela grande maioria dos casos de leishmaniose tegumentar naquele Estado. Os possíveis mecanismos de disseminação das lesões são também discutidos neste trabalho.The authors describe a case of a patient from Barrreirinhas, MA with disseminated cutaneous leishmaniasis, who presented 58 lesions spread all over his body, with many different aspects, but ulcerative lesions were predominant. They discuss the difficulty of laboratorial diagnosis in the beginning of the investigation and its therapeutics implications. The parasite isolated yvas identified as Leishmania viannia braziliensis using monoclonal antibodies in the fluorescent antibody test. This form of the disease is different from the diffuse cutaneous leishmaniasis found in Maranhão, due to Leishmania (Leishmania amazonensis which is responsible for most of the cases of cutaneous leismaniasis in our State. The possible mechanisms of lesions dissemination are discussed.

  16. Leishmania mexicana in Proechimys iheringi denigratus Moojen (Rodentia, Echimyidae in a region endemic for American cutaneous leishmaniasis

    Air C. Barretto

    1985-12-01

    Full Text Available Three isolates of Leishmania were recovered from five of 27 specimens of the rodent Proechimys iheringi denigratus Moojen captured near Três Braços in the Atlantic Forest region of Bahia, Brazil. Two of these isolates were recovered from hamsters inoculated with a pooled triturate of liver, spleen and skin tissue from apparently healthy P. i. denigratus. The third isolate was recovered from a triturate of only skin tissue from another. Metastasis was observed in the inoculated hamsters, the parasites grew abundantly in artificial media and a typical suprapylarial pattern of infection in Lutzomyia longipalpis was produced indicating that the parasites belong to the Leishmania mexicana complex. All isolates reacted with Leishmania mexicana mexicana and Leishmania mexicana amazonensis monoclonal antibodies. The isoenzyme analysis differentiated these isolates from standard isolates of L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami and L. m. ssp.(Goiás-W. Barbosa. These isolates seem to be a subspecies of L. mexicana very closely related to L. m. amazonensis from which they differ by decreased electrophoretic mobility of GPI, PEP and ALAT. This is the first record of the isolation of a parasite of thegenus Leishmania in a rodent captured in the State of Bahia.Três isolados de Leishmania foram obtidos de cinco entre 27 exemplares do roedor Proechimys iheringi denigratus, capturados na região de Três Braços, na mata atlântica do Estado da Bahia, Brasil. O isolamento desse parasito foi feito através de inoculação de triturado de pele, baço e fígado em patas de hamsters. Em pelo menos um dos casos, (MTB-574, o parasito foi isolado da pele. Metas- tase foi observada nos hamsters inoculados, os parasitos cresceram abundantemente em meios artificiais de cultura e um padrão suprapapilario típico foi obtido em Lutzomyia longipalpis, indicando que o parasito pertence ao complexo L. mexicana. Todos os isolados

  17. Tentativa de transmissão da Leishmania donovani pela picada do Lutzomyia longipalpis entre cães

    Ítalo A. Sherlock

    1972-02-01

    Full Text Available Os Autores apresentam dados sôbre tentativas de transmissão experimental da Leishmania donovani pela picada de Lutzomyia longipalpis entre cães. Dois cães jovens sadios foram picados respectivamente por dois e sete flebótomos ricamente infectados e não adquiriram leishmaniose.

  18. Métodos subsidiários para o diagnóstico da Leishmaniose tegumentar americana (LTA: comparação dos resultados do seqüenciamento de DNA e da PCR-RFLP para determinação da espécie de leishmania em amostras cutâneo-mucosas Subsidiary methods for the diagnosis of American tegumentar leishmaniasis (ATL: comparison of sequencing of DNA and PCR-RFLP for identification of leishmania species in skin samples

    Flávio C. Barbosa Garcia

    2005-12-01

    Full Text Available FUNDAMENTOS: Métodos moleculares têm-se mostrados mais eficazes para o diagnóstico da LTA. OBJETIVOS: Comparar os resultados da intradermorreação de Montenegro (IRM, presença de leishmania em biópsia (Bc, reação de imunofluorescência indireta (Rifi, seqüenciamento de DNA e PCR-RFLP (-restriction fragment lenght polymorphism para o diagnóstico da LTA. MÉTODOS: Foram estudados 152 pacientes com LTA. Para a PCR em Bc, utilizaram-se primers específicos para seqüência de 120bp do kDNA do minicírculo comum a todas as espécies de leishmanias. O produto da PCR, utilizado para seqüenciamento e para restrição enzimática com Hae III, foi comparado às culturas L. (L. amazonensis e L. (V. braziliensis. RESULTADOS: Houve predomínio do sexo masculino (75%, da cor branca (80% e da profissão urbana (48%. A idade variou de três a 77 anos, com 56,5% entre 21 e 50 anos. 65,8% eram do Estado de São Paulo, prevalecendo a forma cutânea (79,6%. A IRM foi positiva em 73,4%, e a Rifi em 59,7%, enquanto a Bc evidenciou presença de leishmania em 30,6%. A PCR foi positiva em 81,6%, e a PCR-RFLP identificou L. (V. braziliensis como espécie predominante (66%, o que também ocorreu com o seqüenciamento. Comparando PCR-RFLP e seqüenciamento, houve 61% de concordância entre os resultados, mostrando significância da PCR-RFLP para L. (V. braziliensis. CONCLUSÕES: A IRM e a PCR foram estatisticamente equivalentes como métodos subsidiários para o diagnóstico da LTA, a PCR-RFLP e o seqüenciamento também o foram na identificação das espécies de leishmania, o primeiro apresentando menores custo e tempo de execução comparado ao seqüenciamento de DNA.BACKGROUND: ATL is endemic in Brazil, and molecular methods have been shown more effective for its diagnosis. OBJECTIVES: Our purpose was to compare the results of Montenegro’s skin reaction (MR, presence of leishmania in skin biopsy (Bx, indirect immunofluorescence (IIF for leishmania in sera

  19. Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission Formas infectante de Leishmania no vetor flebotomíneo e algumas observações sobre o mecanismo de transmissão

    Ralph Lainson

    1987-09-01

    Full Text Available Infective stages of Leishmania (Leishmania amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L. chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of [quot ]metacyclic[quot ] promastigotes in the mouthparts of the vector is discussed.Foi demonstrado através de infecção experimental, que estágios infectivos de Leishmania (L. amazonensis, capazes de produzir infecção na pele do hamster, encontram-se presentes no vetor flebotomíneo Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 e 120 horas após o inseto ter recebido sua refeição sangüínea infectiva. Da mesma maneira, foi comprovada a presença de estágios infectivos de L. (L. chagasi em exemplares do vetor Lu. longipalpis, examinados 38, 50, 63, 87, 110, 135, 171 e 221 horas após o repasto sangüíneo infectivo - através da inoculação em hamster por via intraperitoneal dos flagelados obtidos desses fle botomíneos. A questão sobre a transmissão do gênero Leishmania pelo flebotomíneo ser ou não dependente da presença de promastigotos "metacíclios" na proboscis do vetor, é discutida.

  20. Componentes antiinflamatórios na saliva do Lutzomyia longipalpis, vetor da Leishmania chagasi

    Marta Chagas Monteiro

    2005-06-01

    Full Text Available A inoculação da saliva de vetores na pele do hospedeiro é importante tanto para a alimentação do inseto quanto para a transmissão e estabelecimento de várias infecções. Em leishmaniose, vários estudos demonstram que a saliva dos vetores Lutzomyia e Phlebotomus contém substâncias com atividades imunossupressoras, imunomodulatórias, vasodilatadoras, anti-plaquetárias e anticoagulantes. Os componentes salivares auxiliam a alimentação do inseto através do aumento do fluxo sanguíneo, assim como induzem a imunossupressão no hospedeiro, o que é fundamental para o estabelecimento da infecção por Leishmania. Neste trabalho foi observado que a saliva induz a produção de IL-10, citocina antiinflamatória, não alterando a produção de IFN-g , citocina próinflamatória, no foco da inflamação. Além disso, a saliva potencializa o edema induzido por carragenina.

  1. 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

    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)

  2. Gamma radiation affects the anti-Leishmania activity of Bothrops moojeni venom and correlates with L-amino acid oxidase activity

    Tempone, A.G.; Lourenco, C.O.; Spencer, P.J.; Rogero, J.R.; Nascimento, N. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Div. de Radiobiologia; Andrade Junior, H.F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical

    1999-11-01

    Leishmania causes human disfiguring skin disease in endemic areas of Amazon and North Eastern Brazil. Those parasites present a remarkable resistance to most treatments, except those using toxic antimonial salts. We detected a specific anti-Leishmania activity in snake venoms, using an in vitro promastigote assay. In this report, we analyzed the activity of Bothrops moojeni venom against L. Amazonensis, using whole venom or fractions of L-amino acid oxidase (L-AO). Crude venom of B.moojeni, was fractionated by molecular exclusion chromatography. Activity against promastigotes was detected by respiratory oxidative conversion of MTT in a colorimetric assay and L-AO activity was detected by a colorimetric assay with peroxidase and OPD as revealing reagents. Crude venom was irradiated with 500, 1000, and 2000 Gy in a {sup 60} Co gamma radiation source. The venom had an anti-Leishmania activity of 33 pg/promastigote and the active fraction migrates as 100-150 kDa, close to the size described for L-AOs, and also presented L-AO activity. The radiation reduces both the L-AO and anti-Leishmania activity in a dose dependent effect. Those data suggests the anti-Leishmania activity in this venom is closely related to the L-amino acid oxidase activity and also that radiation could be used as a tool to detect specific activities reduction in water solutions, similarly to observed in dry preparations. (author) 13 refs., 3 figs.

  3. Glucantime resistant Leishmania promastigotes are sensitive to pentostam

    Elizabeth Spangler Andrade Moreira

    1992-12-01

    Full Text Available Growth inhibition in vitro tests were used to study the susceptibility to pentostam of different Leishmania strains involved in cutaneous and mucocutaneos leishmaniasis - one glucantime sensitive strain, three naturally glucantime resistant strains and one glucantime resistant line developed by in vitro drug exposure. Contrasting with the high degree , of glucantime resistance, all strains were sensitive to pentostam. These differences suggest that there is some relationship between chemical structure and in vitro activity for these antimonial compounds. These data justify a clinical re-evaluation to compare therapeutic efficacy of glucantime and pentostam in the treatment of leishmaniasis.Diferentes amostras de Leishmania foram analisadas quanto à susceptibilidade in vitro ao pentostam - uma cepa de L. (V braziliensis considerada sensível ao glucantime, três cepas (duas L. (V braziliensis e uma L. (L amazonensis consideradas naturalmente resistentes ao glucantime, uma linhagem resistente (L. (V guyanensis selecionada in vitro pela exposição em alta concentração de droga. A elevada sensibilidade destas amostras em contraposição à resistência observada para o glucantime sugere existir relação entre a estrutura química e a atividade destes compostos. Estes dados indicam a necessidade de ima avaliação comparativa de atividade clínica do pentostam e do glucantime no tratamento da leishmaniose.

  4. Estudos da correlação entre estrutura e função da enzima fumarato hidratase em Leishmania major

    Patrícia Rosa Feliciano

    2013-01-01

    Leishmania é um protozoário parasito flagelado responsável pelas Leishmanioses, classificadas como doenças negligenciadas, que causam um risco a 350 milhões de pessoas em todo o mundo. As fumarato hidratases (FHs) são enzimas que catalisam a hidratação reversível da molécula de fumarato em S-malato e estudos recentes em tripanosomatídeos, utilizando Trypanosoma brucei como modelo, apontam essas enzimas como potenciais alvos para o planejamento de compostos com ação tripanossomicida e leishman...

  5. [PCR-RFLP/Hsp70 for identification and tipification of leishmania from the tropical region].

    Margarita Montalvo, Ana; Fraga, Jorge; Aylema Romero, Jaqueline; Monzote, Lianet; Ivon Montano, Ing; Dujardin, Jean Claude

    2006-01-01

    The optimization of the PCR conditions for amplification of the gene coding for the 70 kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, Lamazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosornma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

  6. Cura espontânea da leishmaniose causada por Leishmania Viannia Braziliensis em lesões cutâneas

    Jackson Maurício Lopes Costa

    1990-12-01

    Full Text Available Os autores relatam que durante 14 anos de trabalho clínico em campo, realizado nas comunidades de Três Braços e Corte de Pedra, Bahia, acompanharam 1.416 pacientes portadores de Leishmaniose Tegumentar Americana, cuja espécie envolvida na transmissão, é predominantemente a Leishmania Viannia brasilienses. A terapêutica utilizada rotineiramente nos casos é o antimoniato-N-metilglucamina (Glucantime. Contudo, 16 pacientes do sexo masculino recusaram-se a utilizar a medicação e 6 do sexo feminino encontravam-se em período gestacional, portanto não utilizaram o medicamento. Estes pacientes foram acompanhados por um período entre 4 a 12 anos, a partir do diagnóstico. Observou-se que em 9 pacientes (40,9% desta casuística, o tempo de cicatrizaçâo após o aparecimento da lesão, pode ser calculado em 6 meses de evolução. Quando se eleva a observação para 12 meses, temos que 19 pacientes (86,3% cicatrizaram suas lesões neste período. Em 3 casos (13,6% as lesões permaneceram ativas por mais de 12 meses. Conclui-se que os determinantes da cicatrizaçâo natural das lesões produzidas por Leishmania Viannia Braziliensis permanecem desconhecidos, dificultando para nós entendermos e compararmos aos efeitos das drogas utilizadas no tratamento da leishmaniose tegumentar.

  7. Identification, using isoenzyme electrophoresis and monoclonal antibodies, of Leishmania isolated from humans and wild animals of Ecuador.

    Mimori, T; Grimaldi, G; Kreutzer, R D; Gomez, E A; McMahon-Pratt, D; Tesh, R B; Hashiguchi, Y

    1989-02-01

    Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.

  8. Effects of seco-steroids purified from Physalis angulata L., Solanaceae, on the viability of Leishmania sp Efeitos de seco-esteróides purificados de Physalis angulata L., Solanaceae na viabilidade de Leishmania sp

    Elisalva T. Guimarães

    2010-12-01

    Full Text Available Physalis angulata L., Solanaceae, is an annual herb commonly used in popular medicine in many tropical and subtropical countries. P. angulata extracts contain a variety of substances, but little is known about their pharmacological activities. In this work we investigated the in vitro antileishmanial activity of seco-steroids (physalins purified from P. angulata. Addition of physalins B, F, and G caused a concentration-dependent inhibition in the growth of L. amazonensis promastigotes, being the IC50 values were 6.8, 1.4, and 9.2 μM, respectively. Physalin D was less active and had an IC50 value of 30.5 μM. Physalins were also active in cultures of other Leishmania species (L. major, L. braziliensis, and L. chagasi. Our results demonstrate the potent antileishmanial activity of physalins in cultures of Leishmania species of the New and Old Worlds and suggest the therapeutic potential of these seco-steroids in leishmaniasis.Physalis angulata L., Solanaceae, é uma erva anual utilizada na medicina popular em muitos países tropicais e subtropicais. Apesar dos extratos da P. angulata apresentarem uma grande variedade de substâncias, pouco é conhecido sobre a sua atividade farmacológica. Neste trabalho foi investigado a atividade antileishmania in vitro de seco-esteroides (fisalinas purificados da P. angulata. O tratamento com as fisalinas B, F e G causou uma inibição concentração-dependente do crescimento de promastigotas de Leishmania amazonensis em cultura axênica, com valores de IC50 de 6,8, 1,4, e 9,2 μM respectivamente. A fisalina D foi menos ativa, com valores de IC50 de 30,5 μM. Foi também observada uma atividade leishmanicida em culturas de outras espécies de Leishmania (L. major, L. braziliensis e L. chagasi. Nossos resultados demonstram que as fisalinas inibem o crescimento dos promastigotas com o tratamento de espécies de Leishmania do Velho e do Novo Mundos e sugerem o potencial terapêutico destas moléculas na

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

    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.

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

    Michalsky, Erika M; Fortes-Dias, Consuelo L; Pimenta, Paulo F P; Secundino, Nágila F C; Dias, Edelberto S

    2002-01-01

    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).

  11. Leishmania spp. identification by polymerase chain reaction-restriction fragment length polymorphism analysis and its applications in French Guiana.

    Simon, Stéphane; Veron, Vincent; Carme, Bernard

    2010-02-01

    Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism-polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.

  12. Manipulação da fisiologia digestiva de Lutzomyia longipalpis (Diptera: Psychodidae): efeito da Galactosamina na atividade tripsinolítica intestinal do principal vetor de Leishmania infantum nas Américas.

    Tatiana Lima da Silva

    2015-01-01

    As leishmanioses, consideradas antropozoonoses, são um complexo de doenças causadas por espécies de protozoários parasitos pertencentes ao gênero Leishmania, sendo transmitidos aos seus hospedeiros vertebrados por insetos pequenos denominados flebotomíneos. O agente etiológico da leishmaniose visceral, Leishmania infantum, é transmitido no Brasil aos hospedeiros principalmente pela espécie Lutzomyia longipalpis (Diptera, Psychodidae). As fêmeas de flebotomíneos devem realizar a hematofagia pa...

  13. The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

    Fernando Real

    Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

  14. In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic chalcones

    2010-01-01

    The in vitro screening of 43 polysubstituted chalcones against Leishmania amazonensis axenic amastigotes, led to the evaluation of 9 of them in a macrophage-infected model with the two other most infectious Leishmania species prevalent in Peru (L. braziliensis and L. peruviana). The five most active and selective chalcones were studied in vivo, resulting on the identification of two chalcones with high reduction parasite burden percentages.

  15. Cura espontânea da leishmaniose causada por Leishmania Viannia Braziliensis em lesões cutâneas

    Jackson Maurício Lopes Costa

    1990-12-01

    Full Text Available Os autores relatam que durante 14 anos de trabalho clínico em campo, realizado nas comunidades de Três Braços e Corte de Pedra, Bahia, acompanharam 1.416 pacientes portadores de Leishmaniose Tegumentar Americana, cuja espécie envolvida na transmissão, é predominantemente a Leishmania Viannia brasilienses. A terapêutica utilizada rotineiramente nos casos é o antimoniato-N-metilglucamina (Glucantime. Contudo, 16 pacientes do sexo masculino recusaram-se a utilizar a medicação e 6 do sexo feminino encontravam-se em período gestacional, portanto não utilizaram o medicamento. Estes pacientes foram acompanhados por um período entre 4 a 12 anos, a partir do diagnóstico. Observou-se que em 9 pacientes (40,9% desta casuística, o tempo de cicatrizaçâo após o aparecimento da lesão, pode ser calculado em 6 meses de evolução. Quando se eleva a observação para 12 meses, temos que 19 pacientes (86,3% cicatrizaram suas lesões neste período. Em 3 casos (13,6% as lesões permaneceram ativas por mais de 12 meses. Conclui-se que os determinantes da cicatrizaçâo natural das lesões produzidas por Leishmania Viannia Braziliensis permanecem desconhecidos, dificultando para nós entendermos e compararmos aos efeitos das drogas utilizadas no tratamento da leishmaniose tegumentar.In field clinics in the comunities of Três Braços and Corte de Pedra, Bahia, we have attended 1.416 patients with tegumentary leishmaniasis in fourteen years, the predomi nant species in transmission is Leishmania Viannia brasiliensis (LVB. Because of the danger of metastasis with this infection treatment was routinely recomended with Glucantime. However sixteen patients refused injection therapy and six women were pregnant when seen and not treated. All patients were followed up in our clinic. All these patients closed their skin ulcers although one subsequently relapsed. Patients were followed up for variable periods (four to twelve years, after the diagnosis. In

  16. ALTERAÇÕES DA MATRIZ EXTRACELULAR ESPLÊNICA EM CÃES NATURALMENTE INFECTADOS COM LEISHMANIA (LEISHMANIA INFANTUM CHAGASI

    Nathálya dos Santos Martins

    2015-01-01

    Full Text Available The aim of this study was to study the changes in the splenic extracellular matrix of dogs naturally infected with Leishmania (Leishmania infantum chagasi and its correlation to clinical, histopathological, and parasitological aspects. Eighteen dogs were used, separated into three groups: six non-infected animals (control group and twelve infected animals. The dogs had undefined breed and age, from the township region of São Luís-MA. Paraffined slices of the spleen were stained with Hematoxilin and Eosin (H&E; Gomori’s ammoniacal Silver, to mark reticular fibers; and the Immunohistochemistry technique of streptavidin peroxidase to detect amastigote forms of Leishmania. The morphometrical analyses were done using the KS300 program and the images analysis system Kontron Elektronic/Carl Zeiss, Germany. The results showed that there is a significant increase in the deposition of collagen fibers in the spleen, compared to control animals, showing significant differences for symptomatic and asymptomatic animals. Positive correlations were found between the presence of the parasite in the tissue and collagen deposition. Symptomatic animals showed higher collagen deposition in the spleen, which can be associated to the high parasitism found in the tissue. The results showed that there is an intense fibrogenesis in the spleen in the canine visceral leishmaniasis, thus being associated to the parasitism of the tissue and the degenerative processes of the disease.

  17. An agent-based model for Leishmania major infection

    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].

  18. Occurrence of anti-Leishmania spp., Neospora caninum, and Toxoplasma gondii antibodies in dog sera from Veterinary Hospital from Universidade Estadual de LondrinaOcorrência de anticorpos contra Leishmania spp., Neospora caninum E Toxoplasma gondii em soros de cães atendidos no Hospital Veterinário da Universidade Estadual de Londrina-Pr

    Dauton Luiz Zulpo

    2012-10-01

    Full Text Available The aim of this study was to detect the presence of IgG antibodies anti-Leishmania spp., Toxoplasma gondii and Neospora caninum in dogs from a Veterinary Hospital from Universidade Estadual de Londrina. Blood samples from 112 animals were obtained by jugular venipuncture to obtain sera. The samples were tested by indirect immunofluorescence to detect antibodies anti-Leishmania spp., anti-N. caninum and anti-T. gondii. Thirteen (11.61%, 25 (22.32%, and 57 (50.89% samples were positive for Leishmania spp., N. caninum, and T. gondii, respectively. The co-presence of anti-Leishmania spp. and N. caninum was observed in 6 (5.36%, anti-Leishmania spp. and anti-T. gondii in 8 (14.7%, and anti-N. caninum and anti-T. gondii in 18 (16.07% samples. The co-presence of anti-Leishmania spp., anti-N. caninum and anti-T. gondii was observed in 5 (4.46% dogs. There was a higher prevalence of Leishmania in Toxoplasma and Neospora positive animals, however, these results were not statistically significant (range p = 0.052 p = 0.06. The dogs have an important role in the epidemiological cycle of these diseases, which are important in animal and public health. The northern state of Paraná is an endemic area for human cutaneous leishmaniasis, therefore, studies should be conducted to uncover the real role of dogs as reservoirs of Leishmania to humans in the state. O objetivo do presente trabalho foi detectar a presença de anticorpos contra Leishmania spp., Neospora caninum e Toxoplasma gondii em cães atendidos no Hospital Veterinário, da Universidade Estadual de Londrina. Amostras de 112 animais foram obtidas por venopunção jugular ou cefálica com posterior obtenção dos soros. Estas foram submetidas à técnica de imunofluorescência indireta para detecção de anticorpos da classe IgG anti-Leishmania spp, anti-N. caninum e anti-T. gondii. Dos 112 soros examinados, 13 (11,61%, 25 (22,32% e 57 (50,89% foram positivos para Leishmania spp., N. caninum e T

  19. Evaluation of HIV-Leishmania co-infection in patients from the northwestern Paraná State, Brazil = Avaliação da co-infecção HIV-Leishmania em pacientes da região noroeste do Estado do Paraná, Brasil

    Élide Aparecida Oliveira

    2011-01-01

    Full Text Available Leishmaniasis occurs throughout the world and is one of the opportunistic infections that attack HIV-infected individuals. Few data are available on American cutaneous leishmaniasis (ACL in HIV-infected patients. Current research investigates the occurrence ofHIV-Leishmania co-infection in HIV-infected individuals in an endemic region in Southern of Brazil. A non-randomized transversal investigation, molecular and serum epidemiologic type, on the occurrence of ACL in 169 HIV-infected patients was undertaken. The patients were followed up at the Integrated Nucleus of Health of the city Maringá, Southern of Brazil. Results showed that 13 (7.7% of the HIV-infected patients also presented Leishmania (Viannia DNA, detectable in blood by PCR. Serology, direct research, culture and PCR in skin material produced negative results. PCR positiveness for Leishmania was not associated with CD4 T lymphocytes count, opportunistic disease, treatment, use of proteases inhibitors, tattooing/piercing or use of injectable drugs, residential environment or previous ACL history. Results show that HIVinfected patients who live in endemic areas may reveal Leishmania DNA in the blood without any ACL symptoms. Above findings may be attributed to anti-retrovirus medicine that controls viral replication and maintains the functionality of the immune system and to a possible anti- Leishmania activity of these drugs.As leishmanioses ocorrem em todo o mundo e são infecções oportunistas que afetam indivíduos portadores do vírus HIV. Este estudo investigou a ocorrência da co-infecção HIV-Leishmania em portadores do HIV numa região endêmica para LTA do Sul do Brasil. Foi realizado estudo transversal, não randomizado, utilizando metodologia molecular e sorológica, sobre a ocorrência de LTA em 169 portadores do HIV. Foram estudados pacientes atendidos no Núcleo Integrado de Saúde de Maringá, Paraná, Sul do Brasil. Observou-se que 13 (7,7% dos pacientes infectados

  20. Estado atual da leishmaniose cutânea difusa (LCD no Estado do Maranhão: II. aspectos epidemiológicos, clínico-evolutivos

    Jackson Maurício Lopes Costa

    1992-06-01

    Full Text Available Os Autores fazem um estudo retrospectivo e prospectivo de 6 pacientes portadores de leishmaniose cutânea difusa, observados no Estado do Maranhão a partir de 1974. Os casos abordados são oriundos de diversas regiões do estado, observando-se em todos eles o envolvimento da leishmania (Leishmania amazonensis, sendo que 5 (84% dos pacientes apresentaram início de doença na 1ª década de vida. Em todos os pacientes envolvidos no estudo, houve relato de lesão inicial nodular única, que, posteriormente, em período variável de tempo, disseminou-se adquirindo outros aspectos. Evolutivamente apresentaram múltiplas lesões nodulares e ulceradas, intradermorreação de Montenegro(- e refratariedade aos esquemas terapêuticos utilizados até ao presente momento.The authors describe a retrospective and prospective study of 6 patients with diffuse cutaneous leishmaniasis observed in the State of Maranhão, since 1974. The patients comefromdifferentruralregions of the state and in all of them Leishmania (Leishmania amazonensis was the cause five of the patients initiated their disease in the first decade of life. All the patients first had a solitary, nodular lesion, that after a variable period of time, disseminated and acquired other aspects. Sequentially the patients presented multiple nodular and ulcerative lesions, negative leishmania skin-lests and a refractory response to the therapeutic schedules used up to the present.

  1. Mefloquina no tratamento da leishmaniose cutânea em uma área endêmica de Leishmania (Viannia braziliensis Efficacy of mefloquine in the treatment of skin leishmaniasis in an endemic area of Leishmania (Viannia braziliensis

    Victor Alberto Laguna-Torres

    1999-10-01

    Full Text Available O objetivo deste trabalho foi avaliar a eficácia da mefloquina numa região endêmica de leishmaniose cutânea por Leishmania (Viannia braziliensis, considerando que esta droga de administração oral, eficaz no tratamento da malária, com meia vida prolongada e efeitos colaterais pouco freqüentes poderia ser menos tóxica e de mais fácil administração, quando comparada com os antimoniais pentavalentes. Em Corte de Pedra, no litoral sul do Estado da Bahia, foram tratados, aleatoriamente, dez pacientes portadores de lesões leishmanióticas, subdivididos em dois grupos. O primeiro grupo recebeu mefloquina pela via oral, dose de 250mg/dia, durante seis dias, repetindo-se o mesmo esquema após intervalo de três semanas. O segundo grupo recebeu antimoniato de meglumina (Glucantime® diariamente, pela via endovenosa, na dose de 20mg/kg por 20 dias. Do grupo da mefloquina só um paciente apresentou cicatrização depois do segundo ciclo. Um desses, com quatro lesões apresentou nova lesão durante o primeiro ciclo de tratamento. A evolução dos outros três foi lenta sendo que em nove semanas nenhum deles tinha cicatrizado as úlceras que permaneciam com grande infiltração e sinais evidentes de atividade. O grupo tratado com Glucantime® apresentou evidente melhora.The aim of this study was to evaluate the efficacy of mefloquine in the treatment of skin leishmaniasis in patients infected with Leishmania (Viannia braziliensis at an endemic region. Mefloquine is an oral drug effective against malaria with a prolonged half-life, less toxicity and easier administration than pentavalent antimonials. At Corte de Pedra in the Southern litoral of Bahia State, two randomized groups of ten patients with leishmaniasis were treated. The first group was treated with oral mefloquine, 250mg per day in a single dose for six days and repeated three weeks later. The second group received meglumine antimoniate (Glucantime®, 20mg/kg daily administered

  2. Targeting host syntaxin-5 preferentially blocks Leishmania parasitophorous vacuole development in infected cells and limits experimental Leishmania infections.

    Canton, Johnathan; Kima, Peter E

    2012-10-01

    Our previous observations established a role for syntaxin-5 in the development of Leishmania parasitophorous vacuoles (LPVs). In this study, we took advantage of the recent identification of Retro-2, a small organic molecule that can cause the redistribution of syntaxin-5; we show herein that Retro-2 blocks LPV development within 2 hours of adding it to cells infected with Leishmania amazonensis. In infected cells incubated for 48 hours with Retro-2, LPV development was significantly limited; furthermore, infected cells harbored four to five times fewer parasites than infected cells incubated in vehicle alone. In vivo studies revealed that Retro-2 curbed experimental L. amazonensis infections in a dose-dependent manner. Retro-2 did not have any appreciable effect on the host cell physiological characteristics; furthermore, it had no apparent toxicity in experimental animals. An unexpected, but welcome, finding was that Retro-2 inhibited the replication of Leishmania parasites in axenic cultures. This study is significant because it identifies an endoplasmic reticulum/Golgi SNARE as a potential target for the control of Leishmania infections; moreover, it suggests that small organic molecules can be identified that can selectively disrupt the vesicle fusion machinery that promotes the development of pathogen-containing compartments without exerting toxic effects on the host.

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

    Murat, Paula Guerra; de Medeiros, Márcio José; Souza, Alda Izabel; de Oliveira, Alessandra Gutierrez

    2017-01-01

    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

  4. Histopatologia da leishmaniose tegumentar por Leishmania braziliensis braziliensis: 1. Padrões histopatológicos e estudo evolutivo das lesões

    Albino Verbosa de Magalhães

    1986-08-01

    Full Text Available Os autores analisam material de biópsias de 378 casos de Leishmaniose Tegumentar, causada por Leishmania braziliensis braziliensis, da localidade endêmica de Três Braços (Estado da Bahia. O parásitos, embora escassos, foram encontrados em 63,7% dos casos da forma cutânea e em 37,5% dos casos da forma mucosa. As alterações dérmicas ou do córion da mucosa permitiram identificar cinco padrões histopatológicos: 1 Reação Exsudativa Celular, constituída por um infiltrado histiolinfoplasmocitário; 2 Reação Exsudativa e Necrótica, na qual ocorre uma necrose no seio do infiltrado inflamatório; 3 Reação Exsudativa e Necrótico-Granulomatosa, que corresponde ao quadro descrito como inflamação crônica granulomatosa com necrose; 4 Reação Exsudativa e Granulomatosa, onde se observa uma reação granulomatosa desorganizada, sem que esteja presente necrose tecidual; 5 Reação Exsudativa e Tuberculóide, caracterizada pelo granuloma tuberculóide. O estudo evolutivo realizado em 49 casos, mostrou que houve uma mudança de padrão histopatológico observada, em biópsias sucessivas, em 63,2% dos casos da forma cutânea e em 45,4% dos casos da forma mucosa. Através desse estudo, é possível afirmar-se que o padrão de Reação Exsudativa Celular constitui o quadro inicial e final da lesão, com os demais padrões aparecendo interposto durante a evolução da doença.

  5. Leishmania mexicana in Proechimys iheringi denigratus Moojen (Rodentia, Echimyidae in a region endemic for American cutaneous leishmaniasis

    Air C. Barretto

    1985-12-01

    Full Text Available Three isolates of Leishmania were recovered from five of 27 specimens of the rodent Proechimys iheringi denigratus Moojen captured near Três Braços in the Atlantic Forest region of Bahia, Brazil. Two of these isolates were recovered from hamsters inoculated with a pooled triturate of liver, spleen and skin tissue from apparently healthy P. i. denigratus. The third isolate was recovered from a triturate of only skin tissue from another. Metastasis was observed in the inoculated hamsters, the parasites grew abundantly in artificial media and a typical suprapylarial pattern of infection in Lutzomyia longipalpis was produced indicating that the parasites belong to the Leishmania mexicana complex. All isolates reacted with Leishmania mexicana mexicana and Leishmania mexicana amazonensis monoclonal antibodies. The isoenzyme analysis differentiated these isolates from standard isolates of L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami and L. m. ssp.(Goiás-W. Barbosa. These isolates seem to be a subspecies of L. mexicana very closely related to L. m. amazonensis from which they differ by decreased electrophoretic mobility of GPI, PEP and ALAT. This is the first record of the isolation of a parasite of thegenus Leishmania in a rodent captured in the State of Bahia.

  6. Epidemiovigilância das infeções por Leishmania spp. no efetivo cinotécnico da Guarda Nacional Republicana e em mesocarnívoros silvestres

    Rocha, Hugo Manuel Bernardo

    2013-01-01

    Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Genética Molecular e Biomedicina As leishmanioses são um grupo de doenças de transmissão essencialmente vetorial, causadas por protozoários intracelulares do género Leishmania, que afetam humanos, animais domésticos e silvestres. A leishmaniose canina tem elevada incidência e distribuição alargada em Portugal continental, onde a espécie Leishmania infantum é o age...

  7. Ensayos metodologicos para la investigacion de reservorios de Leishmania spp en los Andes venezolanos Methodological assay for research of reservoirs of Leishmania spp. in the Venezuelan Andes

    Ana Lugo Yarbuh

    1982-12-01

    Full Text Available Se describen dos técnicas, presuntiva y confirmativa, para la investigación de mamíferos que pudieran ser reservorios de Leishmania que parasitan al hombre. Se investigan los cambios en los títulos de inmovilización y aglutinación de promastigotos de cultivo por los sueros de animales normales y expuestos una o varias veces a la inoculación intradérmica de pequeñas dosis de promastigotos vivos. Se registra una caída de los títulos de aglutinación en los sueros de hamsteres, de Holochilus venezuelae y de Didelphis marsupialis después de la inoculación con L. mexicana mexicana de Panamá y de L. gamhami de la región de los Andes venezolanos. Se discute la natureza de estos fenómenos. Se han hecho xenodiagnósticos con Lutzomyia townsendi en Holochilus venezuelae y Sigmodon hispidus infectados experimentalmente com L. mexicana mexicana, L. mexicana amazonensis, L. braziliensis y L. garnhami. Las pruebas fueron leidas mediante el examen microscópico de las gotitas de heces excretadas entre las 108 y 132 horas después de la ingesta infectante, tras colorearlas con Giemsa. Se obtuvieron resultados positivos en 23% de los experimentos usando mamíferos con lesiones localizadas, dejando a los flebótomos ingurgitarse libremente sobre animales anestesiados que poseian una hasta varias lesiones localizadas.Presumptive and confirmative techniques for searching mammals which could be reservoirs for Leishmania parasites from man are described. The changes of immobilising and agglutinating titers for promastigotes from culture by sera from normal and exposed mammals after single or repeated intradermal inoculation of promastigotes are described. A fall in titers of agglunation is observed in sera from hamsters, Holochilus venezuelae and Didelphis marsupialis after inoculation with L. mexicana mexicana from Panama and L. garnhami from the Venezuelan Andes region. The nature of this phenomenon is discussed. Xenodiagnoses were made with

  8. Leishmania metacyclogenesis is promoted in the absence of purines.

    Tiago Donatelli Serafim

    Full Text Available Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

  9. Monoclonal antibody affinity purification of a 78 kDa membrane protein of Leishmania donovani of Indian origin and its role in host–parasite interaction

    Mandira Mukherjee; Anindita Bhattacharyya; Swadesh Duttagupta

    2002-12-01

    Monoclonal antibodies were raised against pathogenic promastigotes of Leishmania donovani of Indian origin. Among these, one was used for immuno-affinity purification of a 78 kDa membrane protein present in both the amastigote and promastigote forms of the parasite. Results of immunoblot experiments with the anti-78 kDa antibody revealed that the protein was present only in parasites belonging to the L. donovani complex. The expression of the protein was observed to be the same during different phases of growth of the promastigotes. Therefore, the 78 kDa protein is neither stage-specific nor differentially regulated. Surface iodination and subcellular fractionation of the promastigotes indicated that the protein was localized on the cell surface. The 78 kDa protein was found to inhibit the binding of promastigotes to macrophages significantly, suggesting that it may play a role in the process of infection. Thus, here we report the purification of a surface protein of L. donovani of Indian origin, which may play an important role in the process of infection.

  10. Studies on cocktails of 31-kDa, 36-kDa and 51-kDa antigens of Leishmania donovani along with saponin against murine visceral leishmaniasis.

    Kaur, H; Thakur, A; Kaur, S

    2015-04-01

    A substantial number of antigens of Leishmania donovani have been described in the past. However, identifying candidate antigens is not enough. Appropriate antigen delivery to induce the right type of immune response against leishmaniasis (i.e. induction of a strong antigen-specific Th1 type of immune response) is another crucial component of an effective vaccine. Therefore, 'cocktail' vaccines are proposed based on the assumption that such cocktails will show enhanced efficacy. Studies have been carried out on LD31 and LD51 polypeptides from L. donovani promastigotes, which have proven to be potential vaccine candidates. This study was designed to check the protective efficacy of various cocktails of low molecular weight antigens alone and along with saponin as adjuvant. Mice were sacrificed on different post-challenge days for evaluation of parasite load and other immunological parameters. Protective efficacy of different vaccine formulations was revealed by significant decline in parasite burden and increased DTH Delayed Type Hypersenstivity responses. The antibody response was of IgG type with elevated IgG2a and decreased production of IgG1, whereas cytokine levels pointed towards the generation of protective Th1 type of immune response. Among all vaccine formulations, cocktail of 31+51+saponin was found to be highly immunogenic and imparted maximum protection.

  11. Advanced Development of Leishmania Topical Skin Test Antigen

    2012-09-28

    tropica promastigotes is a complex mixture of substances, including proteins in the range of 8 kDa to 70 kDa. In Leishmania naïve adult humans, the lysate...humans. 15. SUBJECT TERMS LtSTA = Leishmania tropica Skin test Antigen 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF... tropica (LtSTA) developed by Allermed was intended to be used to screen military and civilian personnel for infection with Leishmania during and

  12. Characterization of Leishmania (Leishmania) tropica axenic amastigotes.

    Nasereddin, Abedelmajeed; Schweynoch, Carola; Schonian, Gabriele; Jaffe, Charles L

    2010-01-01

    Optimum conditions for generating Leishmania (Leishmania) tropica axenic amastigotes (AxA) in culture were determined, pH 5.5/36 degrees C, and the parasites characterized by different techniques, including light microscopy, macrophage infection, stage specific antigen expression and differential display. AxA were morphologically similar to amastigotes and 15.5-fold more infective than stationary phase promastigotes for mouse peritoneal macrophages. Western blotting with promastigote stage specific monoclonal antibodies to either lipophosphoglycan (T2) or a 60 kDa flagella antigen (F3) showed a dramatic decrease in antigen expression when AxA were compared to promastigotes. Similarly F3 gave strong immune fluorescent staining of the promastigote flagellum, but no fluorescence was detected when AxA were examined. Conversely, Western blotting with the amastigote specific monoclonal antibody (T16) showed that this antigen is more highly expressed in AxA than promastigotes. Differential display-PCR was used to identify several parasite genes showing stage specific expression. One gene selectively expressed by AxA was partially sequenced and identified as Leishmania (L.) tropicaamastin. Amastigote specific expression of this gene was further confirmed by reverse transcriptase-PCR (RT-PCR) using AxA and infected macrophages. No amastin expression was observed with promastigotes. Expression of the cysteine protease B (cpb) and protein kinase A catalytic isoform 1 subunit (pkac1) in promastigotes and AxA was also examined by RT-PCR. Pkac1 was strongly expressed by promastigotes, while cpb expression was only seen with AxA or infected macrophages. L. (L.) tropica AxA will prove useful for further studies on parasite differentiation and gene regulation, as well as for drug screening.

  13. Ação leishmanicida de extratos de plantas no desenvolvimento de promastigotas de Leishmania amazonensis e estudo do perfil metabólico utilizando a técnica de cromatografia líquida de alta eficiência (CLAE).

    2006-01-01

    Dois milhões de novos casos de Leishmaniose ocorrem anualmente nas áreas tropicais e subtropicais do globo com uma estimativa de doze milhões de pessoas atualmente infectadas no mundo e trezentos e cinqüenta milhões sob risco de infecção O gênero Leishmania responsável pela doença compreende muitas espécies diversas e complexas que apresentam resistência aos fármacos utilizados no seu tratamento além das características indesejáveis A eficácia do controle e tratamento de uma doença parasitári...

  14. Action of Bothrops moojeni venom and its L-amino acid oxidase fraction, treated with {sup 60}Co gamma rays, in Leishmania spp; Acao do veneno de Bothrops moojeni e sua fracao L-aminoacido oxidase, submetida ao tratamento com raios gama de {sup 60}Co, em Leishmania spp

    Cardoso, Andre Gustavo Tempone

    1999-07-01

    Bothrops moojeni venom showed an anti leishmania activity in vitro, as determined by a cell viability assay using the reduction of MTT. After venom purification, by chromatography techniques, the fractions with anti leishmania and L-amino acid oxidase activities, eluted in the same positions. The molecular weight of the enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa, by SDS-PAGE, migrating as a single band, with an isoelectric point of 4.8 as determined by isoelectric focusing. The purified LAO from B. moojeni venom, 135-fold more active than crude venom, showed homo dimeric constitution, and was active against Leishmania spp from the New World, with an effective concentration against L(L). amazonensis of 1.80 {mu}g/ml (EC{sub 50}), L.(V.) panamensis (0.78 |{mu}g/ml) and L.(L.) chagasi (0.63 ({mu}g/ml). Ultrastructural studies of promastigotes affected by LAO demonstrated cell death, with edema in several organelles such as mitochondria and nuclear membrane, before cell disruption and necrosis. The action of LAO was demonstrated to be hydrogen peroxide-dependent. Studies with LLCMK-2 cells, treated with LAO, showed a toxic effect, with an EC{sub 50} of 11|{mu}g/ml. Irradiation of LAO with 6{sup 0C}o gamma rays, did not affect its whole oxidative activity, neither detoxified the enzyme. Amastigotes treated with LAO were not affected by its hydrogen peroxide, otherwise, the exogenous product, killed amastigotes with an EC{sub 50} of 0.67mM. These data could be of help in the development of alternative therapeutic approaches to the treatment of leishmaniasis. (author)

  15. Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

    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.

  16. Behavior of Leishmania major metacyclic promastigotes during the course of infection and immune response development in resistant versus susceptible hosts Comportamento de promastigoteas metacíclicos de Leishmania major durante o curso da infecção e da resposta imune em hospedeiros resistentes versus suscetíveis

    Regina Coeli Cunha Dórea

    2003-11-01

    Full Text Available Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH in resistant versus susceptible inbred mice. Non-infective (procyclic promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible and C57BL/6 (resistant mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro

  17. Identification of Leishmania chagasi from skin in Leishmania/HIV co-infection: a case report Identificação de Leishmania chagasi na pele em co-infecção Leishmania/HIV: relato de caso

    Marcela Orsini

    2002-06-01

    Full Text Available A case of HIV/Leishmania co-infection presenting both visceral and cutaneous manifestations is reported. Leishmania infection was confirmed by conventional methods (parasitological approach and serology and by PCR. Leishmania chagasi isolated from the skin lesion was characterized by enzyme electrophoresis and by restriction fragment length polymorphism of the internal transcribed spacer of the ribosomal gene.É descrito um caso de co-infecção leishmania/HIV com manifestações cutâneas e visceral. Infecção pela leishmania foi confirmada através de métodos convencionais (parasitológicos e sorológicos e através da PCR. A espécie Leishmania chagasi isolada da pele foi caracterizada por eletroforese enzimática e por polimorfismo de fragmento obtido por enzima de restrição.

  18. ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast

    Amalia Monroy-Ostria

    2014-01-01

    Full Text Available American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L. mexicana, 5% showed a mixed pattern compatible with L. (L. mexicana and L. (V. braziliensis, eight with L. (L. amazonensis and L. (L. mexicana, and one to L. (V. braziliensis. Most of the clinical samples, 109/116 (94%, gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L. mexicana and L. (V. braziliensis or L. (L. mexicana and L. (L. amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

  19. Análise histomorfométrica da matriz extracelular do linfonodo poplíteo de cães naturalmente infectados por Leishmania (L. chagasi

    Kris Régia J. Kondo

    2009-08-01

    Full Text Available Nas Américas, a leishmaniose visceral canina é causada por Leishmania (Leishmania chagasi, um protozoário intracelular obrigatório do sistema fagocítico mononuclear; as principais alterações histológicas associadas a essa doença ocorrem nos em órgãos linfóides. Apesar de o cão ser considerado o principal mantenedor e disseminador da leishmaniose no ambiente urbano, são escassos estudos dos aspectos histopatológicos e histomorfométricos, em cães naturalmente infectados com L. chagasi, que investiguem a interação entre o parasito e a matriz extracelular. Este estudo visou caracterizar e quantificar as alterações dos componentes celulares e da matriz extracelular (colágenos I e III do linfonodo poplíteo de 22 cães com infecção natural por L. chagasi detectada através da reação de imunofluorescência indireta (RIF e compará-las com as alterações encontradas no linfonodo poplíteo de 10 cães não-infectados, negativos na RIF e clinicamente saudáveis. Fragmentos dos linfonodos foram seccionados longitudinalmente, processados rotineiramente para exame histológico e corados por hematoxilina-eosina. Cortes adicionais do mesmo linfonodo incluídos em glicol metacrilato foram corados pelo azul de toluidina para histomorfometria. Linfonodos de cães infectados apresentaram linfadenopatia generalizada, aumento do tamanho e do número dos folículos linfóides, hipertrofia da cápsula e hiperplasia linfóide significativa. Nos linfonodos de cães do grupo infectado, a análise quantitativa de fibras colágenas mostrou significativo predomínio do colágeno I sobre o colágeno III. Esses resultados demonstram que cães infectados por L. chagasi apresentam degradação dos constituintes da matriz extracelular e conseqüente destruição do arcabouço linfóide, alterando a morfologia do órgão.In the Americas, canine visceral leishmaniasis is caused by Leishmania (Leishmania chagasi, an obligatory intracellular parasite of

  20. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  1. The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

    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.

  2. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

    Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

    2015-05-01

    Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

  3. Purification and Biochemical Characterization of Three Myotoxins from Bothrops mattogrossensis Snake Venom with Toxicity against Leishmania and Tumor Cells

    Andréa A. de Moura

    2014-01-01

    Full Text Available Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s, one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like, BmatTX-II (Lys49 PLA2-like, and BmatTX-III (Asp49 PLA2. The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T and SK-BR-3 (breast adenocarcinoma cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

  4. Mucosal Leishmaniasis caused by Leishmania (Viannia braziliensis and Leishmania (Viannia guyanensis in the Brazilian Amazon.

    Jorge Augusto de Oliveira Guerra

    Full Text Available BACKGROUND: Leishmania (Viannia braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V. panamensis, L. (V. guyanensis and L. (L. amazonensis. METHODOLOGY: Leishmania species were characterized by polymerase chain reaction (PCR of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. RESULTS: This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V. braziliensis and 16 cases by L. (V. guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V. guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V. braziliensis in the state of Rondônia. CONCLUSIONS/SIGNIFICANCE: L. (V. braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V. guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

  5. Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

    de Oliveira Guerra, Jorge Augusto; Prestes, Suzane Ribeiro; Silveira, Henrique; Coelho, Leila Inês de Aguiar Raposo Câmara; Gama, Pricila; Moura, Aristoteles; Amato, Valdir; Barbosa, Maria das Graças Vale; de Lima Ferreira, Luiz Carlos

    2011-01-01

    Background Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia. Conclusions/Significance L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River. PMID:21408116

  6. Evaluation of Leishmania (Leishmania chagasi strains isolated from dogs originating from two visceral leishmaniasis-endemic areas in Brazil using multilocus enzyme electrophoresis Avaliação de amostras de Leishmania (Leishmania chagasi isoladas de cães oriundos de duas áreas endêmicas de leishmaniose visceral no Brasil através da eletroforese de isoenzimas

    Carlos Eduardo Ribeiro Coutinho

    2011-10-01

    Full Text Available INTRODUCTION: Domestic dogs are the most important reservoir in the peridomestic transmission cycle of Leishmania (Leishmania chagasi. The genetic variability of subpopulations of this parasite circulating in dogs has not been thoroughly analyzed in Brazil, even though this knowledge has important implications in the clinical-epidemiological context. METHODS: The objective of this study was to evaluate and compare the phenotypic variability of 153 L. chagasi strains isolated from dogs originating from the municipalities of Rio de Janeiro (n = 57 and Belo Horizonte (n = 96, where the disease is endemic. Strains isolated only from intact skin were selected and analyzed by multilocus enzyme electrophoresis using nine enzyme systems (6PG, GPI, NH1 and NH2, G6P, PGM, MDH, ME, and IDHNADP. RESULTS: The electrophoretic profile was identical for all isolates analyzed and was the same as that of the L. chagasi reference strain (MHOM/BR/74/PP75. Phenetic analysis showed a similarity index of one for all strains, with the isolates sharing 100% of the characteristics analyzed. CONCLUSIONS: The results demonstrate that the L. chagasi populations circulating in dogs from Rio de Janeiro and Belo Horizonte belong to a single zymodeme.INTRODUÇÃO: Cães domésticos são considerados os reservatórios mais importantes no ciclo peridoméstico de transmissão de Leishmania (Leishmania chagasi. No entanto, a variabilidade genética de sub-populações que circulam neste hospedeiro é ainda pouco explorada no Brasil, sendo tal conhecimento de grande importância no contexto clínico-epidemiológico. MÉTODOS: O objetivo deste estudo foi avaliar e comparar a variabilidade fenotípica de 153 amostras de L. chagasi isoladas de cães oriundos dos municípios do Rio de Janeiro (n = 57 e Belo Horizonte (n = 96, onde a doença é endêmica. Foram selecionadas somente amostras isoladas de pele íntegra e analisadas por eletroforese de isoenzimas (MLEE empregando nove

  7. Differentiation of Leishmania species by FT-IR spectroscopy

    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.

  8. Histopatologia da leishmaniose tegumentar por Leishmania braziliensis braziliensis. 4. Classificação histopatológica

    1986-01-01

    Os A A. analisaram as alterações histológicas encontradas em 162 casos de Leishmaniose Tegumentar da localidade de Três Braços, Estado da Bahia, dos quais 131 (80,9%) eram de portadores de lesões cutâneas e 31 (19,1%) de portadores de lesões mucosas. Analisaram, também, o comportamento clínico dos cinco padrões histopatológicos, já antes descritos, em relação à terapêutica. O melhor prognóstico esteve sempre ligado ao padrão de Reação Exsudativa e Granulomatosa, ou seja, a uma fase na qual o ...

  9. Histopatologia da leishmaniose tegumentar por Leishmania braziliensis braziliensis: 4. Classificação histopatológica

    Albino Verçosa de Magalhães

    1986-12-01

    Full Text Available Os A A. analisaram as alterações histológicas encontradas em 162 casos de Leishmaniose Tegumentar da localidade de Três Braços, Estado da Bahia, dos quais 131 (80,9% eram de portadores de lesões cutâneas e 31 (19,1% de portadores de lesões mucosas. Analisaram, também, o comportamento clínico dos cinco padrões histopatológicos, já antes descritos, em relação à terapêutica. O melhor prognóstico esteve sempre ligado ao padrão de Reação Exsudativa e Granulomatosa, ou seja, a uma fase na qual o organismo, tendo lançado mão de um mecanismo endógeno de lise parasitária, já circunscreveu a área de necrose por uma reação granulomatosa, e esta é agora apenas o elemento residual. A ação terapêutica nessa fase somente acelera a resolução natural do caso. O grupo seguinte é amplo, e compreende os casos em que a lesão pertence aos padrões de Reação Exsudativa Celular (formas cutâneas, Reação Exsudativa e Necrótica e Reação Exsudativa e Necrótico-Granulomatosa. Nesses casos, o mecanismo de auto-controle da lesão encontra-se ainda em curso, e a ação terapêutica encurta o período de evolução natural. Os f.asos do padrão de Reação Exsudativa e Tuberculóide tiveram um prognóstico variável. Houve boa resposta à terapêutica quando o granuloma tuberculóide característico desse padrão surgiu em pacientes jovens, com curto tempo de evolução da doença e intradermorreação não exacerbada. Nos demais casos tuberculóides —. principalmente em pacientes adultos, com longo tempo de evolução da doença e intradermorreação exacerbada —, a resposta foi menos satisfatória. Em último lugar, com prognóstico reservado, ficaram os casos da forma mucosa que apresentaram o padrão de Reação Exsudativa Celular, onde o infiltrado pode estar desempenhando papel de auto-agressão. O presente estudo evoluiu para a proposição de uma classificação da Leishmaniose Tegumentar, baseada nos padrões histopatol

  10. Histopatologia da leishmaniose tegumentar por Leishmania braziliensis braziliensis. 3. Reação celular nos tecidos

    Albino Verçosa de Magalhães

    1986-10-01

    Full Text Available Os A.A. analisam as alterações histopatológicas observadas em 378 casos de Leishmaniose Tegumentar da localidade de Três Braços Estado da Bahia, dos quais 307 eram de portadores de lesões exclusivamente cutâneas, 54 de portadores de lesões exclusivamnte mucosas e 17 de portadores de lesões cutâneo-mucosas. A infiltração histiolinfoplasmocitária, na maioria dos casos, parece desempenhar o papel de resposta celular inespecífica à presença de um irritante tecidual, porém, nos casos de forma mucosa, não se pode afastar a possibilidade de que esse infiltrado esteja participando de uma reação de tipo autoagressivo. O plasmócito constitui um elemento quase constante nas lesões desenvolvidas, mas não tem sido observado nas lesões residuais, quer em via de cura ou já cicatrizadas; sua presença nestes casos denota, quase sempre, tendência à recidiva. Os mastdcitos foram observados em lesões tanto da forma cutânea como da forma mucosa, mas predominavam nas primeiras. Seu número foi significantemente maior no padrão de Reação Exsudativa e Neerótico Granulomatosa, onde os fenômenos necróticos são bem desenvolvidos. Os eosinófilos apresentaram associação significativa com os mastócitos, confirmando a existência de um eixo bidirecional entre estás duas células, o qual deve participar da modulação inflamatória, na Leishmaniose Tegumentar. Dois tipos de reação granulomatosa foram observados: um desorganizado, em relação, muitas vezes, com a necrose tissular, e outro organizado, mais raro, do tipo tuberculóide. O primeiro foi interpretado como de origem pós-necrótica, surgindo com a redução da carga parasitária, propiciada pelos fenômenos necróticos: eliminado o antígeno e mantidos os níveis de anticorpos, surgem as condições necessárias ao estabelecimento do granuloma, semelhante àquele observado nas lesões por imunocomplexo em excesso de anticorpos. O outro tipo de reação foi o granuloma de c

  11. Leishmania-mediated inhibition of iron export promotes parasite replication in macrophages.

    Rym Ben-Othman

    2014-01-01

    Full Text Available Leishmania parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of Leishmania, but how the parasites compete with the iron export function of their host cell is unknown. Here, we show that infection with Leishmania amazonensis inhibits ferroportin expression in macrophages. In a TLR4-dependent manner, infected macrophages upregulated transcription of hepcidin, a peptide hormone that triggers ferroportin degradation. Parasite replication was inhibited in hepcidin-deficient macrophages and in wild type macrophages overexpressing mutant ferroportin that is resistant to hepcidin-induced degradation. Conversely, intracellular growth was enhanced by exogenously added hepcidin, or by expression of dominant-negative ferroportin. Importantly, dominant-negative ferroportin and macrophages from flatiron mice, a mouse model for human type IV hereditary hemochromatosis, restored the infectivity of mutant parasite strains defective in iron acquisition. Thus, inhibition of ferroportin expression is a specific strategy used by L. amazonensis to inhibit iron export and promote their own intracellular growth.

  12. Patogenia da leishmaniose cutânea experimental: a importância da necrose na eliminação dos parasitos das lesões

    Zilton A. Andrade

    1984-12-01

    Full Text Available Um estudo histopatológico e ultraestrutural das lesões da leishmaniose cutânea causada pela Leishmania mexicana amazonensis em duas cepas isogênicas de camundongo, uma susceptível (Balb/c e outra resistente (A/J, demonstrou que os amastigotas ficavam bem preservados nos vacúolos parasitóforos dos macrófagos, igualmente em ambas as cepas. A reação de imunofluorescência revelou antigenos parasitários no interior e na membrana dos macrófagos de maneira idêntica para ambas as cepas. A diferença ocorria quando os macrófagos apareciam destruídos e as leishmanias ficavam livres ou fagocitadas por polimorfonucleares, neutrófilos e eosinófilos. Estes parasitos exibiam então graus variáveis de nítidas alterações degenerativas. No camundongo resistence, a necrose, de tipo caseoso ou fibrinóide, era mais disseminada e mais freqüente que no animal susceptível. Os achados observados indicaram que as leishmanias não são destruídas no interior dos macrófagos e sim fora deles, especialmente quando fagocitadas por leucócitos polimorfonucleares. A necrose apareceu como o mecanismo mais saliente através do qual o hospedeiro elimina os parasitos das lesões, sendo a mesma um aspecto importante da reação de hipersensibilidade tardia que ocorre nos animais resistentes.Amastigotes of Leshmania mexicana amazonensis appeared healthy and well preserved within the parasitophorous vacuoles of macrophages during the infection of a susceptible (Balb/c or a relatively resistant (A/J inbred strain of mice. Immunofluorescence showed the presence of leishmanial antigens within infected macrophages and in their external membranes similar for both strains. When the amastigotes were found extracellularly or within polymorphonuclear neutrophils or eosinophils, marked ultrastructural degenerative changes were observed in them. Such necrotic alterations were seen frequently in the resistant mice and rarely in the susceptible ones. Thus, necrosis of

  13. Patogenia da leishmaniose cutânea experimental: a importância da necrose na eliminação dos parasitos das lesões

    Zilton A. Andrade

    1984-12-01

    Full Text Available Um estudo histopatológico e ultraestrutural das lesões da leishmaniose cutânea causada pela Leishmania mexicana amazonensis em duas cepas isogênicas de camundongo, uma susceptível (Balb/c e outra resistente (A/J, demonstrou que os amastigotas ficavam bem preservados nos vacúolos parasitóforos dos macrófagos, igualmente em ambas as cepas. A reação de imunofluorescência revelou antigenos parasitários no interior e na membrana dos macrófagos de maneira idêntica para ambas as cepas. A diferença ocorria quando os macrófagos apareciam destruídos e as leishmanias ficavam livres ou fagocitadas por polimorfonucleares, neutrófilos e eosinófilos. Estes parasitos exibiam então graus variáveis de nítidas alterações degenerativas. No camundongo resistence, a necrose, de tipo caseoso ou fibrinóide, era mais disseminada e mais freqüente que no animal susceptível. Os achados observados indicaram que as leishmanias não são destruídas no interior dos macrófagos e sim fora deles, especialmente quando fagocitadas por leucócitos polimorfonucleares. A necrose apareceu como o mecanismo mais saliente através do qual o hospedeiro elimina os parasitos das lesões, sendo a mesma um aspecto importante da reação de hipersensibilidade tardia que ocorre nos animais resistentes.

  14. TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

    Tetaud, Emmanuel; Lefebvre, Michèle; M'Bang-Benet, Diane-Ethna; Crobu, Lucien; Blancard, Corinne; Sterkers, Yvon; Pages, Michel; Bastien, Patrick; Merlin, Gilles

    2016-07-01

    TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

  15. Pipunculidae (Diptera da região neotropical: I. Redescrição de Chalarus chilensis Collin, comb. n. e descrição de duas espécies novas da Amazônia

    J. A. Rafael

    1988-07-01

    Full Text Available Chalarus chilensis Collin, comb. n. é redescrito a partir do tipo e duas novas espécies da Bacia Amazônica, C. amazonensis e C. connexus, são descritas.Chalarus chilensis, comb. n. , is redescribed from the type and two species from the Amazon Basin, C. amazonensis and C. connexus, are described.

  16. Leishmania Skin Test

    2010-03-01

    2009, a dose of 50µg will be used in the design of a phase III clinical trial. 15. SUBJECT TERMS LtSTA = Leishmania tropica Skin Test Antigen 16...2010 on a Leishmania Skin Test (LtSTA) developed from the promastigotes of Leishmania tropica . During this period a phase IIB study was in progress...diluent. The final product is referred to as Leishmania tropica Skin Test Antigen (LtSTA). Figure 3 is a schematic diagram of the Drug Product

  17. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-01-01

    Background Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. Methodology/Principal findings After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. Conclusions/Significance This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how

  18. Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test Estudo da reatividade cruzada em amostras de soro de cães positivos para Leishmania sp., Babesia canis e Ehrlichia canis, pelo ensaio imunoenzimático indireto e pela reação de imunofluorescência indireta

    Trícia Maria F. de Sousa Oliveira

    2008-03-01

    Full Text Available To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT and Enzyme-linked immunosorbent assay (ELISA. All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA.Para verificar a existência de reação cruzada entre leishmaniose visceral, erliquiose e babesiose, nos testes sorológicos utilizados em programas de controle da leishmaniose visceral humana, amostras de soro canino provenientes de áreas endêmicas e não endêmicas para essa enfermidade, foram testadas pela Reação de Imunofluorescência (RIFI e Ensaio imunoenzimático (ELISA. Todos os soros provenientes de área endêmica foram positivos para Leishmania sp pelo ELISA e RIFI, 51% para Babesia canis e 43% para Ehrlichia canis pela RIFI. Pela RIFI, nenhum dos soros provenientes de área não endêmica foi positivo para Leishmania sp, sendo 67% positivos para B. canis e 78% para E. canis pelo mesmo teste. Quando testados pelo ELISA para Leishmania sp., quatro soros da área não endêmica foram positivos. Os cães foram localizados

  19. Estudo clínico randomizado comparando antimoniato de meglumina, pentamidina e anfotericina B para o tratamento da leishmaniose cutânea ocasionada por Leishmania guyanensis A randomized clinical trial comparing meglumine antimoniate, pentamidine and amphotericin B for the treatment of cutaneous leishmaniasis by Leishmania guyanensis

    Leandro Ourives Neves

    2011-12-01

    Full Text Available FUNDAMENTOS: O tratamento da leishmaniose tegumentar americana (LTA ainda constitui desafio, pois a maioria dos medicamentos é injetável e têm-se poucos ensaios clínicos randomizados comparando a eficácia das drogas. Além disso, é provável que as espécies de Leishmania tenham influência nas respostas terapêuticas. OBJETIVOS: Avaliar e comparar a eficácia e a segurança dos esquemas de tratamento na LTA, ocasionada por Leishmania (Viannia guyanensis. MÉTODOS: 185 pacientes foram selecionados, conforme critérios de elegibilidade, e distribuídos, aleatoriamente, em 3 grupos - 2 com 74 enfermos e outro com 37 - que receberam, respectivamente, antimoniato de meglumina, isotionato de pentamidina e anfotericina B em doses, períodos e vias de administração padronizados. Os enfermos foram reexaminados um, dois e seis meses após o final dos tratamentos. RESULTADOS: Não houve diferença entre os grupos terapêuticos em relação ao sexo, idade, número ou local das lesões. A análise por intenção de tratar (ITT mostrou eficácias de 58,1% para a pentamidina e 55,5% para o antimoniato (p=0,857. O grupo da anfotericina B foi analisado separadamente, pois 28 (75,7% pacientes negaram-se a continuar no estudo após a randomização. Eventos adversos leves ou moderados foram relatados por 74 (40% pacientes, principalmente artralgia (20,3%, para o grupo do antimoniato, e dor (35,1% ou enduração (10,8% no local das injeções para a pentamidina. CONCLUSÕES: A pentamidina tem eficácia similar ao antimonial pentavalente para o tratamento da LTA ocasionada por L. guyanensis. Face aos baixos resultados de eficácia apresentados por ambas as drogas, necessita-se, com urgência, investigar novas opções terapêuticas para esta enfermidade.FUNDAMENTALS: American tegumentary leishmaniasis (ATL treatment remains a challenge, since most available drugs are injectable and only a small number of comparative, randomized clinical trials have been

  20. Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

    Macedo-Silva, Roger Magno; dos Santos, Carina de Lima Pereira; Diniz, Vanessa Alvaro; de Carvalho, Jorge José; Guerra, Camila; Côrte-Real, Suzana

    2013-01-01

    Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis. PMID:24626303

  1. Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

    Roger Magno Macedo-Silva

    2014-02-01

    Full Text Available Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L. amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internaliseLeishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

  2. Target Oriented Drugs against Leishmania.

    1980-01-31

    the leishmanial source. Leishmanial strains L32 Leishmania tropica LRC L32 L137 Leishmania tropica LRC L137 L52 Leishmania donovani LRC L52 These...RESOLUTION TEST CHAR] 0REPORT NUMBER I TARGET ORIENTED DRUGS AGAINST LEISHMANIA (First Annual Summary Report) 0URI ZEHAVI, PhD and JOSEPH EL-ON, PhD...GOVT ACCESSION NO. 3. RE PIENT.S CATALOG NUMBER A....*( - ) S. TYPE OF REPORT & PERIOD COVERED TARGET ORIENTED DRUGS AGAINST LEISHMANIA 6 FIRST

  3. Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species

    Sato, Camila Massae; Sanchez, Maria Carmen Arroyo; Celeste, Beatriz Julieta; Duthie, Malcolm S.; Guderian, Jeffrey; Reed, Steven G.; de Brito, Maria Edileuza Felinto; Campos, Marliane Batista; de Souza Encarnação, Helia Valeria; Guerra, Jorge; de Mesquita, Tirza Gabrielle Ramos; Pinheiro, Suzana Kanawati; Ramasawmy, Rajendranath; Silveira, Fernando Tobias; de Assis Souza, Marina

    2016-01-01

    ABSTRACT American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania. The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L. (Viannia) braziliensis, and L. (V.) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL. PMID:27927927

  4. Avaliação da co-infecção por Leishmania em pessoas vivendo com HIV/Aids acompanhadas no Hospital Universitário de Brasília : estudo transversal com ênfase na leishmaniose visceral

    Tamayo, César Omar Carranza

    2009-01-01

    Foi realizado um estudo descritivo transversal com o objetivo de estimar a magnitude da co-infecção Leishmania/HIV em pessoas vivendo com HIV/aids atendidos no Ambulatório de Doenças Infecciosas e Parasitárias do Hospital Universitário de Brasília. Foram incluídos no estudo 163 indivíduos, dos quais, sete apresentavam a síndrome clínica sugestiva de leishmaniose visceral e 156 eram assintomáticos em relação a essa síndrome. Após aceitação e assinatura do termo de consentimento, os pacientes f...

  5. The transmission of suprapylarian Leishmania by bite of experimentally infected sand flies (Diptera: Psychodidae A trasnmissão de Leishmania suprapilária pela picada do flebotomíneo infectado experimentalmente

    L. Ryan

    1987-09-01

    Full Text Available Lutzomyia furcata transmitted Leishmania chagasi to a hamster 10 days after being experimentally fed on an infected spleen. An individual female Psychodopygus carrerai carrerai that had fed on a hamster lesion caused by Leishmania mexicana amazonensis transmitted this parasite 6 days later to another hamster. Transmission electron microscopy of this fly's head revealed a small number of degenerate promastigotes in the foregut, but only a few were attached.O protozoário Leishmania (L. chagasi foi transmitido experimentalmente a um hamster pela picada do flebotomíneo Lutzomyia furcata. Os insetos foram infectados através de uma membrana (pele de pinto, utilizando-se formas amastigotas provenientes do baço de um hamster infectado. O baço foi triturado em sangue de coelho. A L. (L. amazonensis foi transmitida a um hamster pela picada do flebotomíneo Psychodopygus c. carrerai, previamente alimentado em lesão de pele de um outro hamster infectado com o parasita. O exame desse flebotomíneo, através de microscópio eletrônico, revelou um número pequeno de flagelados degenerados, livres no lumen do intestino anterior.

  6. Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp.

    Igor A. Rodrigues

    2014-01-01

    Full Text Available Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC. In addition, the effect of the most active fraction (B2 on parasite’s ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents.

  7. Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp.

    Rodrigues, Igor A.; Azevedo, Mariana M. B.; Chaves, Francisco C. M.; Alviano, Celuta S.; Alviano, Daniela S.; Vermelho, Alane B.

    2014-01-01

    Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite's ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents. PMID:24818162

  8. Intranasal vaccination with leishmanial antigens protects golden hamsters (Mesocricetus auratus against Leishmania (Viannia Braziliensis infection.

    Luzinei da Silva-Couto

    2015-01-01

    Full Text Available Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania amazonensis antigens (LaAg partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection.Golden hamsters vaccinated with either two intranasal (IN doses of LaAg (10 µg or two intramuscular doses of LaAg (20 µg were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM immunisation failed to provide protection.These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.

  9. The experimental chemotherapy of leishmaniasis, VI. The development of rodent models for cutaneous infection with L. major and L. mexicana amazonensis.

    Trotter, E R; Peters, W; Robinson, B L

    1980-06-01

    Two models have been established of leishmanial infections in random-bred TFW albino mice, one with Leishmania major LV39 and the other with L. mexicana amazonensis LV38. These parasites produce consistent lesions when injected subcutaneously a short distance anterior to the base of the tail. Consistent infections could not be produced in the mice with other lines of the L. mexicana group of skin-dwelling protozoa, nor with a line of L. braziliensis guyanensis (LV475). The development of procedures for the maintenance of these parasites, the routine infection of mice and drug testing are described. L. major inocula consist of 1--5 X 10(6) promastigotes derived from NNN culture. L. m. amazonensis inocula contain approximately 10(7) amastigotes obtained from lesions in hamsters. The effects of drugs on the rodent lesions are assessed by two methods. Method A provides a simple lesion score from 0 to 3 made when lesions in control animals begin to reach maximum development (about six weeks after infection). Method B summates earlier 'mean lesion scores' to provide a graphic analysis of doses giving 50 and 90% lesion suppression at the end of seven weeks after the start of treatment which is for five consecutive days (SD 50 and SD90). The activities of several compounds used for the treatment of human leishmanial infections (organic antimonials, diamidines, amphotericin B and primaquine) are compared in mice infected with L. major LV39, L. m. amazonensis LV78 and 'L. infantum LV9' (the latter as reported earlier). It is concluded that the rodent models provide a good parallel with the response to these compounds in man. The visceral organism is significantly more responsive to Pentostam and Glucantime than are L. major or L. m. amazonensis. Primaquine is active only against 'L. infantum LV9'. All three parasites in mice respond to Berenil, but not to pentamidine which is used in the treatment of kala-azar. Amphotericin B is effective against cutaneous and visceral

  10. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

  11. Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

    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 (Tm) 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 Tm 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.

  12. Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania

    Adade, Camila M.; Carvalho, Ana Lúcia O.; Tomaz, Marcelo A.; Costa, Tatiana F. R.; Godinho, Joseane L.; Melo, Paulo A.; Lima, Ana Paula C. A.; Rodrigues, Juliany C. F.; Zingali, Russolina B.; Souto-Padrón, Thaïs

    2014-01-01

    Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases. PMID:25330220

  13. Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

    Mimori Tatsuyuki

    2006-09-01

    Full Text Available Abstract Background Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL from Ecuador were analyzed. Methods Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia panamensis (21 isolates, 7 zymodemes, L. (V. guyanensis (7 isolates, 4 zymodemes, L. (V. braziliensis (5 isolates, 3 zymodemes, L. (Leishmania mexicana (11 isolates, 4 zymodemes, L. (L. amazonensis (10 isolates, 2 zymodemes and L. (L. major (2 isolates, 1 zymodeme. L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL; eight cases of leishmaniasis recidiva cutis (LRC found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

  14. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    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.

  15. The yeast Scheffersomyces amazonensis is an efficient xylitol producer.

    Cadete, Raquel M; Melo-Cheab, Monaliza A; Viana, Adriana L; Oliveira, Evelyn S; Fonseca, César; Rosa, Carlos A

    2016-12-01

    This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493(T), cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04 g g(-1) in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD(+) respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD(+) needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493(T) is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.

  16. Expresión heteróloga de ARN mensajeros de Leishmania en ovocitos de anfibios

    Arroyo Olarte Rubén Darío

    2006-06-01

    Full Text Available La técnica de expresión heteróloga en ovocitos de Xenopus laevis ha sido ampliamente utilizada para la caracterización funcional de canales iónicos. En este estudio se reporta el uso del modelo nativo de ovocitos de Bufo marinus para la expresión heteróloga de ARN poliadenilado (ARNm poli(A+ de Leishmania amazonensis y la posterior identificación de canales iónicos mediante registros electrofisiológicos utilizando la técnica de voltaje controlado (voltage-clamp. Se logró realizar la maduración, extracción y cultivo de ovocitos de B. marinus bajo condiciones similares a las estandarizadas para X. laevis. El potencial de reposo promedio así como las corrientes endógenas mostraron ser definidas por canales de cloruro dependientes de calcio y rectificadores de salida, de manera similar a lo reportado en X. laevis. La inyección de ARNm poli(A+ de L. amazonensis generó la expresión de corrientes de cloruro de  amplitud, cinética y características farmacológicas diferentes a las corrientes endógenas, reportadas en estudios previos con X. laevis como posible resultado de la expresión heteróloga de canales iónicos de L. amazonensis.

  17. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Nour Hammoudeh

    2014-12-01

    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  18. Avaliação diagnóstica para Leishmania spp. e Trypanosoma cruzi em gatos domésticos procedentes da associação protetora dos animais do município de Ilha Solteira, SP, Brasil

    Martin, Maria Fernanda Alves [UNESP

    2013-01-01

    As leishmanioses são zoonoses que acometem o homem e outras espécies de mamíferos silvestres e domésticos. É uma doença causada por protozoários intracelulares do gênero Leishmania. O agente causador da leishmaniose visceral no Novo Mundo é Leishmania infantum (syn. L. chagasi), sendo Lutzomya longipalpis o principal vetor responsável pela sua transmissão. O gato doméstico também desenvolve a infecção, geralmente de forma assintomática, podendo atuar também como reservatório destes protozoári...

  19. Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp.

    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.

  20. The Brown Alga Stypopodium zonale (Dictyotaceae: A Potential Source of Anti-Leishmania Drugs

    Deivid Costa Soares

    2016-09-01

    Full Text Available This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 μg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 μM (9 μg/mL and 22.9 μM (10 μg/mL, and selectivity indexes of 8.4 μM and 11.5 μM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO production, but the generation of reactive oxygen species (ROS may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.

  1. The Brown Alga Stypopodium zonale (Dictyotaceae): A Potential Source of Anti-Leishmania Drugs

    Soares, Deivid Costa; Szlachta, Marcella Macedo; Teixeira, Valéria Laneuville; Soares, Angelica Ribeiro; Saraiva, Elvira Maria

    2016-01-01

    This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 μg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 μM (9 μg/mL) and 22.9 μM (10 μg/mL), and selectivity indexes of 8.4 μM and 11.5 μM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen species (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs. PMID:27618071

  2. The Brown Alga Stypopodium zonale (Dictyotaceae): A Potential Source of Anti-Leishmania Drugs.

    Soares, Deivid Costa; Szlachta, Marcella Macedo; Teixeira, Valéria Laneuville; Soares, Angelica Ribeiro; Saraiva, Elvira Maria

    2016-09-08

    This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 μg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 μM (9 μg/mL) and 22.9 μM (10 μg/mL), and selectivity indexes of 8.4 μM and 11.5 μM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen species (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.

  3. Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis Leishmania braziliensis: isolamento de antígeno contendo carboidrato e a possibilidade de sua aplicação no imunodiagnóstico da Leishmaniose Tegumentar Americana

    T. G. V. Silveira

    1995-06-01

    Full Text Available Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL. The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.A Leishmania braziliensis é um dos agentes causadores da Leishmaniose Tegumentar Americana (LTA. A cepa 034-JCG, isolada de paciente da região norte do estado do Paraná, Brasil, foi cultivada em meio Blood Agar Base, liofilizada e submetida a extração com fenol-água e o extrato obtido foi tratado com RNase I. O antígeno contendo carboidrato (Ag-CHO mostrou-se imunogênico para coelhos e apresentou pelo menos uma fração com poucas cargas negativas em pH 8,2. Este antígeno apresenta reação cruzada com extrato fenólico do meio de cultura usado para o cultivo de promastigotas e com antígenos de superfície de

  4. A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonesis Infection

    Alves-Silva, Marcus Vinícius; Nico, Dirlei; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa B.

    2017-01-01

    The Leishmania donovani nucleoside hydrolase (NH36) and NH A34480 of Leishmania amazonensis share 93% of sequence identity. In mice, the NH36 induced protection against visceral leishmaniasis is mediated by a CD4+ T cell response against its C-terminal domain (F3). Besides this CD4+ Th1 response, prevention and cure of L. amazonensis infection require also additional CD8+ and regulatory T-cell responses to the NH36 N-terminal (F1 domain). We investigated if mice vaccination with F1 and F3 domains cloned in tandem, in a recombinant chimera, with saponin, optimizes the vaccine efficacy against L. amazonensis infection above the levels promoted by the two admixed domains or by each domain independently. The chimera induced the highest IgA, IgG, and IgG2a anti-NH36 antibody, IDR, IFN-γ, and IL-10 responses, while TNF-α was more secreted by mice vaccinated with F3 or all F3-contaning vaccines. Additionally, the chimera and the F1 vaccine also induced the highest proportions of CD4+ and CD8+ T cells secreting IL-2, TNF-α, or IFN-γ alone, TNF-α in combination with IL-2 or IFN-γ, and of CD4+ multifunctional cells secreting IL-2, TNF-α, and IFN-γ. Correlating with the immunological results, the strongest reductions of skin lesions sizes were determined by the admixed domains (80%) and by the chimera (84%), which also promoted the most pronounced and significant reduction of the parasite load (99.8%). Thus, the epitope presentation in a recombinant chimera optimizes immunogenicity and efficacy above the levels induced by the independent or admixed F1 and F3 domains. The multiparameter analysis disclosed that the Th1-CD4+ T helper response induced by the chimera is mainly directed against its FRYPRPKHCHTQVA epitope. Additionally, the YPPEFKTKL epitope of F1 induced the second most important CD4+ T cell response, and, followed by the DVAGIVGVPVAAGCT, FMLQILDFYTKVYE, and ELLAITTVVGNQ sequences, also the most potent CD8+ T cell responses and IL-10 secretion. Remarkably

  5. Evaluación de la respuesta de isotipos de inmunoglobulina especifica a Leishmania en leishmaniasis tegumentaria Americana Evaluation of specific immunoglobin isotype response to Leishmania in American tegumentary leishmaniasis

    Maricel Labrada

    1989-09-01

    Full Text Available Con el fin de determinar las clases de anticuerpo producido contra el parásito y la cinética de los mismos en relación a la evolución de la infección, se estudiaron los sueros de 133 pacientes infectados con Leishmania del complejo braziliensis. Se utilizó la prueba de inmunofluorescencia indirecta y amastigotas de L. mexicana amazonensis como antígeno. En los sueros obtenidos al momento de consultar para el diagnóstico se encontró IgM en 54 de los sueros absorbidos con Straphylococcus aureus Cowan I y en 5 de los no absorbidos. La IgM sólo se encontro en los sueros de pacientes con tiempo devolución de las lesiones Leishmania-specific immunoglobulin subclass response was evaluated in 133 patients infected with Leishmania braziliensis. The indirect immunofluorescent antibody test (IFAT was employed with amastigotes of L. mexicana amazonensis as antigen. Among the 133 sera obtained at consultation fo rdiagnosis of active lesions, IgM was detected in 54 following absorption with Staphylococcus aureus Cowan strain I, and in 5 sera prior to absorption. IgM reactive with Leishmania antigen was only found in sera from patients whose lesions had envolved over past two months or less. Leishmania-specific IgG was detected in all sera prior to absorption. Sera obtained at the time of recurrence of after complete healing of lesions presented only specific IgC. The combined use of the Montenegro skin test and specific IgM increased the sensitivity of immunodiagnostic methods in patients with lesions of less than 2 months duration. Normal control volunteers were negative for specific IgM and unreactive to Montenegro skin testin. Among 16 patients with non-leishmanial lesion, 3 with sporotrichosis showed IgG reactive with Leishmania; none, including 4 with lesions of less than two months duration, showed specific IgM. We conclude that in patients infected with L. brasiliensis the presence of specific IgG and IgM is associated with the time of

  6. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.O método daperoxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquimicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixação. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana ámazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5 µm de espessura. Os antígenos de T. cruzi foram corados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluições entre 1:1.000 e 1:2.000. Diferentemente, os antígenos dç Leishmania foram revelados pelos soros anti- Leishmania somente em baixas diluições, ou seja, entre 1:60 e 1:160 enquanto que os soros

  7. Detection of Leishmania spp. based on the gene encoding HSP20

    Montalvo, Ana M; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Fraga, Jorge; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Rodríguez, Omaira; Laboratorio de referencia e investigación en enfermedades tropicales de sanidad militar. Bogotá, Colombia.; Blanco, Orestes; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Llanos-Cuentas, Alejandro; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia. Lima, Perú.; García, Ana L.; Universidad de San Simón. Cochabamba, Bolivia.; Valencia, Braulio M; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia. Lima, Perú.; Muskus, Carlos; Programa de Estudio y Control de Enfermedades Tropicales, Universidad de Antioquia. Medellín, Colombia.; Van der Auwera, Gert; Biomedical Sciences Department. Institute of Tropical Medicine of Antwerp. Amberes, Bélgica.; Requena, José M; Centro de Biología Molecular Severo Ochoa. Madrid, España.

    2014-01-01

    Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The anal...

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

    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.

  9. Leishmania(Leishmania) chagasi in captive wild felids in Brazil.

    Dahroug, Magyda A A; Almeida, Arleana B P F; Sousa, Valéria R F; Dutra, Valéria; Turbino, Nívea C M R; Nakazato, Luciano; de Souza, Roberto L

    2010-01-01

    This study used a PCR-RFLP test to determine the presence of Leishmania (Leishmania) chagasi in 16 captive wild felids [seven Puma concolor (Linnaeus, 1771); five Panthera onca (Linnaeus, 1758) and four Leopardus pardalis (Linnaeus, 1758)] at the zoological park of the Federal University of Mato Grosso, Brazil. Amplification of Leishmania spp. DNA was seen in samples from five pumas and one jaguar, and the species was characterized as L. chagasi using restriction enzymes. It is already known that domestic felids can act as a reservoir of L. chagasi in endemic areas, and further studies are necessary to investigate their participation in the epidemiological chain of leishmaniasis.

  10. Sequence Analysis of HSP70 Gene of Leishmania major and Leishmania tropica in Chabahar and Mashhad

    Mansour Dabirzadeh

    2016-04-01

    Full Text Available Background and Objective: Cutaneous leishmaniasis is a parasitic disease and a health problem in different parts of Iran, especially two cities of Mashhad and Chabahar. Due to morphological similarities of most Leishmania species and difference in reservoirs of L. major and L. tropica, it is necessary to determine the parasite specie to combat the disease. Thus, this study used gene sequencing and genotyping of 70-kDa heat shock protein (HSP70 to differentiate the two species of Leishmania. Methods: In this descriptive-analytical study, microscope slides and cultures were prepared from 43 patients suspected of cutaneous leishmaniasis in Chabahar and Mashhad. PCR was performed after genomic DNA extraction and then PCR products were sequenced and analyzed. Results: Of the 43 patients studied, 32 direct smear and culture (74.4% were positive and 11 (25.6% showed negative results, and were therefore excluded from the study. Using HSP70-specific primers, 1962 bp and 1152bp bands were observed for HSP70 of L. major in Chabahar and L. tropica in Mashhad, respectively. Based on the results, there were 18 nucleotide differences between HSP70 of L. major in Chabahar and L. tropica in Mashhad. Conclusion: Due to the morphological similarities between Leishmania species and inability to differentiate species through parasitological methods, the HSP70 gene can be used for identification of the species, and prevention and treatment of the disease.

  11. Effects of linalool and eugenol on the survival of Leishmania (L.) infantum chagasi within macrophages.

    Dutra, Fernando L; Oliveira, Maurício M; Santos, Reinaldo S; Silva, Wagner Seixas; Alviano, Daniela S; Vieira, Danielle P; Lopes, Angela H

    2016-12-01

    The most commonly used drugs against visceral leishmaniasis are based on pentavalent antimonial compounds, which have played a fundamental role in therapy for over 70 years. However, the treatment is painful and has severe toxic side effects that can be fatal. Antimonial resistance is spreading and reaching alarming proportions. Linalool and eugenol have been shown to kill Leishmania (L.) amazonensis and Trypanosoma cruzi at low doses. In the present study, we demonstrate the effects of linalool and eugenol, components of essential oils, on Leishmania (L.) infantum chagasi, one of the causative agents of visceral leishmaniasis. We compared the effects of those compounds to the effects of glucantime, a positive control. In L. infantum chagasi killing assays, the LD50 for eugenol was 220μg/ml, and that for linalool was 550μg/ml. L. infantum chagasi was added to cultures of peritoneal mouse macrophages for four hours prior to drug treatment. Eugenol and linalool significantly decreased the number of parasites within the macrophages. Eugenol and linalool enhanced the activities of the L. infantum chagasi protein kinases PKA and PKC. Linalool also decreased L. infantum chagasi oxygen consumption. In conclusion, both linalool and eugenol promoted a decrease in the proliferation and viability of L. infantum chagasi. These effects were more pronounced during the interaction between the parasites and peritoneal mouse macrophages.

  12. Homologous recombination in Leishmania enriettii.

    1991-01-01

    We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping del...

  13. The leishmania ARL-1 and Golgi traffic.

    Annelise Sahin

    Full Text Available We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1. The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN; N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

  14. Leishmania (Viannia braziliensis em cães naturalmente infectados Leishmania (Viannia braziliensis in naturally infected dogs

    Maria de Fátima Madeira

    2003-10-01

    Full Text Available Foram estudados oito cães provenientes do Município de Maricá (RJ, com lesões sugestivas de leishmaniose tegumentar americana por métodos parasitológicos e sorológicos. Leishmania spp foi encontrada em seis cães através do cultivo in vitro. Anticorpos específicos foram detectados em seis animais pelo ELISA e em dois pela imunofluorescência indireta. Cinco isolados caninos analisados apresentaram zimodema similar a Leishmania (Viannia braziliensis. Sugere-se que cães clinicamente suspeitos sejam acompanhados periodicamente, na tentativa de confirmar o diagnóstico da leishmaniose tegumentar canina.Eight dogs from Maricá Municipality (RJ, with suggestive lesion of american tegumentary leishmaniasis were studied by parasitological and serological methods. Leishmania spp was found in six dogs by in vitro cultivation. Specific antibodies were detected in six dogs by ELISA and in two by indirect immunofluorescence. Five canine isolates were found to belong to the same zymodeme as Leishmania (Viannia braziliensis. The authors suggest that clinically suspect dogs should be followed-up in an attempt to confirm the diagnostic of canine tegumentary leishmaniasis.

  15. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C. A. V.; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-01-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  16. Leukodepletion Filters for Prevention of Transfusion Transmission Of Leishmania

    2006-11-01

    untersuchung uber die markierung von Leishmania donovani, Leishmania tropica , und Leishmania braziliensis mit ferritin. Tropenmed Parasitol 1975...LEUKODEPLETION FILTERS FOR PREVENTION OF TRANSFUSION TRANSMISSION OF LEISHMANIA Lisa J. Cardo, Jeanne Salata, Ronald Harman, Juan Mendez, Peter...ABSTRACT BACKGROUND: Leishmania is an intracellular parasite of monocytes transmissible by transfusion. The feasibility of reducing

  17. Estudo histológico e parasitológico do trato gastrintestinal de cães infectados com Leishmania (Leishmania) chagasi

    Aldair Junio Woyames Pinto

    2011-01-01

    São poucas as descrições das alterações patológicas e parasitológicas relacionadas ao envolvimento do trato gastrointestinal (TGI) na leishmaniose visceral canina e, sobretudo considerando-se o TGI de forma sistemática. Assim, neste trabalho objetivou-se um estudo sistemático, clínico, anatomopatológico e parasitológico do TGI de cães naturalmente infectados com Leishmania (Leishmania) chagasi provenientes da região metropolitana de Belo Horizonte, MG. Após confirmação sorológica (RIFI e ELIS...

  18. Molecular Detection of Leishmania in Sand Flies (Diptera: Psychodidae: Phlebotominae) Collected in the Caititu Indigenous Reserve of the Municipality of Lábrea, State of Amazonas, Brazil.

    Silva, T R R; Assis, M D G; Freire, M P; Rego, F D; Gontijo, C M F; Shimabukuro, P H F

    2014-11-01

    Phlebotominae sand flies are of medical importance because they are vectors of human pathogens, such as protozoa of the genus Leishmania Ross, etiological agent of American cutaneous leishmaniasis (ACL). In Lábrea, a municipality in the state of Amazonas, Brazil, ACL is primarily associated with subsistence activities, such as collection and extraction of forest products, undertaken by both indigenous and nonindigenous people. Data on ACL in indigenous populations are scarce, such that there is little information on the identity of the etiologic agent(s), reservoir host(s) and insect vector(s). The aim of this work was to study the sand fly fauna collected during an 8-d surveillance of different habitats in the Indigenous Reserve Caititu, Lábrea. In total, 1,267 sand flies were collected in different habitats for eight consecutive days, of which 819 (64.6%) were females and 448 (35.4%) males, from 10 genera and 32 species. The most abundant genera were Psychodopygus (34.3%), Trichophoromyia (22.9%), and Nyssomyia (15.3%). The most abundant species were Trichophoromyia ubiquitalis (Mangabeira) (n = 235, 18.5%), Psychodopygus davisi (Root) (n = 228, 18.0%) and Nyssomyia antunesi (Coutinho) (n = 135, 10.7%). Direct sequencing of polymerase chain reaction products demonstrated the presence of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis in the following species of sand flies: Evandromyia apurinan (Shimabukuro, Silveira, & Silva), Nyssomyia umbratilis (Ward & Fraiha), Nyssomyia yuilli yuilli (Young & Porter), Ps. davisi, Sciopemyia servulolimai (Damasceno & Causey), and Th. ubiquitalis. The presence of natural infection by Leishmania detected in the sand fly species investigated in this study suggests their possible role in the transmission cycle of ACL in the studied area.

  19. Detection of Leishmania RNA Virus in Leishmania Parasites

    Desponds, Chantal; Kuhlmann, F. Matthew; Robinson, John; Hartley, Mary-Anne; Prevel, Florence; Castiglioni, Patrik; Pratlong, Francine; Bastien, Patrick; Müller, Norbert; Parmentier, Laurent; Saravia, Nancy Gore; Beverley, Stephen M.; Fasel, Nicolas

    2013-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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

  20. Detection of Leishmania RNA virus in Leishmania parasites.

    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.

  1. Características epidemiológicas da leishmaniose tegumentar americana em uma região endêmica do Estado da Bahia: III. Fauna flebotomínica Epidemiological characteristics of American tegumentary leishmaniasis in an endemic region of the State of Bahia: III. Phlebotomine fauna

    Julio A. Vexenat

    1986-09-01

    Full Text Available A fauna flebotomínica da região de Três Braços, uma área endêmica de leishmaniose cutânea-mucosa localizada no sudeste do Estado da Bahia, na região cacaueira, é muito variada. Foram identificadas 30 espécies de Lutzomyia em 13.535 exemplares coletados entre os anos de 1976 e 1984. Lu. withmani foi a espécie altamente predominante no ambiente peridoméstico e no interior das residências, com percentuais de 99,0 e 97,5, respectivamente. Na floresta, as espécies predominantes foram Lu. ayrozai e Lu. yuilli, aparecendo Lu. whitmani com apenas 1,0% do total de exemplares examinados. Lu. flaviscutellata, vetor comprovado da Leishmania mexicana amazonensis, foi também coletada em baixos índices. Lu. wellcomei, vetor da L. braziliensis braziliensis na Serra dos Carajás, Pará, Brasil, não foi encontrada na região de Três Braços onde o parasito causando infecções humanas é predominantemente L. b. braziliensis. Embora não se tenha encontrado infecção natural por promastigotas em 1.832 fêmeas de diversas espécies examinadas, discute-se a possibilidade de Lu. whitmani ser um vetor da L.b. braziliensis na região, mantendo, provavelmente, a transmissão entre o cão e o homem.The phlebotomine fauna is highly varied in três Braços, an endemic area american cutaneous leishmaniasis, situated in the cacao growing region in the southeast of Bahia State, brazil. Thirty species of the Lutzomyia genus were identified in 13,535 spcecimens collected between 1976 and 1984. Lutzomyia whitmani was the dominant species accounting for 99% or flies in the periodomicile and 97.5% of those caught in homes. In the forest the predominant species were Lu. ayrozai and Lu. yulli. Lu whitmani accounted for only 1.0% of the specimens examined. Lu. flaviscutellata, the proven vector of Leishmania mexicana amazonensis, was also collected in small numbers, I.u. wellcomei, a known vector of L. braziliensis braziliensis in the Serra dos Carajás, Par

  2. 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

    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.

  3. A focus of mucocutaneous leishmaniasis in Três Braços, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs.

    Cuba Cuba, C A; Miles, M A; Vexenat, A; Barker, D C; McMahon Pratt, D; Butcher, J; Barreto, A C; Marsden, P D

    1985-01-01

    The characterization and identification to species and subspecies of 20 stocks of Leishmania isolated from the region of Três Braços, Bahia, Brazil, are described: 17 stocks were from patients and three from dogs. The following techniques were used (i) biological (growth in culture, hamster tissues and phlebotomine gut), (ii) biochemical (isoenzyme and kinetoplast DNA analysis) and (iii) immunological (using monoclonal antibodies). All except two stocks belong to the L. braziliensis complex. One of these two corresponded to L. mexicana amazonensis but the other, while clearly in the mexicana complex, showed slight differences from the L. mexicana amazonensis reference strain on isoenzyme analysis. Two stocks from different lesions in the same patient and with different growth characteristics in hamster tissues were both identified as L. braziliensis braziliensis. All the fully characterized stocks of the L. braziliensis complex were identified as L. braziliensis braziliensis. L. braziliensis guyanensis was not identified. Dog and human stocks of L. braziliensis braziliensis were indistinguishable. From these findings and other evidence, L. braziliensis braziliensis seems to be the predominant species transmitted in Três Braços.

  4. An Enhanced Method for the Identification of Leishmania spp. using Real-Time PCR and Sequence Analysis of the 7SL RNA Gene Region

    Stevenson, Lindsay G.; Fedorko, Daniel P.; Zelazny, Adrian M.

    2010-01-01

    The accurate identification of Leishmania species is important for the treatment of infected patients. Molecular methods offer an alternative to time consuming traditional laboratory techniques for species determination. We redesigned a 7SL rRNA gene based PCR and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including: L. major, L. tropica, L. aethiopica, L. guyanensis, and the previously indistinguishable L. brasiliensis and L. panamensis. The L. donovani complex members remain indistinguishable by this method, as are the representatives of L. amazonensis/L. garnhami and L. mexicana/L. pifanoi. PMID:20226334

  5. Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target.

    Castro-Pinto, Denise B; Echevarria, Aurea; Genestra, Marcelo S; Cysne-Finkelstein, Léa; Leon, Leonor L

    2004-02-01

    The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.

  6. Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani

    Jensen, A T; Curtis, J; Montgomery, J

    2001-01-01

    To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identif......To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family...

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

    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.

  8. Flagellate infections of Brazilian sand flies (Diptera: Psychodidae): isolation in vitro and biochemical identification of Endotrypanum and Leishmania.

    Arias, J R; Miles, M A; Naiff, R D; Povoa, M M; de Freitas, R A; Biancardi, C B; Castellon, E G

    1985-11-01

    Flagellate infections were found in 1,063 of 18,895 sand flies collected in the states of Amazonas, Pará, Rondonia and Acre, Brazil. Infection rates were 13.4% (species group Shannoni); 7.5% (subgenus Nyssomyia); 6.7% (subgenus Lutzomyia series Cruciata); 0.5% (genus Psychodopygus) and 3.1% for other sand flies (various subgenera). Leishmania braziliensis guyanensis and L. mexicana amazonensis were isolated, respectively, from the known vectors, Lutzomyia umbratilis and L. flaviscutellata. Single stocks of L. braziliensis-like and L. mexicana-like organisms were isolated, respectively, from L. whitmani and L. yuilli. Thirty-eight flagellate stocks, isolated by direct culture from sand flies were characterized in detail by morphology in culture, behavior in hamsters and mice and by enzyme profiles. Sixteen stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum schaudinni; 8 stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum sp.; 7 stocks from Psychodopygus ayrozai and P. paraensis were identified as Leishmania sp. previously isolated from the armadillo, Dasypus novemcinctus; 2 stocks of Trypanosoma rangeli were isolated from recently fed Lutzomyia sp. (Shannoni group) sand flies; the remaining 5 stocks from L. umbratilis and L. yuilli could not be identified. Observations suggested that Shannoni group sand flies were the natural vectors of Endotrypanum. Leishmania sp. infections in the man-biting flies P. ayrozai and P. paraensis were restricted to the midgut and associated with recent bloodmeals. Unidentified flagellates in L. umbratilis and L. yuilli were distributed throughout the digestive tract with no trace of bloodmeals.

  9. Leishmania mexicana: aspectos taxonómicos y rareza de la infección humana en Três Braços, Bahia, Brasil

    César Augusto Cuba Cuba

    1984-09-01

    Full Text Available Del estúdio de 51 stocks de Leishmania aislados de pacientes humanos de leishmaniasis cutaneomucosa en Três Braços, Bahia, Brasil, los autores describen, en detalle, el análisis de los dos únicos stocks de L. mexicana, identificando uno de ellos como L. mexicana amazonensis. El otro aislado permanece en posición taxonómica no definida pues considerándosele como un miembro de L. mexicana, encuéntranse dificultades para su identificación subespecífica. Evaluan también los parâmetros biológicos e isoenzimáticos y discuten el papel de los anticuerpos monoclonales en la tipificación de éstos stocks. Los autores remarcan la rareza de la transmisión de parásitos del complejo L. mexicana en esta región, aun cuando estudien epidemiológicamente por mais de 8 anos la infección humana.In a study of 51 stocks of Leishmania isolated from patients with cutaneous leishmaniasis in Três Braços, Bahia, Brazil 49 were characterized as L. braziliensis braziliensis. Only two L. mexicana stocks were identified. One was subspeciated as L. mexicana amazonensis. The other differed from this species and to date remains unspeciated. The biological parameters, isoenzyme analysis and monoclonal antibody findings relating to these two strains are discussed. Transmission of parasites of the mexicana complex to man have been rare in this region during the 8 years of study of the human infection.

  10. Antiproliferative and leishmanicidal effect of ajoene on various Leishmania species: ultrastructural study.

    Ledezma, Eliades; Jorquera, Alicia; Bendezú, Herminia; Vivas, Julio; Pérez, Gladinex

    2002-08-01

    Ajoene [(E,Z)-4,5,9 trithiadodeca 1,6,11 triene 9-oxide], the major bioactive compound derived from garlic, shows a potent trypanolytic and antimicotic activity. In this paper we evaluate its effect on Leishmania mexicana(Lm:MHOM/VE/80/NR), L eishmania mexicana venezuelensis (Lmv: MHOM/VE/80/H16), L eishmania mexicana amazonensis (Lma: M112, IFLA/BR/67/PH8) and L eishmania donovani chagasi (Ldch: MHOM/BR/74/PP75). Ajoene showed a potent leishmanicidal activity in vitro against all species studied. Concentrations higher than 0.3 microM led to total inhibition of growth, and 10 microM induced 100% lysis of Leishmaniaafter 96 h of incubation in a chemically defined culture medium. The 50% inhibitory concentration (IC(50)) for lysis, for all species, was about to 2 microM. The effect was dose-dependent and a threefold increase in concentration (30 microM) produced 100% lysis of cultured forms after 72 h. Ultrastructural studies showed a time- and dose-dependent morphological alteration of the mitochondrial membrane and nuclear envelope, as well as the formation of large autophagic vacuoles.

  11. First Report of Heterorhabditis amazonensis from Venezuela and Characterization of Three Populations.

    Morales, Naiyulin; Morales-Montero, Patricia; Puza, Vladimir; San-Blas, Ernesto

    2016-09-01

    During a survey in western Venezuela in 2011, three new populations of Heterorhabditis amazonensis (LPV081, LPV156, and LPV498) were isolated. Some differences were found in terms of morphometry compared with the original description; however, the distance from the anterior end to the excretory pore is the most variable character; significantly shorter in all infective juveniles and in other developmental stages depending on the population. According to a Principal Component Analysis, LPV498 possesses more differences in morphometric characteristics and can be separated from the other two. Those intraspecific differences could be attributed to the geographic origin of the nematode. Molecular studies of ITS regions demonstrated that the sequences of the Venezuelan strains were identical to those of the type species originally isolated in the Brazilian Amazonian forest. This is an interesting fact because in several studies on heterorhabditids, intraspecific variability has been recorded. Herein, we present the first report of H. amazonensis in Venezuela and the characterization of three populations of this species.

  12. Mixed mucosal leishmaniasis infection caused by Leishmania tropica and Leishmania major.

    Shirian, Sadegh; Oryan, Ahmad; Hatam, Gholam Reza; Daneshbod, Yahya

    2012-11-01

    Mixed infections with different Leishmania species could explain differences in the clinical courses of these infections. On identification of Leishmania parasites from Iranian patients with mucosal leishmaniasis (ML), a patient with both oral and nasal lesions was found to be concomitantly infected with Leishmania tropica and L. major. Mixed infection was identified by PCR amplification of Leishmania kinetoplast DNA on scraping of cytological smears and histopathological sections. L. major and L. tropica were isolated from the nasal and oral lesions, respectively. These species were also confirmed by immunohistochemistry. This seems to be the first reported case of concurrent ML infection with two Leishmania species. It indicates that, at least in this patient, previous infection with one of these Leishmania species did not protect against infection with the other. This result has important implications for the development of vaccines against leishmaniases and implies careful attention in the treatment of this infectious disease.

  13. Homologous recombination in Leishmania enriettii.

    Tobin, J F; Laban, A; Wirth, D F

    1991-02-01

    We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping deletions of the chloramphenicol acetyltransferase (CAT) gene, as well as the neomycin-resistance gene, were cotransfected into L. enriettii. Analysis of the DNA from these cells by Southern blotting and plasmid rescue revealed that a full-length or doubly deleted CAT gene could be reconstructed by homologous crossing-over and/or gene conversion between the two deletion plasmids. Additionally, parasites cotransfected with pALT-Neo and pALT-CAT-S, a plasmid containing two copies of the chimeric alpha-tubulin-CAT gene, resulted in G418-resistant parasites expressing high levels of CAT activity. The structure of the DNA within these cells, as shown by Southern blot analysis and the polymerase chain reaction, is that which would be expected from a homologous exchange event occurring between the two plasmids.

  14. 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

    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.

  15. Advanced Developement of Leishmania Tropical Skin Test Antigen

    2011-09-01

    Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Leishmania tropica Skin Test Antigen ( LtSTA) is lysate...31, 2011 on a Leishmania Skin test (LtSTA) made from the promastigotes of Leishmania tropica . During the last reporting period between June 18, 2009...sequences of other Leishmania species, closely related parasites and humans. In as much as the L. tropica genome has not been completely assembled, the

  16. Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice Leishmania spp.: isolamento de parasitos pela inoculação de macerados de biopsias de pacientes em camundongos deficientes em interferon gama

    Milton Adriano Pelli de Oliveira

    2010-04-01

    Full Text Available Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.O isolamento e a identificação da espécie de parasito do gênero Leishmania são importantes para a confirmação e auxiliam na epidemiologia da leishmaniose. Os camundongos são freqüentemente utilizados para isolar patógenos, porém, as linhagens mais comuns de camundongos são resistentes à infecção por parasitos do subgênero Leishmania (Viannia. Neste estudo, avaliamos a inoculação de macerados de biópsias de pacientes infectados em camundongos deficientes do gene do interferon gama (IFNγ KO como um método para aumentar a possibilidade de isolar Leishmania spp. Biópsias de 25 pacientes infectados com Leishmania sp. foram avaliadas para a presença de parasitos pelos métodos de imunohistoqu

  17. Características clínicas da leishmaniose tegumentar em crianças de 0 a 5 anos em uma área endêmica de Leishmania (Viannia braziliensis

    Ampuero Julia

    2006-01-01

    Full Text Available Para caracterizar a clínica da leishmaniose tegumentar em crianças de 0 a 5 anos de idade, foram avaliadas, retrospectivamente, 4.464 fichas clínicas do Centro de Saúde de Corte de Pedra, Presidente Tancredo Neves, Bahia, Brasil, área endêmica de leishmaniose tegumentar americana, entre maio de 1987 e dezembro de 1995. Foram registrados neste período 4.275 casos novos de leishmaniose, dos quais, 491 (11,5% correspondiam a crianças de 0 a 5 anos. A razão entre gênero masculino e feminino nas crianças foi 1,1:1. A forma clínica predominante foi a cutânea (98% e as lesões ulceradas foram as mais freqüentes (99%. A localização das lesões ocorreu, principalmente, acima da cintura (p<0,05, e 35,5% apresentaram lesões múltiplas. A magnitude da doença em crianças, a freqüência semelhante observada em ambos os gêneros e a localização das lesões sugere a possibilidade de transmissão vetorial no domicílio ou peridomicílio.

  18. Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion

    Christensen, Stephen M.; Dillon, Laura A. L.; Carvalho, Lucas P.; Passos, Sara; Novais, Fernanda O.; Hughitt, V. Keith; Beiting, Daniel P.; Carvalho, Edgar M.; Scott, Phillip; El-Sayed, Najib M.

    2016-01-01

    Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes. PMID:27631090

  19. Mechanisms of pathogenesis: differences amongst Leishmania species.

    Colmenares, Maria; Kar, Sujata; Goldsmith-Pestana, Karen; McMahon-Pratt, Diane

    2002-04-01

    One of the features of the genus Leishmania is the diversity of tropism/disease resulting from infection. With notable exceptions, the form (visceral, cutaneous, diffuse cutaneous, mucocutaneous) and severity of disease is a function of the infecting Leishmania species together with host genetics and consequent inflammatory and immune responses. It has become evident from genetic and immunological studies using the murine model that the various members of the genus Leishmania differ in aspects of their 'approach' to the host immune system. We are just beginning to appreciate the complexities of these interactions, which have import for the development of a vaccine against leishmaniasis. In this paper, what is currently understood concerning the mechanisms of leishmanial pathogenesis (based upon studies employing the murine model) is briefly summarized.

  20. Síntese e avaliação biológica de bioisósteros de nitrofural ativos contra Leishmania amazonensis

    Jornada, Daniela Hartmann

    2015-01-01

    Cutaneous leishmaniasis is a world widely distributed disease and it is estimated, by the World Health Organization (WHO), the incidence of 500,000 to one million cases per year. According to the Department of the Unified Health System (DATASUS), in 2005 it was registered more than 180 million cases of cutaneous leishmaniasis in Brazil. Although treatments for leishmaniasis are available, it is a complex process, because of difficulties in administration, once the majority of the drugs are in...

  1. Innate Immunity to Leishmania Infection: Within Phagocytes

    Marcela Freitas Lopes

    2014-01-01

    Full Text Available Infection by Leishmania takes place in the context of inflammation and tissue repair. Besides tissue resident macrophages, inflammatory macrophages and neutrophils are recruited to the infection site and serve both as host cells and as effectors against infection. Recent studies suggest additional important roles for monocytes and dendritic cells. This paper addresses recent experimental findings regarding the regulation of Leishmania major infection by these major phagocyte populations. In addition, the role of IL-4 on dendritic cells and monocytes is discussed.

  2. Role of Leishmania (Leishmania chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

    Kucknoor Ashwini S

    2005-02-01

    Full Text Available Abstract Background The parasitic protozoa belonging to Leishmania (L. donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L. chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L. chagasi within the U937 macrophage cells. Results The amastigote specific Ldccys2 genes of L. (L. chagasi and L. (L. donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis. Conclusions The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L. chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L. chagasi, especially in cases such as this, where a null mutant cannot be achieved by

  3. Susceptibility of spiny rats (Proechimys semispinosus to Leishmania (Viannia panamensis and Leishmania (Leishmania chagasi

    BL Travi

    2002-09-01

    Full Text Available The role of Proechimys semispinosus as reservoir of Leishmania (Viannia panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID in the snout or feet with 10(7 promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7 promastigotes or ID in the ear (10(8 promastigotes. PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i. and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

  4. Estudo, ao microscópio óptico e eletrônico, do rim de caes natural e experimentalmente infectados com Leishmania (Leishmania chagasi

    Washington Luiz Tafuri

    1989-06-01

    Full Text Available Os autores estudam os rins de 4 cães infectados com Leishmania (Leishmania chagasi. Dois animais (um macho e uma fêmea naturalmente infectados foram sacrificados 18 meses após sua permanência no laboratório. Dois machos foram inoculados por via endovenosa, com lxlO6 promastigotas da cepa MHO/BR/70/BH46 e sacrificados após 18 meses e 2 anos, respectivamente. Em todos os animais os rins estavam lesados. As alterações encontradas foram: (1 glomerulonefrite mesangioproliferativa focal ou difusa, com pronunciada hipertrofia e hiperplasia das células mesangiais e com alargamento da matriz; (2 espessamento da membrana basal com depósitos eletrondensos; (3 nefrite intersticial intertubular crônica com exsudação plasmocitária intensa. (4 degeneração albuminosa dos túbulos renais. Baseados nos achados os autores discutem os prováveis mecanismos patogenéticos.

  5. Plants used in the treatment of leishmanial ulcers due to Leishmania (Viannia braziliensis in an endemic area of Bahia, Brazil

    Flávio França

    1996-06-01

    Full Text Available This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia braziliensis (L(Vb among the rural population of a cocoa- producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plant species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale - Anacardiaceae, used by 65% of the population, folha-fogo (Clidemia hirta - Melastomataceae 39%, alfavaca-grossa (Plectranthus amboinicus - Lamiaceae 33%, mastruz (Chenopodium ambrosioides - Chenopodiaceae 31%, erva-de-santa-maria (Solatium americanum - Solanaceae (25% and transagem (Plantago major - Plantaginaceae. 2%.Este trabalho relata as plantas usadas no tratamento da leishmaniose cutânea, causada por Leishmania (Viannia braziliensis (L(Vb, na população rural da faixa litorânea produtora de cacau do estado da Bahia, Brasil. Um inquérito realizado entre 100 pacientes, identificou 49 espécies de plantas usadas para tratar úlceras de pele causadas por esta espécie de Leishmânia. As principais plantas usadas foram o cajueiro-branco (Anacardium occidentale - Anacardiaceae usado por 65% da população, a folha-fogo (Clidemia hirta - Melastomataceae 39%, a alfavaca-grossa (Plectranthus amboinicus - Lamiaceae 33%, o mastruz (Chenopodium ambrosioides - henopodiaceae 31%, a erva-de-santa-maria (Solanum americanum - Solanaceae 25% e a transagem (Plantago major - Plantaginaceae 2%.

  6. First Cases of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) naiffi Infection in Surinam

    P.P.A.M. van Thiel; T. van Gool; P.A. Kager; A. Bart

    2010-01-01

    Cutaneous leishmaniasis in Surinam is generally caused by infection by Leishmania guyanensis. We report three cases of infection with Leishmania (Viannia) naiffi, a Leishmania species not described from Surinam before. Treatment with pentamidine proved to be effective

  7. Developmentally regulated sphingolipid degradation in Leishmania major.

    Ou Zhang

    Full Text Available Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs through de novo synthesis (to produce inositol phosphorylceramide and salvage (to obtain sphingomyelin from the host. A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity and sphingomyelin (the SMase activity. Recent studies of a L. major ISCL-null mutant (iscl(- indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(- mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(- promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-. These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.

  8. Comorbidity of Leishmania major with cutaneous sarcoidosis

    Hamideh Moravvej

    2014-01-01

    Full Text Available Background: leishmaniasis infection might manifest as sarcoidosis; on the other hand, some evidences propose an association between sarcoidosis and leishmaniasis. Most of the times, it is impossible to discriminate idiopathic sarcoidosis from leishmaniasis by conventional histopathologic exam. Aim: We performed a cross-sectional study to examine the association of sarcoidosis with leishmaniasis in histopathologically diagnosed sarcoidal granuloma biopsy samples by polymerase chain reaction (PCR. Materials and Methods: We examined paraffin-embedded skin biopsy samples obtained from patients with clinical and histopathological diagnosis as naked sarcoidal granuloma, referred to Skin Research Center of Shaheed Beheshti Medical University from January 2001 to March 2010, in order to isolate Leishmania parasite. The samples were reassessed by an independent dermatopathologist. DNA extracted from all specimens was analyzed by the commercially available PCR kits (DNPTM Kit, CinnaGen, Tehran, Iran to detect endemic Leishmania species, namely leishmania major (L. major. Results: L. major was positive in PCR of Eight out of twenty-five examined samples. Conclusion: Cutaneous leishmaniasis may be misinterpreted as sarcoidosis; in endemic areas, when conventional methods fail to detect Leishmania parasite, PCR should be utilized in any granulomatous skin disease compatible with sarcoidosis, regardless of the clinical presentation or histopathological interpretation.

  9. Ocorrência de Leishmania spp. em felinos do município de Araçatuba, SP Occurrence de Leishmania spp. in domestic cats from Araçatuba, SP

    Katia Denise Saraiva Bresciani

    2010-06-01

    Full Text Available Este trabalho teve como objetivo comparar a ocorrência de Leishmania spp. em gatos por dois métodos (citológico e sorológico, bem como associar a ocorrência deste protozoário com as variáveis sexo, idade e raça. Amostras séricas de 283 felinos domésticos foram testadas pela Reação de Imunofluorescência Indireta (RIFI, e o exame parasitológico direto de linfonodos também foi realizado para a verificação da positividade para Leishmania spp. Ocorrência de 0,7% (2/283 foi observada nos felinos examinados, por meio de imprint de linfonodos e nenhum animal apresentou títulos de anticorpos para Leishmania spp. As duas fêmeas positivas eram sem raça definida, sendo uma jovem e outra adulta. Por meio dos resultados obtidos, não foi constatada diferença estatisticamente significante em relação às variáveis sexo, raça e idade nos gatos desta pesquisa (p > 0,05. Ocorrência de Leishmania spp. nos gatos deste estudo foi baixa. Devido a esta baixa incidência sugere-se que estes não assumem importância epidemiológica na área do estudo.This study had the purpose to compare the occurrence of Leishmania spp. in felines through two methods (cytological and serological, as well as to associate the occurrence of this protozoan with the sex, age and breed variables. Serum samples from 283 domestic felines were processed by means of Indirect Immunofluorescence Reaction (IIR, and the direct parasitological test for linfonodes was also carried out in order to verify positivity for Leishmania spp. Occurrence of 0.7% (2/283 was observed in the tested felines by means of linfonode imprinting and no animal showed title of antibodies for Leishmania spp. The two positive females were mongrel, a young female and an adult female feline. From the obtained results, no statistically significant difference was observed as regards the sex, breed and age variables in this research (p > 0.05. Occurrence of Leishmania spp. in the cats of this study was

  10. Molecular cloning and expression of the Leishmania tropica KMP-11 gene.

    Meriee, Mouayad; Soukkarieh, Chadi; Abbady, Abdul Qader A

    2014-08-01

    Kinetoplastid membrane protein-11 (KMP-11) is a small protein of 11 kDa present in all kinetoplastid protozoa studded so far. This protein which is highly expressed in all stages of the Leishmania life cycle is considered a potential candidate for a leishmaniasis vaccine against many leishmania species. KMP-11 has been recently described in Leishmania tropica. In the present study, the KMP-11 gene was extracted from L. tropica by PCR using two oligonucleotide primers designed to amplify the entire coding region of this gene. Then, the purified PCR products were successfully ligated into a high expression vector the pRSET-GFP. This expression vector provides the opportunity to clone the desired insert as a fusion protein with a GFP and a tag, polyhistidine region. The GFP use as a carrier to improve immune response and the polyhistidine tag facilitates detection of the expressed protein with anti-His antibodies and also purification of the protein using affinity purification. After wards KMP-11 coding region was sequenced and the recombinant protein was induced and purified from Escherichia coli cultures. The results of the present study will increase our knowledge about molecular cloning and expression of the L. tropica KMP-11 gene, and this may be used as an effective target for controlling cutenous leishmaniasis.

  11. Development and Production of a Leishmania Skin Test

    2009-03-01

    manufacturing process of Leishmania tropica Skin Test Antigen (LtSTA) was made during this contract period to increase the yield and robustness of the...interest group. 15. SUBJECT TERMS LtSTA = Leishmania tropica Skin Test Antigen 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT... tropica Skin Test Antigen (LtSTA), is a sterile injectable microfluidized lysate of Leishmania tropica (WR#1063:C1A) promastigotes. The product is heat

  12. Comparative genomic studies and in-silco strategies on Leishmania brazilensis, Leishmania infantum and Leishmania major: Conserved features, putative functions and potential drug target

    Rakesh N. R.

    2013-06-01

    Full Text Available Leishmaniasis is a parasitic disease found largely in the tropics, which the World Health Organization has estimated infects 12 million people worldwide each year. More recently cases have been reported in Europe among intravenous drug users with HIV. At least 20 Leishmania species infect humans. New world parasite Leishmania. braziliensis is the causative agent of mucocutaneous Leishmaniasis. The old world species Leishmania. major and Leishmania. infantum, which are present in Africa, Europe and Asia, are parasites that cause cutaneous and visceral Leishmaniasis respectively. Aim of this Study is determination of major common genes and Protein identified Gene location on each of the chromosomes, and identification of a common protein drug target Promastigote surface antigen with available lead molecule acetylglucosamine (6-(acetylamino-6-deoxyhexopyranose and docking studies on those considered Leishmania species.

  13. Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

    Pramod K Kushawaha

    Full Text Available Visceral leishmaniasis (VL is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI, a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT, nitric oxide (NO production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%. The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

  14. Nematodes of Amazonian birds, with a description of Hoazinstrongylus amazonensis n.gen.n.sp. (Trichostrongylidae, Libyostrongylinae

    R. Magalhães Pinto

    1985-06-01

    Full Text Available During recent studies of the parasites of birds from the Amazonian Regio, the following nematodes were recovered: Hoazinstrongylus amazonensis n.gen.n.sp. from Opisthocomus hoazin (Muller, 1776; Ascaridia columbae (Gmel., 1790 Travassos, 1913, from Leptotila r. rufaxilla (Richard & Bernard, 1712 representing a new host record; Inglisakis ibanezi Freitas, Vicente & Santos, 1969, Cyrnea (C. semilunaris (Molin, 1860 Seurat, 1914 and Thelazia digitata Travassos, 1918. A compelte description is restrained to the new genus and new species here proposed. The other known and well described species are listed and accounted.Durante recentes estudos dos parasitas de aves capturadas na Região Amazônica, foram identificadas as seguintes espécies de nematóides: Hoazinstrongylus amazonensis n.gen.n.sp, de Opisthocomus hoazin (Muller, 1776, Ascaridia columbae (Gmel., 1790 Travassos, 1912, de Leptotila r. rufaxilla (Richard & Bernard, 1712 que se constitui em um novo hospedeiro para a espécie, Inglisakis ibanezi Freitas, Vicente & Santos, 1969, Cyrnea (C. semilunaris (Molin, 1860 Seurat, 1914 e Thelazia digitata Travassos, 1918.

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

    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.

  16. Detection of Leishmania major and Leishmania tropica in domestic cats in the Ege Region of Turkey.

    Paşa, Serdar; Tetik Vardarlı, Aslı; Erol, Nural; Karakuş, Mehmet; Töz, Seray; Atasoy, Abidin; Balcıoğlu, İ Cüneyt; Emek Tuna, Gülten; Ermiş, Özge V; Ertabaklar, Hatice; Özbel, Yusuf

    2015-09-15

    Leishmaniosis is a group of diseases caused by different species of Leishmania parasites in mammalian species. The aim of the present study was to investigate the presence of Leishmania spp. DNA in cats using real time polymerase chain reaction (RT-PCR) assays targeting internal transcribed spacer (ITS1) and heat-shock protein 70 gene (Hsp70) regions with Leishmania species-specific primers and probes. Blood samples were collected from 147 cats (73 female; 74 male) in the endemic regions for zoonotic visceral leishmaniasis in the western provinces of Turkey and analyzed using two RT-PCR assays. Additionally, Hsp70 RT-PCR products were sequenced. ELISA assays for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) were also carried out for 145 of the 147 samples. Overall, 13/147 (8.84%) cats were positive for Leishmania by RT-PCR (4 L. major and 9 L. tropica). FIV and FeLV antibody and/or antigen was detected in 4 and 5 cats among Leishmania DNA positives, respectively. To the best of our knowledge, this study is the first to investigate and report the presence of L. major and L. tropica infections in a large group of domestic cats in Turkey. The results obtained indicate that species identification of Leishmania is essential for epidemiological understanding and that clinical signs alone are not indicative for leishmaniosis in cats, as it is in dogs. This study suggests that extensive research should be carried out in cat populations in order to fully understand the role of cats in the epidemiology of the disease.

  17. Leishmania infantum and Leishmania braziliensis: Differences and Similarities to Evade the Innate Immune System

    Falcão, Sarah de Athayde Couto; Jaramillo, Tatiana M. G.; Ferreira, Luciana G.; Bernardes, Daniela M.; Santana, Jaime M.; Favali, Cecília B. F.

    2016-01-01

    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 DCs’ biology for 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 of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. 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 24 h. 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 DCs after 24 h. 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. PMID:27536300

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

    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.

  19. The genetic toolbox for Leishmania parasites.

    Roberts, Sigrid C

    2011-01-01

    Leishmania parasites cause a variety of devastating diseases in tropical areas around the world. Due to the lack of vaccines and limited availability of drugs, new therapeutic targets are urgently needed. A variety of genetic tools have been developed to investigate the complex biology of this parasite and its interactions with the host. One of the main techniques is the generation of knock-out parasites via targeted gene replacement, a process that takes advantage of the parasites ability to undergo homologous recombination. Studying the effect of gene deletions in vitro and in infectivity models in vivo allows understanding the function of a target gene and its potential as a therapeutic target. Other genetic manipulations available include episomal and chromosomal complementation and the generation of overproducer strains. However, there are also limitations, such as the lack of RNA interference machinery in most Leishmania species and limited options for inducible expression systems. The genomes of several Leishmania species have now been sequenced and will provide powerful resources in combination with the genetic tools that are available. The increasing knowledge of parasite biology and host parasite interactions derived from these studies will raise the number of potential therapeutic targets, which are sorely needed to combat leishmaniasis.

  20. Herbal extract targets in Leishmania tropica.

    Mohammad, Bassim I; Al Shammary, Maani N; Abdul Mageed, Roaa H; Yousif, Nasser Ghaly

    2015-12-01

    The present study aims to investigate the effect of some herbal extract such as phenolic compounds on the viability of Leishmania tropica promastigotes in vitro. Four tested chemical agents (caffeic acid (CA), ferulic acid (FA), syringic acid (SA) and 4-hydroxybenzoic acid (4-HBA)) were used in this study. The viability of Leishmania tropica promastigotes was investigated under five different concentrations (10, 15, 20, 25 and 30 mg/ml) of each agent after (72 h). CA was the most active agent on the promastigotes viability after 72 h exposure to 30 mg/ml concentration so that the parasiticidal effect reach (53 × 10(4)) promastigote/ml. FA is the second agent in parasiticidal effect that parasiticidal effect reach to (50 × 10(4) promastigote/ml) at a concentration (30 mg/ml), 4-HBA is the third agent in parasiticidal effect that reach to (48 × 10(4) promastigote/ml) at a concentration (30 mg/ml), SA is the weakest agent in parasiticidal activity that reach to (44 × 10(4) promastigote/ml) at a concentration (30 mg/ml). It can be concluded that (CA, FA, SA and 4-HBA) possess acidal effect on the Leishmania tropica promastigotes in vitro.

  1. Leishmania major: Parasite Interactions Suggesting Sexuality

    Sousa Maria Auxiliadora de

    1997-01-01

    Full Text Available In five experiments, Leishmania (Leishmania major (MRHO/SU/59/P-strain grew poorly when seeded in FYTS medium supplemented with 15% fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5%. As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occurred at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2%. Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process

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

    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.

  3. Testing of Experimental Compounds for Efficacy against Leishmania.

    1987-02-01

    protozoan parasites of the family Trypanosomatidae, genus Leishmania, are widely distributed throughout the world and are found on every inhabited...the treatment of human beings infected with protozoan parasites of the genus Leishmania. Several significant developments have been forthcming fram

  4. An improved purification procedure for Leishmania RNA virus (LRV)

    de Souza, Marcos Michel; Manzine, Livia Regina; da Silva, Marcos Vinicius G.; Bettini, Jefferson; Portugal, Rodrigo Vilares; Cruz, Angela Kaysel; Arruda, Eurico; Thiemann, Otavio Henrique

    2014-01-01

    Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations. PMID:25242960

  5. Leishmania mexicana: aspectos taxonómicos y rareza de la infección humana en Três Braços, Bahia, Brasil

    César Augusto Cuba Cuba

    1984-09-01

    Full Text Available Del estúdio de 51 stocks de Leishmania aislados de pacientes humanos de leishmaniasis cutaneomucosa en Três Braços, Bahia, Brasil, los autores describen, en detalle, el análisis de los dos únicos stocks de L. mexicana, identificando uno de ellos como L. mexicana amazonensis. El otro aislado permanece en posición taxonómica no definida pues considerándosele como un miembro de L. mexicana, encuéntranse dificultades para su identificación subespecífica. Evaluan también los parâmetros biológicos e isoenzimáticos y discuten el papel de los anticuerpos monoclonales en la tipificación de éstos stocks. Los autores remarcan la rareza de la transmisión de parásitos del complejo L. mexicana en esta región, aun cuando estudien epidemiológicamente por mais de 8 anos la infección humana.

  6. An overview on Leishmania vaccines: A narrative review article

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

    Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is not totally worthwhile. However, sound immunity due to natural infection, implies that vigor cellular immunity against Leishmania parasites, via their live, attenuated or killed forms, can be developed in dogs and humans. Moreover, genetically conserved antigens (in most of Leishmania species), and components of sand fly saliva confer potential immunogenic molecules for Leishmania vaccination. Vaccines successes in animal studies and some clinical trials clearly justify more researches and investments illuminating opportunities in suitable vaccine designation. PMID:25992245

  7. An overview on Leishmania vaccines: A narrative review article.

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

    Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is not totally worthwhile. However, sound immunity due to natural infection, implies that vigor cellular immunity against Leishmania parasites, via their live, attenuated or killed forms, can be developed in dogs and humans. Moreover, genetically conserved antigens (in most of Leishmania species), and components of sand fly saliva confer potential immunogenic molecules for Leishmania vaccination. Vaccines successes in animal studies and some clinical trials clearly justify more researches and investments illuminating opportunities in suitable vaccine designation.

  8. Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters.

    Verma, Richa; Joseph, Sujith K; Kushwaha, Vikas; Kumar, Vikash; Siddiqi, M I; Vishwakarma, Preeti; Shivahare, Rahul; Gupta, Suman; Murthy, P K

    2015-12-01

    Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

  9. On Leishmania enriettii and Other Enigmatic Leishmania Species of the Neotropics

    Ralph Lainson

    1997-05-01

    Full Text Available There are 20 named species of the genus Leishmania at present recognized in the New World, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have not till now been found to cause human leishmaniasis; namely, Leishmania (Leishmania enriettii, L. (L. hertigi, L. (L. deanei, L. (L. aristidesi, L. (L. forattinii and L. (Viannia equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s and sandfly vector of L. (L. enriettii. Doubt is cast on the validity of the species L. herreri, described in Costa Rican sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors

  10. [Importance of amastigote forms morphology to differentiate Leishmania infantum and Leishmania major species].

    Aoun, K; Chahed, M K; Mokni, M; Harrat, Z; Bouratbine, A

    2003-01-01

    The microscopic study of the dermal smears of 62 cases of cutaneous leishmaniose, 27 infected by Leishmania (L.) infantum and 35 by L. major, showed that the amastigotes of L. infantum are meaningfully smaller (p < 0.001). This criteria is a simple pary alternative to distinguish these 2 species which have completely different epidemiology, recovery delay and prophylactic dispositions.

  11. Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values.

    Heverton Silva Camargos

    Full Text Available Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10(6 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4-9% at their respective IC50 values. For assays with high cell concentrations (2×10(9 parasites/mL, the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

  12. Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values.

    Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

    2014-01-01

    Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10(6) parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4-9%) at their respective IC50 values. For assays with high cell concentrations (2×10(9) parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

  13. Transporte de leishmania do sítio inflamatório para o linfonodo drenante: potenciais fagócitos envolvidos e cinética de disseminação

    Hermida, Micely D' El Rei

    2013-01-01

    A infecção por Leishmania modula a função de integrinas em fagócitos inflamatórios, afetando a migração celular e disseminação do parasito. O conhecimento sobre as populações de fagócitos capazes de transportar Leishmania ou fragmentos de parasitos mortos, e os mecanismos de disseminação da Leishmania do sítio de infecção para os diferentes tecidos ainda está incompleto. Nesse trabalho, nós adaptamos um modelo de migração de células in vivo para estudar o efeito da infecção ...

  14. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  15. Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

    Reza Arjmand

    2015-01-01

    Full Text Available Background: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM. In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Materials and Methods: L. major (MRHO/IR/75/ER from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. Results: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. Conclusion: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

  16. Augmented Oxygen-Dependent Killing of Leishmania.

    1992-06-30

    Leishmania are protozoan parasites which cause debilitating disease in tropical regions of South America, Asia, Africa and the Middle East...these two studies indicate that PO2s in excess of 1700 mmHg may be required to enhance lethal effects of AmB in yeast cells and protozoan parasites . Such...high partial pressures of oxygen (e.g. >240 kPa) might possibly exert detrimental effects in fungi and 15 protozoan parasites treated with AmB in

  17. Notas e descrições em Hesperophanini, Eburiini, Piezocerini e Trachyderini (Coleoptera, Cerambycidae, Cerambycinae) do Brasil e da Bolívia

    Ubirajara R. Martins; Maria Helena M. Galileo

    2010-01-01

    Novos táxons descritos - Hesperophanini: Paraliostola nigramacula sp. nov., do Brasil (Rondônia); Eburiini: Simplexeburia gen. nov., e sua espécie-tipo, S. divisa sp. nov. do Brasil (Amazonas); Piezocerini: Gorybia amazonensis sp. nov. and G. sulcata sp. nov., ambas do Brasil (Amazonas); Trachyderini: Galissus rubiventris sp. nov., da Bolívia (Santa Cruz). Notas e novos registros são apresentados para Liostola nitida Zajciw, 1962 e Ochrus chapadense Napp & Martins, 1982 (Hesperophanini); Unci...

  18. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better...... Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan...

  19. Infecção natural de Lutzomyia longipalpis por Leishmania sp. em Teresina, Piauí, Brasil Natural infection of Lutzomyia longipalpis by Leishmania sp. in Teresina, Piauí State, Brazil

    Jackellyne Geórgia Dutra e Silva

    2007-07-01

    Full Text Available No Brasil, o controle do vetor Lutzomyia longipalpis é uma das principais estratégias utilizadas para limitar a expansão da leishmaniose visceral americana. Entretanto, poucos são os estudos sobre a infecção natural destes flebotomíneos por espécies de Leishmania. Um estudo sobre a infecção natural de Lu. longipalpis por Leishmania sp. foi realizado no bairro Bela Vista, Teresina, Piauí, um dos principais focos urbanos da leishmaniose visceral americana no Brasil. Entre fevereiro de 2004 e janeiro de 2005 realizaram-se 180 capturas usando-se armadilhas luminosas do tipo CDC. Foram dissecadas e examinadas, em média, dez fêmeas por captura para detecção de formas evolutivas de Leishmania sp., identificando-se 1.832 exemplares de Lu. longipalpis e seis de Lu. whitmani. Vinte (1,1% espécimes, todas de Lu. longipalpis, estavam infectadas com as formas procíclica e nectomonada de Leishmania sp., localizadas, principalmente, na porção posterior do trato digestivo. O maior percentual de insetos infectados foi encontrado quatro meses após o período de maior precipitação pluviométrica, sugerindo que variáveis climáticas podem contribuir para a predição não apenas da abundância destes dípteros, mas também do seu grau de infecção natural.In Brazil, control of the vector Lutzomyia longipalpis is one of the main strategies used to limit the expansion of American visceral leishmaniasis. However, studies on the ecology of this sand fly are rare, especially regarding its natural infection with species of Leishmania. A study of the natural infection of Lu. longipalpis by Leishmania sp. was carried out in the Bela Vista neighborhood in the city of Teresina, Piauí State, Brazil, an important area of American visceral leishmaniasis transmission. From February 2004 to January 2005, sand flies were captured with CDC light traps. Approximately 10 female sand flies in each capture were dissected and examined for the presence of

  20. Cloning of Leishmania Major P4 Gene

    Minoo Shaddel

    2008-01-01

    Full Text Available Objective: Leishmania major P4 gene is normally expressed during amastigote form ofthe parasite and can be good candidate for producing an effective vaccine. In this study wecloned this gene in suitable vector (pQE-30 for further vaccine preparation studies.Materials and Methods: Leishmania promastigotes were grown in N.N.N.medium and culturein RPMI 1640 cell culture medium. Total genomic DNA was extracted by centrifugationof promastigotes. The pellet was suspended in lysis buffer and followed by boiling method.PCR was carried out using P4 gene specific primers. PCR product was detected by agarosgel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. Reactionwas transformed into XL1- Blue competent cell and recombinant plasmid screened usingagar plate contained X-gal and IPTG. The product was extracted, digested by restrictionenzyme and electrophoresed on agarose gel.Results: Plasmid was extracted and cloned gene was released by restriction enzyme andsubcloned into pQE-30 expression vector.Conclusion: This construct is ready for protein expression in in-vitro.

  1. Avaliação da produção da vacina LBSapSal contra Leishmaniose visceral canina, frente ao desafio intradérmico experimental com Leishmania chagasi e extrato de glândula salivar de Lutzomyia longipalpis.

    Mendonça, Caroline Amaral de

    2011-01-01

    Programa de Pós-Graduação em Ciências Farmacêuticas. CIPHARMA, Escola de Farmácia, Universidade Federal de Ouro Preto. No Brasil, o controle da leishmaniose visceral (LV) tem sido baseado num “tripé” de ações que preconiza o tratamento de casos humanos, o combate ao vetor e a eliminação de cães soropositivos, que infelizmente não tem alcançado bons resultados na redução dos casos de LV humana e canina. Assim, a imunoprofilaxia surge como uma das alternativas mais importantes para o control...

  2. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    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

  3. The first case report of Leishmania (leishmania chagasi in Panthera leo in Brazil

    Magyda AA Dahroug

    2011-06-01

    Full Text Available We reported here the first known case of natural infection of a lion (Panthera leo-Linnaeus, 1758 with Leishmania (Leishmania chagasi (L. chagasi in Brazil. The specimen was created by a circus handler in the state of Mato Grosso and was donated to the zoological park of the Federal University of Mato Grosso. Infection by L. chagasi was detected using a PCR-RFLP test. It was known that the domestic felids can act as reservoir of infection of L. chagasi in endemic areas, making it important that studies demonstrate their participation in the epidemiological chain. We demonstrate in this work that wild animals can have an important role in the epidemiological chain and must be considered in order to plan methods of control of this zoonosis.

  4. The first case report of Leishmania (leishmania) chagasi in Panthera leoin Brazil

    Magyda AA Dahroug; Arleana BPF Almeida; Valéria RF Sousa; Valéria Dutra; Luciana D Guimarães; César E Soares; Luciano Nakazato; Roberto L de Souza

    2011-01-01

    We reported here the first known case of natural infection of a lion (Panthera leo-Linnaeus, 1758) with Leishmania (Leishmania) chagasi (L. chagasi) in Brazil. The specimen was created by a circus handler in the state of Mato Grosso and was donated to the zoological park of the Federal University of Mato Grosso. Infection by L. chagasi was detected using a PCR-RFLP test. It was known that the domestic felids can act as reservoir of infection of L. chagasi in endemic areas, making it important that studies demonstrate their participation in the epidemiological chain. We demonstrate in this work that wild animals can have an important role in the epidemiological chain and must be considered in order to plan methods of control of this zoonosis.

  5. Antiproliferative and ultrastructural effects of phenethylamine derivatives on promastigotes and amastigotes of Leishmania (Leishmania) infantum chagasi.

    Brasil, Paula Ferreira; de Freitas, Júlia Araújo; Barreto, Anna Léa Silva; Adade, Camila Marques; Reis de Sá, Leandro Figueira; Constantino-Teles, Pamella; Toledo, Fabiano Travanca; de Sousa, Bruno A; Gonçalves, Augusto Cesar; Romanos, Maria Teresa Villela; Comasseto, João V; Dos Santos, Alcindo A; Tessis, Ana Claudia; Souto-Padrón, Thais; Soares, Rosangela Maria A; Ferreira-Pereira, Antonio

    2017-04-01

    Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis.

  6. The first case report of Leishmania (leishmania) chagasi in Panthera leo in Brazil.

    Dahroug, Magyda A A; Almeida, Arleana B P F; Sousa, Valéria R F; Dutra, Valéria; Guimarães, Luciana D; Soares, César E; Nakazato, Luciano; de Souza, Roberto L

    2011-06-01

    We reported here the first known case of natural infection of a lion (Panthera leo-Linnaeus, 1758) with Leishmania (Leishmania) chagasi (L. chagasi) in Brazil. The specimen was created by a circus handler in the state of Mato Grosso and was donated to the zoological park of the Federal University of Mato Grosso. Infection by L. chagasi was detected using a PCR-RFLP test. It was known that the domestic felids can act as reservoir of infection of L. chagasi in endemic areas, making it important that studies demonstrate their participation in the epidemiological chain. We demonstrate in this work that wild animals can have an important role in the epidemiological chain and must be considered in order to plan methods of control of this zoonosis.

  7. Identificação e caracterização molecular das espécies de Leishmania spp. em pacientes com HIV/AIDS em Pernambuco

    SILVA, Elis Dionisio da

    2015-01-01

    A coinfecção Leishmania - HIV/aids é considerada uma doença emergente, devido à sobreposição geográfica das duas infecções em várias regiões no mundo. Tem sido observado em pacientes coinfectados uma diversidade clínica importante, pobre eficácia aos tratamentos leishmanicidas convencionais, além da dificuldade de um diagnóstico preciso. O objetivo desse estudo foi avaliar o uso da PCR-RFLP para identificação das espécies de Leishmania em pacientes com HIV/aids, fornecendo dados que auxiliem ...

  8. Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis.

    Daniel R Abánades

    Full Text Available BACKGROUND: Protection and recovery from visceral leishmaniasis (VL have been associated with cell-mediated immune (CMI responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals. METHODOLOGY AND PRINCIPAL FINDINGS: VL patients with negative delayed-type hypersensitivity (DTH were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+, were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110-130 kDa consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+ reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA, with less cross-reactivity against Chagas disease patients' sera. SIGNIFICANCE: Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.

  9. Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications

    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

    2015-01-01

    Background: 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. Methods: Thirty-five L. killicki and 25 L. tropica strain...

  10. Implications of a Neotropical Origin of the Genus Leishmania

    Noyes Harry

    1998-01-01

    Full Text Available The hypothesis of a Neotropical origin of the Leishmania/Endotrypanum clade is reviewed. The position of the L. (Sauroleishmania external to the subgenus L. (Leishmania is not consistent with the Neotropical origin of the latter subgenus. It is suggested that this may be a consequence of a faster evolutionary rate in the L. (Sauroleishmania. The implications for the classsification of the phlebotomine sandflies of the hypothesis for a Neotropical origin of the Leishmania is also considered. The classification of Galati (1995 is proposed to be most consistent with the hypothesis of a Neotropical origin of the Leishmania, whilst classifications which place the New and Old World species in separate taxa are inconsistent with this hypothesis.

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

    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.

  12. Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs.

    Marcondes, M; Biondo, A W; Gomes, A A D; Silva, A R S; Vieira, R F C; Camacho, A A; Quinn, John; Chandrashekar, R

    2011-01-10

    Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.

  13. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    Ryuichi Miura

    Full Text Available Canine distemper virus (CDV vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively. Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

  14. The genome of the kinetoplastid parasite, Leishmania major

    Ivens, AC; Peacock, CS; Worthey, EA; Murphy, L; Aggarwal, G; Berriman, M.; Sisk, E; Rajandream, MA; Adlem, E; Aert, Rita; Anupama, A; Myler, PJ; Apostolou, Z; Attipoe, P; Bason, N.

    2005-01-01

    Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjuga...

  15. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    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 infl...

  16. An overview on Leishmania vaccines: A narrative review article

    REZVAN, Hossein; MOAFI, Mohammad

    2015-01-01

    Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is...

  17. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    Patricia Barrera; Valeria P Sülsen; Esteban Lozano; Mónica Rivera; María Florencia Beer; Carlos Tonn; Martino, Virginia S.; Sosa, Miguel A.

    2015-01-01

    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 the host’s cells. In this work, we have Investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promasti...

  18. Characterization of a Leishmania Stage Specific Mitochondrial Membrane Protein that Enhances the Activity of Cytochrome C Oxidase and Its Role in Virulence

    Dey, Ranadhir; Meneses, Claudio; Salotra, Poonam; Kamhawi, Shaden; Nakhasi, Hira L.; Duncan, Robert

    2010-01-01

    Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27kDa protein encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than i...

  19. Infecção por Leishmania (Leishmania chagasi em crianças de uma área endêmica de leishmaniose visceral americana na Ilha de São Luis-MA, Brasil

    Caldas Arlene J.M.

    2001-01-01

    Full Text Available Realizou-se estudo prospectivo com 648 crianças de zero a cinco anos no município da Raposa-MA, de julho/97 a junho/98, com o objetivo de avaliar as características da infecção por L.(L.chagasi e verificar se existe associação entre desnutrição e infecção assintomática. Utilizou-se questionário com dados socioeconômicos, ambientais e hábitos de vida; realizou-se Intradermorreação de Montenegro(IDRM com antígeno de L. amazonensis e Enzyme Linked Immunosorbant Assay(ELISA para detectar infecção, e exame antropométrico. A prevalência inicial, final e incidência da infecção foram 18,6%, 20,6% e 10,8% pelo IDRM, e 13,5%, 34,4% e 28% pelo ELISA, respectivamente. A prevalência da desnutrição crônica (altura/idade foi 26%. Não houve associação estatisticamente significante entre desnutrição e infecção assintomática por L. (L. chagasi. A forma assintomática da doença está presente nas áreas estudadas, necessitando de medidas de controle mais efetivas.

  20. Canine inflammatory myopathy associated with Leishmania Infantum infection.

    Paciello, Orlando; Oliva, Gaetano; Gradoni, Luigi; Manna, Laura; Foglia Manzillo, Valentina; Wojcik, Slawomir; Trapani, Francesca; Papparella, Serenella

    2009-02-01

    Inflammatory myopathy associated with several infectious diseases occurs in dogs including those caused by Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Hepatozoon canis. However, muscle disease due to Leishmania infection has been poorly documented. The aim of this study was to examine the distribution and types of cellular infiltrates and expression of MHC class I and II in muscle biopsies obtained from 15 male beagle dogs from a breeder group with an established diagnosis of leishmaniasis. Myopathic features were characterized by necrosis, regeneration, fibrosis and infiltration of mononuclear inflammatory cells consisting of lymphocytes, plasma cells and histiocytes. The predominant leukocyte populations were CD3+, CD8+ and CD45RA+ with lesser numbers of CD4+ cells. Many muscle fibers had MHC class I and II positivity on the sarcolemma. There was a direct correlation between the severity of pathological changes, clinical signs, and the numbers of Leishmania amastigotes. Our studies provided evidence that: 1) Leishmania should be considered as a cause of IM in dogs; 2) Leishmania is not present within muscle fibers but in macrophages, and that 3) the muscle damage might be related to immunological alterations associated with Leishmania infection. Leishmania spp. should also be considered as a possible cause in the pathogenesis of human myositis.

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

    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

  2. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2007-01-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of the...

  3. Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

    Patrícia Flávia Quaresma

    2012-06-01

    Full Text Available Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB polymerase chain reaction (PCR was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs. We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human, respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

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

    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.

  5. Delayed culture of Leishmania in skin biopsies.

    Dedet, J P; Pratlong, F; Pradinaud, R; Moreau, B

    1999-01-01

    Between January 1997 and October 1998, 16 skin biopsies collected from 13 patients with cutaneous leishmaniasis in French Guiana were inoculated in culture medium after travel for 3-17 days from the place of biopsy to the culture laboratory in France. Each biopsy fragment was introduced near the flame of a Bunsen burner into the transport medium (RPMI medium supplemented with 10% fetal calf serum) which was maintained at ambient temperature during postal delivery to France. In France the biopsies were ground in sterile saline before being inoculated into NNN culture tubes. The cultures were incubated at 25 degrees C and subcultured every week until the 5th week. The cultures were positive in 9 cases, remained negative in 4, and were contaminated in 3 cases. Positive results were obtained at all seasons and for 3 different Leishmania species. The study indicates that delayed culture can yield useful results from biopsies taken in field conditions.

  6. Time-course analysis of the Shewanella amazonensis SB2B proteome in response to sodium chloride shock

    Parnell, John J.; Callister, Stephen J.; Rompato, Giovanni; Nicora, Carrie D.; Pasa-Tolic, Ljiljana; Williamson, Ashley; Pfrender, Michael E.

    2011-06-29

    Organisms in the genus Shewanella have become models for response to environmental stress. One of the most important environmental stresses is change in osmolarity. In this study, we experimentally determine the response mechanisms of Shewanella amazonensis SB2B during osmotic stress. Osmotic stress in SB2B was induced through exposure to NaCl, and the time-course proteomics response was measured using liquid chromatography mass spectrometry. Protein trends were qualitatively compared to gene expression trends and to phenotypic characterization. Osmotic stress affects motility, and has also been associated with a change in the membrane fatty acid composition (due to induction of branched chain amino acid degradation pathways); however, we show this is not the case for SB2B. Although proteins and genes involved with branched chain amino acid degradation are induced, fatty acid degradation pathways are not induced and no change in the fatty acid profile occurs in SB2B as a result of osmotic shock. The most extensive response of SB2B over the time course of acclimation to high salt involves an orchestrated sequence of events comprising increased expression of signal transduction associated with motility and restricted cell division and DNA replication. After SB2B has switched to increased branched chain amino acid degradation, motility, and cellular replication proteins return to pre-perturbed levels.

  7. Molecular Identification of Leishmania Species Causing Cutaneous Leishmaniasis In Mashhad area, Iran

    Mohammad Karimian Shirazi

    2014-08-01

    Results: In first step of PCR, all of sampled were positive for Leishmania spp and in second step Leishmania tropica and L.major were detected in 94% and 6% in positive –PCR amplicon , respectively. Conclusion: Based on the results, Leishmania tropica is more prevalent than L.major in Mashhad area

  8. Identification of geographically distributed sub-populations of Leishmania (Leishmania major by microsatellite analysis

    Schwenkenbecher Jan

    2008-06-01

    Full Text Available Abstract Background Leishmania (Leishmania major, one of the agents causing cutaneous leishmaniasis (CL in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L. major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT based on 10 different microsatellite markers was applied to 106 strains of L. (L. major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA; the Middle East (ME; and Africa (AF. This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L. major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host.

  9. Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

    José Arturo Gutiérrez

    2001-08-01

    Full Text Available Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (aprox. 40 kDa. The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

  10. Polymorphisms of cpb multicopy genes in the Leishmania (Leishmania) donovani complex.

    Hide, M; Bañuls, A L

    2008-02-01

    In leishmaniasis, cysteine protease b (cpb) multicopy genes have been extensively studied because of their implication in host-parasite interactions. In the Leishmania donovani complex, responsible for visceral leishmaniasis, a set of interesting polymorphisms has been revealed, such as copy sequence or expression according to the parasite's life stage. The single nucleotide polymorphisms observed among these copies could be related to clinical characteristics such as dermotropic versus viscerotropic status. CPB COOH-terminal extension (CTE) is mainly responsible for genetic variability among the copies and appears highly immunogenic. These results suggest that further study of the role of CPBs, especially CTE in clinical outcome, is warranted.

  11. Cryptic Leishmania infantum infection in Italian HIV infected patients

    Rubino Raffaella

    2009-12-01

    Full Text Available Abstract Background Visceral leishmaniasis (VL is a protozoan diseases caused in Europe by Leishmania (L. infantum. Asymptomatic Leishmania infection is more frequent than clinically apparent disease. Among HIV infected patients the risk of clinical VL is increased due to immunosuppression, which can reactivate a latent infection. The aims of our study were to assess the prevalence of asymptomatic L. infantum infection in HIV infected patients and to study a possible correlation between Leishmania parasitemia and HIV infection markers. Methods One hundred and forty-five HIV infected patients were screened for the presence of anti-Leishmania antibodies and L. infantum DNA in peripheral blood. Statistical analysis was carried out by using a univariate regression analysis. Results Antibodies to L. infantum were detected in 1.4% of patients. L. infantum DNA was detected in 16.5% of patients. Significant association for PCR-Leishmania levels with plasma viral load was documented (p = 0.0001. Conclusion In our area a considerable proportion of HIV infected patients are asymptomatic carriers of L. infantum infection. A relationship between high HIV viral load and high parasitemic burden, possibly related to a higher risk of developing symptomatic disease, is suggested. PCR could be used for periodic screening of HIV patients to individuate those with higher risk of reactivation of L. infantum infection.

  12. Cross-protective efficacy of Leishmania infantum LiHyD protein against tegumentary leishmaniasis caused by Leishmania major and Leishmania braziliensis species.

    Lage, Daniela Pagliara; Martins, Vívian Tamietti; Duarte, Mariana Costa; Costa, Lourena Emanuele; Tavares, Grasiele de Sousa Vieira; Ramos, Fernanda Fonseca; Chávez-Fumagalli, Miguel Angel; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2016-06-01

    Vaccination can be considered the most cost-effective strategy to control neglected diseases, but nowadays there is not an effective vaccine available against leishmaniasis. In the present study, a vaccine based on the combination of the Leishmania-specific hypothetical protein (LiHyD) with saponin was tested in BALB/c mice against infection caused by Leishmania major and Leishmania braziliensis species. This antigen was firstly identified in Leishmania infantum and showed to be protective against infection of BALB/c mice using this parasite species. The immunogenicity of rLiHyD/saponin vaccine was evaluated, and the results showed that immunized mice produced high levels of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with rLiHyD, as well as by using L. major or L. braziliensis protein extracts. After challenge, vaccinated animals showed significant reductions in the infected footpad swellings, as well as in the parasite burden in the infection site, liver, spleen, and infected paws draining lymph nodes, when compared to those that were inoculated with the vaccine diluent (saline) or immunized with saponin. The immunization of rLiHyD without adjuvant was not protective against both challenges. The partial protection obtained by the rLiHyD/saponin vaccine was associated with a parasite-specific IL-12-dependent IFN-γ secretion, which was produced mainly by CD4(+) T cells. In these animals, a decrease in the parasite-mediated IL-4 and IL-10 responses, associated with the presence of high levels of LiHyD- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a hypothetical protein that was firstly identified in L. infantum, when combined to a Th1 adjuvant, was able to confer a cross-protection against highly infective stationary-phase promastigotes of two Leishmania species causing tegumentary leishmaniasis.

  13. FIRST REPORT OF CUTANEOUS LEISHMANIASIS CAUSED BY Leishmania (Leishmania infantum chagasi IN AN URBAN AREA OF RIO DE JANEIRO, BRAZIL

    Marcelo Rosandiski LYRA

    2015-10-01

    Full Text Available SUMMARY American tegumentary leishmaniasis (ATL is an infectious disease caused by protozoa of the genus Leishmania, and transmitted by sandflies. In the state of Rio de Janeiro, almost all of the cases of American tegumentary leishmaniasis (ATL are caused by Leishmania (Viannia braziliensis, while cases of visceral leishmaniasis (VL are caused by Leishmania (Leishmania infantum chagasi. The resurgence of autochthonous VL cases in Rio de Janeiro is related to the geographic expansion of the vector Lutzomyia longipalpis and its ability to adapt to urban areas. We report the first case of leishmaniasis with exclusively cutaneous manifestations caused by L. (L. infantum chagasi in an urban area of Rio de Janeiro. An eighty-one-year-old woman presented three pleomorphic skin lesions that were not associated with systemic symptoms or visceromegalies. Multilocus enzyme electrophoresis identified L. (L. infantum chagasi, but direct smear and PCR of bone narrow were negative for Leishmania sp. (suggesting exclusively cutaneous involvement. We discuss the different dermatological presentations of viscerotropic leishmaniasis of the New and Old World, and the clinical and epidemiological importance of the case. Etiologic diagnosis of ATL based upon exclusive clinical criteria may lead to incorrect conclusions. We should be aware of the constant changes in epidemiological patterns related to leishmaniases.

  14. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major.

    Ezatpour, Behrouz; Saedi Dezaki, Ebrahim; Mahmoudvand, Hossein; Azadpour, Mojgan; Ezzatkhah, Fatemeh

    2015-01-01

    The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0-100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL.

  15. Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice.

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2011-02-01

    Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4(+) lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4(-) lymphocyte up to one month post-challenge suggesting that CD4(-) lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.

  16. Rattus norvegicus (Rodentia: Muridae) Infected by Leishmania (Leishmania) infantum (syn. Le. chagasi) in Brazil.

    Lara-Silva, Fabiana de Oliveira; Barata, Ricardo Andrade; Michalsky, Erika Monteiro; Ferreira, Eduardo de Castro; Lopes, Maria Olímpia Garcia; Pinheiro, Aimara da Costa; Fortes-Dias, Consuelo Latorre; Dias, Edelberto Santos

    2014-01-01

    In the present study we surveyed the fauna of phlebotomine sand flies and small mammals in peridomestic areas from a Brazilian municipality where the American cutaneous leishmaniasis (ACL) is endemic. A total of 608 female phlebotomine sand flies were captured during nine months in 2009 and 2010. Seven different species were represented with 60% of them being Lutzomyia intermedia and Lu. whitmani, both incriminated vectors of ACL. Lu. longipalpis, a proven vector of visceral leishmaniasis (VL) was also captured at high proportion (12.8%). Genomic DNA analysis of 136 species-specific pools of female sand flies followed by molecular genotyping showed the presence of Leishmania infantum DNA in two pools of Lu. longipalpis. The same Leishmania species was found in one blood sample from Rattus norvegicus among 119 blood and tissue samples analysed. This is the first report of Le. infantum in R. norvegicus in the Americas and suggests a possible role for this rodent species in the zoonotic cycle of VL. Our study coincided with the reemergence of VL in Governador Valadares.

  17. Seasonal transmission of Leishmania (Leishmania mexicana in the state of Campeche, Yucatan Peninsula, Mexico

    Andrade-Narvaez Fernando J

    2003-01-01

    Full Text Available In the Yucatan Peninsula, Mexico, localized cutaneous leishmaniasis (LCL caused by Leishmania (Leishmania mexicana is a typical wild zoonosis restricted to the forest, and humans are only accidentally involved. The transmission of L. (L. mexicana has been related to the patient's occupation: "chicleros"(gum collectors and agricultural workers. The objective of this study was to document L. (L. mexicana seasonally of transmission in endemic areas of LCL in the state of Campeche, Yucatan Peninsula, Mexico. The timing of incidence of LCL in humans during 1993-1994, as well as the rate and time of infection in rodents and sand flies between February 1993 and March 1995 were analyzed. Rodents and sand flies were found infected between November and March, when men carried out their field activities and are exposed. Based on results analyzed, it is concluded that L. (L. mexicana in the endemic area of LCL in the state of Campeche, Yucatan Peninsula, Mexico, presents a seasonal transmission restricted to the months of November to March. The knowledge of the timing of the transmission cycle in an endemic area of leishmaniasis is very important because intervention measures on the high-risk focus and population might be restricted.

  18. Seasonal transmission of Leishmania (Leishmania) mexicana in the state of Campeche, Yucatan Peninsula, Mexico.

    Andrade-Narvaez, Fernando J; Canto Lara, Silvia B; Van Wynsberghe, Nicole R; Rebollar-Tellez, Eduardo A; Vargas-Gonzalez, Alberto; Albertos-Alpuche, Nelly E

    2003-12-01

    In the Yucatan Peninsula, Mexico, localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana is a typical wild zoonosis restricted to the forest, and humans are only accidentally involved. The transmission of L. (L.) mexicana has been related to the patient's occupation: "chicleros" (gum collectors) and agricultural workers. The objective of this study was to document L. (L.) mexicana seasonally of transmission in endemic areas of LCL in the state of Campeche, Yucatan Peninsula, Mexico. The timing of incidence of LCL in humans during 1993-1994, as well as the rate and time of infection in rodents and sand flies between February 1993 and March 1995 were analyzed. Rodents and sand flies were found infected between November and March, when men carried out their field activities and are exposed. Based on results analyzed, it is concluded that L. (L.) mexicana in the endemic area of LCL in the state of Campeche, Yucatan Peninsula, Mexico, presents a seasonal transmission restricted to the months of November to March. The knowledge of the timing of the transmission cycle in an endemic area of leishmaniasis is very important because intervention measures on the high-risk focus and population might be restricted.

  19. Rattus norvegicus (Rodentia: Muridae Infected by Leishmania (Leishmania infantum (syn. Le. chagasi in Brazil

    Fabiana de Oliveira Lara-Silva

    2014-01-01

    Full Text Available In the present study we surveyed the fauna of phlebotomine sand flies and small mammals in peridomestic areas from a Brazilian municipality where the American cutaneous leishmaniasis (ACL is endemic. A total of 608 female phlebotomine sand flies were captured during nine months in 2009 and 2010. Seven different species were represented with 60% of them being Lutzomyia intermedia and Lu. whitmani, both incriminated vectors of ACL. Lu. longipalpis, a proven vector of visceral leishmaniasis (VL was also captured at high proportion (12.8%. Genomic DNA analysis of 136 species-specific pools of female sand flies followed by molecular genotyping showed the presence of Leishmania infantum DNA in two pools of Lu. longipalpis. The same Leishmania species was found in one blood sample from Rattus norvegicus among 119 blood and tissue samples analysed. This is the first report of Le. infantum in R. norvegicus in the Americas and suggests a possible role for this rodent species in the zoonotic cycle of VL. Our study coincided with the reemergence of VL in Governador Valadares.

  20. Clinical picture of cutaneous leishmaniases due to Leishmania (Leishmania mexicana in the Yucatan peninsula, Mexico

    Andrade-Narváez Fernando J

    2001-01-01

    Full Text Available Localized cutaneous leishmaniasis (LCL, known as "chiclero's ulcer" in southeast Mexico, was described by Seidelin in 1912. Since then, the sylvatic region of the Yucatan peninsula has been identified as an endemic focus of LCL. The purpose of the present work was to describe the clinical picture of LCL caused by Leishmania (Leishmania mexicana in the Yucatan peninsula. A total of 136 cases of LCL, based on isolation and characterization of L. (L. mexicana by isoenzymes and/or monoclonal antibodies, were selected. Some variability of clinical features regarding number, type, size, form, location and time of evolution of the lesions was observed. The most frequently observed presentation was a single, ulcerated, rounded small lesion, located on the ear, with an evolution time of less than three months, with neither cutaneous metastases nor lymphatic nor mucosal involvement. This picture corresponds to previous studies carried out in the same endemic area where an organism of the L. mexicana complex has been incriminated as a major aetiological agent of classical "chiclero's ulcer", confirming that in the Yucatan peninsula LCL due to L. (L. mexicana when located on the pinna of the ear is a remarkable characteristic.

  1. Molecular crosstalks in Leishmania-sandfly-host relationships

    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.

  2. Toll-Like Receptors in Leishmania Infections: Guardians or Promoters?

    Marilia S. Faria

    2012-01-01

    Full Text Available Protozoa of the genus Leishmania cause a wide variety of pathologies ranging from self-healing skin lesions to visceral damage, depending on the parasite species. The outcome of infection depends on the quality of the adaptive immune response, which is determined by parasite factors and the host genetic background. Innate responses, resulting in the generation of mediators with anti-leishmanial activity, contribute to parasite control and help the development of efficient adaptive responses. Among those, the potential contribution of members of the Toll-like receptors (TLRs family in the control of Leishmania infections started to be investigated about a decade ago. Although most studies appoint a protective role for TLRs, there is growing evidence that in some cases, TLRs facilitate infection. This review highlights recent advances in TLR function during Leishmania infections and discusses their potential role in restraining parasite growth versus yielding disease.

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

    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

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

    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.

  5. Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi.

    Bhatia, A; Daifalla, N S; Jen, S; Badaro, R; Reed, S G; Skeiky, Y A

    1999-08-20

    We report here the molecular cloning and characterization of two related hydrophilic antigens of Leishmania chagasi. These two antigens have predicted molecular weights of approximately 9 and 26 kDa and detect antibodies in sera of patients with kala-azar (k). Thus, to maintain consistency with nomenclature of the previously described 39 kDa diagnostic antigen of L. chagasi (k39 [1]), these antigens are being referred to as k9 and k26. A significant difference between k9 and k26 is the presence of 11 copies of a 14 amino acid repeat in the open reading frame of k26. The region flanking the repeats of k26 shares a 69% identity with the open reading frame of k9. The recombinant proteins encoded by both antigens are very hydrophilic and show aberrant migration on SDS PAGE. Results of Southern blot analysis reveal that k9 and k26 are conserved to varying degrees among various Leishmania species. Interestingly, the repeat region of k26 is specific to L. chagasi and L. donovani while the flanking region is conserved among several other species. Transcript levels of k26 are significantly upregulated in the amastigote stage of the parasite. Our results show that recombinant K26 is specific in detecting antibodies in infection sera from visceral leishmaniasis (VL) patients. Thus rK26 may complement rK39 in a more accurate diagnosis of VL in the old and the new world.

  6. Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana Detección de DNA de Leishmania braziliensis en pacientes de leishmaniose tegumentaria americana Detection of Leishmania braziliensis DNA in American tegumentary leishmaniasis patients

    Leila Martins

    2010-06-01

    Full Text Available Foi realizado diagnóstico para leishmaniose tegumentar americana a partir de sangue de pacientes residentes em dois municípios endêmicos do estado de Pernambuco. O DNA de 119 amostras de sangue foi extraído e submetido a reação em cadeia da polimerase. Utilizaram-se primers do minicírculo do DNA do cinetoplasto (kDNA de Leishmania braziliensis, circulante em Pernambuco, cuja seqüência-alvo gera um fragmento de 750 pares de bases. No total 58 (48,7% indivíduos apresentaram amplificação positiva e 61 (51,3% negativa. Das amostras positivas para a PCR, 37 (≅ 64% pertenciam a indivíduos tratados e sem lesão. Conclui-se que a técnica de PCR é eficaz para identificar o DNA de leishmânia em material de biópsias e em sangue venoso.Fue realizado diagnóstico para leishmaniosis tegumentaria americana a partir de sangre de pacientes residentes en dos municipios endémicos del estado de Pernambuco (Noreste de Brasil. El DNA de 119 muestras de sangre fue extraído y sometido a la reacción en cadena de la polimerasa. Se utilizaron primers del minicírculo del DNA del cinetoplasto (kDNA de Leishmania braziliensis, circulante en Pernambuco, cuya secuencia blanco genera un fragmento de 750 pares de bases. En total 58 (48,7% individuos presentaron amplificación positiva y 61 (51,3% negativa. De las muestras positivas para la PCR, 37 (≅64% pertenecían a individuos tratados y sin lesión. Se concluyó que la técnica de la PCR es eficaz para identificar el DNA de Leishmania en material de biopsias y en sangre venosa.Diagnostic tests for American tegumentary leishmaniasis were performed on blood samples of patients living in two endemic municipalities in the state of Pernambuco, Northeastern Brazil. DNA was extracted from 119 samples and used as template for polymerase chain reaction (PCR analysis. The tests used primers specific for the kinetoplast mini-circle DNA (kDNA of Leishmania braziliensis, a species circulating in Pernambuco, which

  7. CD8+ T cells in Leishmania infections: friends or foes?

    Simona eStager

    2012-01-01

    Full Text Available Host protection against several intracellular pathogens requires the induction of CD8+ T cell responses. CD8+ T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8+ T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8+ T cells during various types Leishmania infections, following vaccination, and as potential immunotherapeutic targets.

  8. Lutzomyia longipalpis naturally infected by Leishmania (L. chagasi in Várzea Grande, Mato Grosso State, Brazil, an area of intense transmission of visceral leishmaniasis Lutzomyia longipalpis naturalmente infectado por Leishmania (L. chagasi em Várzea Grande, Mato Grosso, Brasil, uma área de transmissão intensa de leishmaniose visceral

    Nanci A. Missawa

    2010-12-01

    Full Text Available The American visceral leishmaniasis (AVL is caused by parasites belonging to the genus Leishmania (Trypanosomatidae and is transmitted to humans through the bite of certain species of infected phlebotomine sand flies. In this study, we investigated the natural infection ratio of Lutzomyia longipalpis, the main vector species of AVL in Brazil, in Várzea Grande, Mato Grosso State. Between July 2004 and June 2006, phlebotomine sand flies were captured in peridomestic areas using CDC light-traps. Four hundred and twenty (420 specimens of Lu. longipalpis were captured. 42 pools, containing 10 specimens of Lu. longipalpis each, were used for genomic DNA extraction and PCR (polymerase chain reaction amplification. Leishmania spp. DNA was detected in three out of the 42 pools tested, resulting in a minimal infection ratio of 0.71%. Restriction fragment length polymorphism (RFLP analysis indicated that Leishmania (L. chagasi was the infective agent in the positive pools.A leishmaniose visceral americana (LVA é causada por parasitos pertencentes ao gênero Leishmania (Trypanosomatidae e transmitida ao homem através da picada de certas espécies de flebotomíneos, previamente infectados. Neste trabalho, investigamos o índice de infecção natural de Lutzomyia longipalpis, principal vetor da LVA no Brasil, em Várzea Grande, Estado do Mato Grosso. De julho de 2004 a junho de 2006, foram feitas capturas de flebotomíneos em áreas peridomésticas utilizando armadilhas de luz CDC. Foram capturadas 420 espécimens de Lu. longipalpis. Quarenta e dois grupos, formados por 10 espécimens de Lu. longipalpis cada um, foram submetidos à extração de DNA genômico e amplificação por PCR (reação em cadeia da polimerase. DNA de Leishmania spp. foi detectado em 3 dos 42 grupos testados, resultando em um índice mínimo de infecção de 0,71%. A análise de polimorfismos de fragmentos de restrição (RFLP indicou Leishmania (L. chagasi como a esp

  9. Occurrence of Leishmania (Leishmania) chagasi in a domestic cat (Felis catus) in Andradina, São Paulo, Brazil: case report.

    Coelho, Willian Marinho Dourado; Lima, Valéria Marçal Felix de; Amarante, Alessandro Francisco Talamini do; Langoni, Helio; Pereira, Virgínia Bodelão Richini; Abdelnour, Aziz; Bresciani, Katia Denise Saraiva

    2010-01-01

    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.

  10. Morphology and small subunit rDNA-based phylogeny of Ceratomyxa amazonensis n. sp. parasite of Symphysodon discus, an ornamental freshwater fish from Amazon.

    Mathews, Patrick D; Naldoni, Juliana; Maia, Antonio A; Adriano, Edson A

    2016-10-01

    The specious genus Ceratomyxa Thélodan, 1892, infect mainly gallbladder of marine fishes, with only five species reported infecting species from freshwater environment. This study performed morphological and phylogenetic analyses involving a new Ceratomyxa species (Ceratomyxa amazonensis n. sp.) found in gallbladder of Symphysodon discus Heckel, 1840 (Perciformes: Cichlidae), an important ornamental fish endemic to Amazon basin. Mature spores were strongly arcuate shaped and measured 7.0 ± 0.3 (6.2-7.6) μm in length, 15.8 ± 0.4 (15.0-16.7) μm in thickness, and polar capsules 3.22 ± 0.34 (2.4-3.6) μm in length and 2.63 ± 0.17 (2.4-2.9) μm in width. This was the first small subunit ribosomal DNA (SS rDNA) sequencing performed to Ceratomyxa species parasite of freshwater fish, and the phylogenetic analysis showed C. amazonensis n. sp. clustering in the early diverging subclade of the ceratomyxids, together with species of parasites of amphidromous/estuaries fishes, suggesting some role of the transition of the fishes between marine/freshwater environments in the evolutionary history of these parasites.

  11. Retention of Leishmania (Leishmania Mexicana in naturally infected rodents from the State of Campeche, Mexico

    Nicole R Van Wynsberghe

    2000-10-01

    Full Text Available In the State of Campeche, Mexico, zoonotic cutaneous leishmaniasis is mainly due to Leishmania (L. mexicana. The parasite population is maintained in a mammalian species, a reservoir in which the ideal course of infection should be long and relatively nonpathogenic. The objective of the present study was to document the retention of L. (L. mexicana in 29 naturally infected rodents. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite, by cultures of needle aspirates from the base of the tail. Peromyscus yucatanicus and Ototylomys phyllotis were incriminated as the primary reservoir hosts. The finding that the multiplication of parasites in P. yucatanicus might be triggered by temperature, suggests that this animal would be a good choice for further research on L. (L. mexicana.

  12. Retention of Leishmania (Leishmania) mexicana in naturally infected rodents from the State of Campeche, Mexico.

    Van Wynsberghe, N R; Canto-Lara, S B; Damián-Centeno, A G; Itzá-Ortiz, M F; Andrade-Narváez, F J

    2000-01-01

    In the State of Campeche, Mexico, zoonotic cutaneous leishmaniasis is mainly due to Leishmania (L.) mexicana. The parasite population is maintained in a mammalian species, a reservoir in which the ideal course of infection should be long and relatively nonpathogenic. The objective of the present study was to document the retention of L. (L.) mexicana in 29 naturally infected rodents. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite, by cultures of needle aspirates from the base of the tail. Peromyscus yucatanicus and Ototylomys phyllotis were incriminated as the primary reservoir hosts. The finding that the multiplication of parasites in P. yucatanicus might be triggered by temperature, suggests that this animal would be a good choice for further research on L. (L.) mexicana.

  13. Molecular detection of Leishmania infantum in naturally infected

    C. Maia, M.O. Afonso, L. Neto, L. Dionísio & L. Campino

    2009-12-01

    Full Text Available Background & objectives: In Portugal, Phlebotomus perniciosus and P. ariasi, (SubgenusLarroussius; Diptera: Psychodidae are the proven vectors of leishmaniasis caused by Leishmaniainfantum. The Algarve Region in southern Portugal has been considered an endemic focus ofleishmaniasis since 1980s. The main objective of the present study was to validate a molecularapproach to detect Leishmania infection in phlebotomines based on DNA extraction from thefemale sandfly whole body, minus genitalia, followed by PCR for application on epidemiologicalsurveys.Methods: In Algarve Region, from early May until early November 2006, sandflies were capturedby CDC miniature light-traps. kDNA-PCR and ITS1-PCR were used to screen the presence ofLeishmania DNA in female sandflies after species identification by entomological keys.Results: A total of 474 sandflies were collected in 108 biotopes. One female of P. perniciosus, thepredominant species, was found infected with L. infantum reflecting an overall infection rate of0.47%.Interpretation & conclusion: PCR associated with morphological characterization of the sandflieswill be a powerful epidemiological tool for the determination of the number of phlebotominesinfected with Leishmania spp in nature. In addition, the simultaneous occurrence of dogs and P.perniciosus infected with L. infantum shows that Algarve continues to be an endemic focus ofcanine leishmaniasis. Furthermore, as P. sergenti and P. papatasi which transmit L. tropica and L.major, respectively were present, the future introduction of these two Leishmania species in southernregion of Portugal should not be neglected.

  14. Sand fly evolution and its relationship to Leishmania transmission

    PD Ready

    2000-08-01

    Full Text Available The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis. Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

  15. Vector transmission of leishmania abrogates vaccine-induced protective immunity.

    Nathan C Peters

    2009-06-01

    Full Text Available Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is "leishmanization," in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.

  16. Leishmania (Viannia) naiffi: rare enough to be neglected?

    Fagundes-Silva, Giselle Aparecida; Romero, Gustavo Adolfo Sierra; Cupolillo, Elisa; Yamashita, Ellen Priscila Gadelha; Gomes-Silva, Adriano; Guerra, Jorge Augusto de Oliveira; Da-Cruz, Alda Maria

    2015-01-01

    In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and presents a wide spectrum of clinical manifestations due, in part, to the circulation of at least seven Leishmania species. Few reports of Leishmania (Viannia) naiffi infection suggest that its occurrence is uncommon and the reported cases present a benign clinical course and a good response to treatment. This study aimed to strengthen the clinical and epidemiological importance of L. (V.) naiffi in the Amazon Region (Manaus, state of Amazonas) and to report therapeutic failure in patients infected with this species. Thirty Leishmania spp samples isolated from cutaneous lesions were characterised by multilocus enzyme electrophoresis. As expected, the most common species was Leishmania (V.) guyanensis (20 cases). However, a relevant number ofL. (V.) naiffi patients (8 cases) was observed, thus demonstrating that this species is not uncommon in the region. No patient infected withL. (V.) naiffi evolved to spontaneous cure until the start of treatment, which indicated that this species may not have a self-limiting nature. In addition, two of the patients experienced a poor response to antimonial or pentamidine therapy. Thus, either ATL cases due to L. (V.) naiffi cannot be as uncommon as previously thought or this species is currently expanding in this region. PMID:26517660

  17. Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

    W.R. Faber; J. Wonders; A.J. Jensema; E. Chocholova; P.A. Kager

    2009-01-01

    Summary We describe a case of cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani, which was successfully treated with oral miltefosine. Given the increased prevalence of travelling, patients presenting with lymph-node enlargement should have leishmaniasis included in the differe

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

    Dostálová Anna

    2012-12-01

    Full Text Available Abstract 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.

  19. Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infantum in stray cats of İzmir, Turkey.

    Can, Hüseyin; Döşkaya, Mert; Özdemir, H Gökhan; Şahar, Esra Atalay; Karakavuk, Muhammet; Pektaş, Bayram; Karakuş, Mehmet; Töz, Seray; Caner, Ayşe; Döşkaya, Aysu Değirmenci; İz, Sultan Gülce; Özbel, Yusuf; Gürüz, Yüksel

    2016-08-01

    Leishmaniasis caused by more than 20 species of genus Leishmania is transmitted by the bite of infected phlebotomine sand flies. The studies on Leishmania infection in cats is very few in Turkey and therefore we aimed to screen stray cats living in city of İzmir located in western Turkey using nested PCR targeting kinetoplast DNA and serological techniques (ELISA and IFA). Leishmania DNA positive samples were also studied by ITS1 real time PCR. Whole blood and serum samples were obtained from stray cats (n: 1101) living in different counties of İzmir. In serological assays, a serum sample was considered positive in 1:40 dilution in IFA and for ELISA a serum sample was accepted positive when the absorbance value (AV) exceeded the mean AV + Standard Deviation (SD) of the negative control serum samples. According to the results, the seropositivity rates were 10.8% (119/1101) and 15.2% (167/1101) by in house ELISA and IFA, respectively. Among serology coherent samples, the seropositivity rate was 11.1% (116/1047) as detected by both assays after discordant samples (n: 54) were discarded. Of the 1101 stray cats, six (0.54%) were positive by nested PCR while only one of these six samples was positive by ITS1 real time PCR. During PCR, three controls designated as Leishmania infantum, Leishmania tropica, and Leishmania major were used for species identification. According to nested PCR results, L. tropica was identified in two cats (no.76 and 95). In another cat (no. 269), there were two bands in which one of them was well-matched with L. infantum and the other band had ∼850 bp size which does not match with any controls. Remaining three cats (no. 86, 514, and 622) also had the ∼850 bp atypical band size. ITS1 real time PCR detected L. tropica in only one cat (no. 622) which showed an atypical band size in nested PCR. These results indicated that three cats with only one atypical band (no. 86, 514, and 622) and the cat with mixed infection (no. 269) were

  20. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  1. Easy identification of leishmania species by mass spectrometry.

    Oussama Mouri

    2014-06-01

    Full Text Available BACKGROUND: Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management. METHODOLOGY: We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL. As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST at the National Reference Center for Leishmania. PRINCIPAL FINDINGS: Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level. CONCLUSIONS/SIGNIFICANCE: Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.

  2. Dissecting Leishmania infantum Energy Metabolism - A Systems Perspective.

    Abhishek Subramanian

    Full Text Available Leishmania infantum, causative agent of visceral leishmaniasis in humans, illustrates a complex lifecycle pertaining to two extreme environments, namely, the gut of the sandfly vector and human macrophages. Leishmania is capable of dynamically adapting and tactically switching between these critically hostile situations. The possible metabolic routes ventured by the parasite to achieve this exceptional adaptation to its varying environments are still poorly understood. In this study, we present an extensively reconstructed energy metabolism network of Leishmania infantum as an attempt to identify certain strategic metabolic routes preferred by the parasite to optimize its survival in such dynamic environments. The reconstructed network consists of 142 genes encoding for enzymes performing 237 reactions distributed across five distinct model compartments. We annotated the subcellular locations of different enzymes and their reactions on the basis of strong literature evidence and sequence-based detection of cellular localization signal within a protein sequence. To explore the diverse features of parasite metabolism the metabolic network was implemented and analyzed as a constraint-based model. Using a systems-based approach, we also put forth an extensive set of lethal reaction knockouts; some of which were validated using published data on Leishmania species. Performing a robustness analysis, the model was rigorously validated and tested for the secretion of overflow metabolites specific to Leishmania under varying extracellular oxygen uptake rate. Further, the fate of important non-essential amino acids in L. infantum metabolism was investigated. Stage-specific scenarios of L. infantum energy metabolism were incorporated in the model and key metabolic differences were outlined. Analysis of the model revealed the essentiality of glucose uptake, succinate fermentation, glutamate biosynthesis and an active TCA cycle as driving forces for parasite

  3. Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection.

    Tatiana R de Moura

    Full Text Available BACKGROUND: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. METHODS AND FINDINGS: A cDNA library from the Salivary Glands (SGs of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. CONCLUSIONS: We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

  4. Detección de Leishmania spp. en base al gen que codifica la proteína HSP20

    Ana M Montalvo

    2014-10-01

    Full Text Available Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20 para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR.Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (κ = 0,731, lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

  5. Detección de Leishmania spp. en base al gen que codifica la proteína HSP20

    Ana M. Montalvo

    Full Text Available Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20 para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR.Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (κ = 0,731, lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

  6. Post mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil.

    de Fátima Madeira, Maria; de O Schubach, Armando; Schubach, Tânia M P; Pereira, Sandro A; Figueiredo, Fabiano B; Baptista, Cibele; Leal, Cristianni A; Melo, Cíntia X; Confort, Eliame M; Marzochi, Mauro C A

    2006-06-15

    A parasitological study was conducted on 66 dogs seroreactive for Leishmania captured as a control measure of visceral leishmaniasis in the State of Rio de Janeiro, Brazil. Biological samples from different anatomical sites were collected during autopsy of the animals and cultured on biphasic medium (NNN/Schneider). The Leishmania isolates were characterized by isoenzyme electrophoresis. Leishmania was isolated from 80.3% of the animals: 12 animals with Leishmania (Viannia) braziliensis isolated exclusively from cutaneous lesions, 39 with L. (L.) chagasi isolated from different sites in the same animal, and 2 with simultaneous isolation of L. (V.) braziliensis from cutaneous lesions and L. (L.) chagasi from different sites. Isolation in culture revealed the absence of Leishmania parasites in 13 animals. The results obtained confirm the existence of mixed infections in dogs in Rio de Janeiro and indicate the need to complement the investigation of seroreactive dogs using methods for the parasitological diagnosis and identification of Leishmania species.

  7. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

  8. Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937

    Cerqueira Elúzio José Lima

    2003-01-01

    Full Text Available Quatro Equus asinus foram inoculados com promastigotas de Leishmania chagasi Cunha & Chagas, 1937 e acompanhados durante 12 meses através de: pesquisa de amastigotas em esfregaços e culturas de sangue periférico em fragmentos de tecido do lábio inferior, medula óssea, baço e fígado e de testes de ELISA e TRALd. Estes foram positivos nos 8º, 10º e 12º meses após a inoculação. O exame histopatológico pós necropsia, demonstrou discreto número de amastigotas no fígado de dois dos eqüídeos inoculados. Apesar de desafiados com elevado número de promastigotas, os animais não desenvolveram infecções patentes e não infectaram experimentalmente a vetora Lutzomya longipalpis. Os resultados induzem a acreditar que os eqüídeos são desprovidos de importância como reservatórios na cadeia de transmissão da leishmaniose visceral, embora sirvam como boa fonte de alimentação sangüínea e proliferação da vetora Lutzomyia longipalpis.

  9. Absence of Metalloprotease GP63 Alters the Protein Content of Leishmania Exosomes

    Kasra Hassani; Marina Tiemi Shio; Caroline Martel; Denis Faubert; Martin Olivier

    2014-01-01

    Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm ...

  10. Immunization with the cysteine proteinase Ldccys1 gene from Leishmania (Leishmania) chagasi and the recombinant Ldccys1 protein elicits protective immune responses in a murine model of visceral leishmaniasis.

    Ferreira, Josie Haydée L; Gentil, Luciana Girotto; Dias, Suzana Souza; Fedeli, Carlos Eduardo C; Katz, Simone; Barbiéri, Clara Lúcia

    2008-01-30

    The gene Ldccys1 encoding a cysteine proteinase of 30 kDa from Leishmania (Leishmania) chagasi, as well as the recombinant cysteine proteinase rLdccys1, obtained by cloning and expression of the Ldccys1 gene in the pHIS vector, were used to evaluate their ability to induce immune protective responses in BALB/c mice against L. (L.) chagasi infection. Mice were immunized subcutaneously with rLdccys1 plus Bacille Calmette Guerin (BCG) or Propionibacterium acnes as adjuvants or intramuscularly with a plasmid carrying the Ldccys1 gene (Ldccys1/pcDNA3) and CpG ODN as the adjuvant, followed by a booster with rLdccys1 plus CpG ODN. Two weeks after immunization the animals were challenged with 1 x 10(7) amastigotes of L. (L.) chagasi. Both immunization protocols induced significant protection against L. (L.) chagasi infection as shown by a very low parasite load in the spleen of immunized mice compared to the non-immunized controls. However, DNA immunization was 10-fold more protective than immunization with the recombinant protein. Whereas rLdccys1 induced a significant secretion of IFN-gamma and nitric oxide (NO), animals immunized with the Ldccys1 gene increased the production of IgG2a antibodies, IFN-gamma and NO. These results indicated that protection triggered by the two immunization protocols was correlated to a predominant Th1 response.

  11. Qualidade espermática de sêmen de cães naturalmente infectados por Leishmania sp: Semen quality of dogs naturally infected by Leishmania sp

    É. Labat

    2010-06-01

    Full Text Available Avaliaram-se alterações espermáticas associadas à infecção por leishmaniose no sêmen de cães naturalmente infectados, utilizando-se, durante oito semanas consecutivas, ejaculados de seis cães soronegativos e seis cães soropositivos. As amostras foram colhidas uma vez por semana e avaliadas quanto ao volume, concentração, motilidade, vigor, morfologia espermática, integridade da cromatina, avaliação simultânea da integridade da membrana plasmática, acrossoma e potencial mitocondrial. Concomitantemente foram dosadas a proteína total do plasma seminal e sanguíneo. A leishmaniose visceral causou aumento dos defeitos maiores e menores nos espermatozoides dos animais acometidos pelo estágio moderado a severo da doença. Em estágios mais avançados da enfermidade, a integridade das membranas acrossomal e plasmática foi afetada negativamente. Não foi possível estabelecer um critério quanto à avaliação do potencial mitocondrial. A incidência de alterações morfológicas nos animais acometidos não promoveu aumento de injurias à cromatina. Todos os animais com leishmaniose apresentaram hiperproteinemia do sêmen.The spermatic changes associated with the natural infection in dogs by Leishmania sp was evaluated during eight consecutive weeks, using ejaculates of six seronegative and six seropositive dogs. The samples were collected once a week and evaluated for volume, concentration, motility, vigor, sperm morphology, chromatin integrity, simultaneous evaluation of the plasmatic membrane integrity, acrosome, and mitochondrial potential. The total proteins of the seminal plasma and blood were measured. The visceral leishmaniasis caused increase of major and minor defects in spermatozoa of animals attacked by moderate to severe stages of the disease. In more advanced stages of the illness, the acrosomal and plasmatic membranes integrity was adversely affected. It was not possible to establish a pattern refering the evaluation of

  12. The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

    Campino, Lenea; Cortes, Sofia; Dionísio, Lídia; Neto, Luís; Afonso, Maria Odete; Maia, Carla

    2013-01-01

    Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World. PMID:23828004

  13. The FML (Fucose Mannose Ligand of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

    Claris B. Palatnik de Sousa

    1996-04-01

    Full Text Available The Fucose-Mannose Ligand (FML of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p O FML (Ligame de Fucose-Manose de Leishmania donovani é uma fração glicoproteica complexa. O seu potencial no diagnóstico da leishmaniose visceral humana foi testado com soros provenientes de Natal, Rio Grande do Norte, Brasil. O teste de FML-ELISA mostrou 100% de sensibilidade e 96% de especificidade, identificando pacientes com calazar declarado (p<0.001, comparados com soros normais e indivíduos com infecção subclínica. Mais de 20% dos sororreativos assimptomáticos desenvolveram a doença no prazo de 10 meses. Na análise de doadores de sangue, 5% de sororeativos, atingindo até 17% num único dia foram detectados. A glicoproteínaGP36 do FHL é reconhecida especificamente por soros de pacientes com calazar. O potencial imunoprotetor do FML no calazar experimental foi testado no modelo swiss albino em combinação com saponina pelas vias subcutâneas e/ou intraperitoneal seguido de desafio com 2x 10(7 amastigolas de Leishmania donovani. Um aumento de 80.0% na resposta de anticorpos específicos (p<0.001 e a redução de 85.5 % da carga parasitária no fígado (p<0.001 foi detectado nos animais vacinados com FML e saponina, independentemente da via de administração.

  14. Sandfly saliva of Lutzomyia ovallesi (Diptera: Psychodidae as a possible marker for the transmission of Leishmania in Venezuela Andes region

    E. Nieves , Y. Sánchez , H. Sánchez , M. Rondón, N. González & J. Carrero

    2012-03-01

    Full Text Available Background & objectives: The saliva of the Phlebotominae is highly immunogenic to the vertebrate host and isa determining factor in the Leishmania infection. The aim of this work was to study the saliva of Lutzomyiaovallesi as a possible risk marker for the transmission of Leishmania.Methods: Two populations of L. ovallesi from different geographical areas and subjected to different environmentalconditions were compared by geometric morphometry of the wings, by protein profile analysis of salivary glandsand by assessing the presence of anti-saliva protein in human sera confronted with laboratory L. ovallesi saliva.Results: The results showed differences in the isometric size and structure of the wings but no allometric effects.Protein profiles of salivary glands of both the L. ovallesi populations studied were found to be similar, based on11 protein bands with molecular weights ranging from 16 to 99 kDa. Anti-saliva antibodies were present inhuman sera, but human sera infected and uninfected with leishmaniasis could not be differentiated.Interpretation & conclusion: We conclude that the saliva of laboratory-reared L. ovallesi is representative ofthat of the wild population. It is suggested to study the presence of anti-saliva antibodies in other species ofsandflies and mosquitoes

  15. Towards an unbiased metabolic profiling of protozoan parasites : optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis

    t'Kindt, Ruben; Jankevics, Andris; Scheltema, Richard A.; Zheng, Liang; Watson, David G.; Dujardin, Jean-Claude; Breitling, Rainer; Coombs, Graham H.; Decuypere, Saskia; Kindt, Ruben t’

    2010-01-01

    Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of

  16. Prevalência de adultos infectados por Leishmania leishmania chagasi entre doadores de sangue do Hemocentro Regional de Montes Claros, Minas Gerais, Brasil Prevalence of adults infected with Leishmania (Leishmania chagasi among blood donors of the Hemominas Foundation in Montes Claros, Minas Gerais, Brazil

    Elaine V. R. Urias

    2009-01-01

    Full Text Available O objetivo deste trabalho foi estudar a prevalência de adultos infectados por L. L. chagasi entre doadores de sangue do Hemocentro Regional de Montes Claros/MG .Realizou-se estudo epidemiológico, transversal e quantitativo, no período de 16/09/08 a 13/11/08. Participaram da pesquisa 421 doadores aptos na triagem clínica, sendo realizada imunofluorescência indireta para L.L.chagasi. Aqueles que apresentaram resultados positivos foram submetidos ao teste rápido antígeno-específico para Leishmania donovani. A análise das variáveis gênero, faixa etária, procedência, número de doações, resultados sorológicos para leishmaniose e chagas, foi realizada pelos testes estatísticos qui-quadrado (x2, x2 com tendência linear e teste Fisher. Foi considerado o nível de significância de 5% (pThe objective of this work was to study the prevalence of adults infected by Leishmania (Leishmania chagasi among blood donors of the Hemominas Foundation in Montes Claros, Brazil. A cross-sectional, quantitative epidemiological study was performed of 421 blood donors from September 16 2008 to November 13 2008. The L. l. chagasi indirect immunofluorescence test (RIFI was utilized. Donors that presented with positive results in RIFI were retested using the fast immunochromatographic test (Trald. The gender, age, place of origin, number of donations, leishmania and chagas disease serum results were studied with statistical correlations being analyzed utilizing the chi-square test (x2, x2 with linear tendency and the Fisher test; a level of significance of 5% (p <0.05 was considered acceptable. The profile of the study sample was similar to the overall donor profile. The participants were mostly donors from urban areas (92.7%, living in Montes Claros (67.9%, men (61.3% and with ages between 18 and 29 years old. In relation to the serum results, 23 (5.5% were positive according to the RIFI however none of them were positive by the Trald. On comparing the

  17. Estudo in silico dos domínios protéicos presentes em uma amostra de 86 clusters de Leishmania chagasi

    Araújo de Queiroz, Rafael

    2005-01-01

    A Leishmania chagasi, um parasita protozoário, é o agente etiológico da Leishmaniose visceral nas Américas. Foi realizado um estudo do transcriptoma desse organismo utilizando-se 17.921 etiquetas de genes expressos (ESTs) gerados a partir de uma biblioteca de cDNA de promastigota. A montagem de todas as seqüências utilizando o CAP3 resultou em 1.449 clusters (utilizando 49% dos ESTs). Aproximadamente 35% dos clusters apresentaram similaridade com alguma seqüência depositada em bancos públicos...

  18. Drug resistance in Leishmania: similarities and differences to other organisms.

    Papadopoulou, B; Kündig, C; Singh, A; Ouellette, M

    1998-01-01

    The main line of defense available against parasitic protozoa is chemotherapy. Drug resistance has emerged however, as a primary obstacle to the successful treatment and control of parasitic diseases. Leishmania spp., the causative agents of leishmaniasis, have served as a useful model for studying mechanisms of drug resistance in vitro. Antimonials and amphotericin B are the first line drugs to treat Leishmania followed by pentamidine and a number of other drugs. Parasites resistant against all these classes of drugs have been selected under laboratory conditions. A multiplicity of resistance mechanisms has been detected, the most prevalent being gene amplification and transport mutations. With the tools now available, it should be possible to elucidate the mechanisms that govern drug resistance in field isolates and develop more effective chemotherapeutic agents.

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

    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.

  20. Acute cysticercosis favours rapid and more severe lesions caused by Leishmania major and Leishmania mexicana infection, a role for alternatively activated macrophages.

    Rodríguez-Sosa, Miriam; Rivera-Montoya, Irma; Espinoza, Arlett; Romero-Grijalva, Miriam; López-Flores, Roberto; González, Jorge; Terrazas, Luis I

    2006-08-01

    Parasitic helminths have developed complex mechanisms to modulate host immunity. In the present study we found that previous infection of mice with the cestode Taenia crassiceps favours parasitemia and induces larger cutaneous lesions during both Leishmania major and Leishmania mexicana co-infections. Analysis of cytokine responses into draining lymph nodes indicated that co-infection of T. crassiceps-Leishmania did not inhibit IFN-gamma production in response to Leishmania antigens, but significantly increased IL-4 production. Additionally, anti-Leishmania-specific IgG1 antibodies and total IgE increased in co-infected mice, whereas, IgG2a titers remained similar. Macrophages from Taenia-infected mice displayed increased mRNA transcripts of arginase-1, Ym1, and Mannose Receptor, as well as greater production of urea (all markers for an alternate activation state) compared to macrophages from Leishmania-infected mice. In contrast, lower mRNA transcripts for IL-12p35, IL-12p40, IL-23p19, and iNOS were detected in macrophages obtained from cestode-infected mice compared to uninfected and Leishmania-infected mice after LPS stimulation. The presence of cestode also generated impaired macrophage anti-leishmanicidal activity in vitro, as evidenced by the inability of these macrophages to prevent Leishmania growth compared to macrophages from uninfected mice. This was observed despite the fact that both groups of cells were exposed to IFN-gamma. Flow cytometry showed high IFN-gammaR expression on Taenia-induced macrophages. Thus, lack of response to IFN-gamma is not associated with the absence of its receptor. Our data suggest that cestode infection may favour Leishmania installation by inducing alternatively activated macrophages rather than inhibiting Th1-type responses.

  1. Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica.

    Al-Qahtani, Ahmed; Alkahtani, Saad; Kolli, Bala; Tripathi, Pankaj; Dutta, Sujoy; Al-Kahtane, Abdullah A; Jiang, Xiong-Jie; Ng, Dennis K P; Chang, Kwang Poo

    2016-04-01

    Photodynamic inactivation ofLeishmaniaspp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency againstLeishmania tropicapromastigotes and axenic amastigotesin vitro The uptake of these PCs by bothLeishmaniastages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation ofLeishmaniaspp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitizedLeishmania tropicastrains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm(-2) Quantitative fluorescence assays based on the loss of GFP/CFSE from liveLeishmania tropicashowed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.Leishmania tropicastrains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation ofLeishmaniaspp. for use as vaccines or vaccine carriers.

  2. The activity of ozonated olive oil against Leishmania major promastigotes

    Omid Rajabi; Ameneh Sazgarnia; Fatemeh Abbasi; Pouran Layegh

    2015-01-01

    Objective(s): Cutaneous Leishmaniasis is a common and endemic disease in Khorasan province in North-East of Iran. The pentavalant antimony (Sb V) is the mainstay of treatment that has many side effects and resistance to the drug has been reported. The microbicidal effect of ozone was proven in different microorganisms. Since there is no study in this respect and to achieve a low cost and effective treatment, we decided to evaluate the efficacy of ozone against promastigotes of Leishmania majo...

  3. Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major.

    Lye, Lon-Fye; Kang, Song Ok; Nosanchuk, Joshua D; Casadevall, Arturo; Beverley, Stephen M

    2011-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H(4)B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah(-) null mutant and an episomal complemented overexpressing derivative (pah-/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah(-) parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah(-) showed an increased sensitivity to Tyr deprivation, while the pah(-)/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah(-) showed no alterations in H(4)B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

  4. Testing of Experimental Compounds for Efficacy Against Leishmania

    1986-02-01

    leishmaniases, the group of diseases caused by protozoan parasites of the family Trypanosomatidae, genus Leishmania, are widely distributed throughout...species of phlebotomine flies and in most areas the leishmaniases are zoonoses with canines, rodents, or other mammals serving as reservoir hosts. These... parasites are a significant health hazard to humans in these areas. Visceral leishmaniasis, the most severe type, is endemic in many areas where

  5. A Randomized Controlled Trial of Local Heat Therapy Versus Intravenous Sodium Stibogluconate for the Treatment of Cutaneous Leishmania Major Infection

    2010-01-01

    Dermatotrophic Leishmania species such as L. major, L. tropica , and L. mexicana are thermosensitive with higher temperatures limiting amastigote replication...Wahid M, Bismullah M, Quinnell RJ, et al. (2005) Efficacy of thermotherapy to treat cutaneous leishmaniasis caused by Leishmania tropica in Kabul...A Randomized Controlled Trial of Local Heat Therapy Versus Intravenous Sodium Stibogluconate for the Treatment of Cutaneous Leishmania major

  6. Transmission of Leishmania in coffee plantations of Minas Gerais, Brazil

    Bruce Alexander

    2002-07-01

    Full Text Available Transmission of Leishmania was studied in 27 coffee plantations in the Brazilian State of Minas Gerais. Eighteen females and six males (11.6% of the people tested, aged between 7-65 gave a positive response to the Montenegro skin test. Awareness of sand flies based on the ability of respondents to identify the insects using up to seven predetermined characteristics was significantly greater among inhabitants of houses occupied by at least one Mn+ve individual. Five species of phlebotomine sand fly, including three suspected Leishmania vectors, were collected within plantations under three different cultivation systems. Four of these species i.e., Lu. fischeri (Pinto 1926, Lu. migonei (França 1920, Lu. misionensis (Castro 1959 and Lutzomyia whitmani (Antunes & Coutinho 1939 were collected in an organic plantation and the last of these was also present in the other two plantation types. The remaining species, Lu. intermedia (Lutz & Neiva 1912, was collected in plantations under both the "adensado" and "convencional" systems. The results of this study indicate that transmission of Leishmania to man in coffee-growing areas of Minas Gerais may involve phlebotomine sand flies that inhabit plantations.

  7. Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

    Oliva Glaucius

    2007-09-01

    Full Text Available Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT (EC 2.4.2.8 is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

  8. Leishmania (Viannia naiffi: rare enough to be neglected?

    Giselle Aparecida Fagundes-Silva

    2015-09-01

    Full Text Available In the Brazilian Amazon, American tegumentary leishmaniasis (ATL is endemic and presents a wide spectrum of clinical manifestations due, in part, to the circulation of at least seven Leishmaniaspecies. Few reports of Leishmania (Viannia naiffiinfection suggest that its occurrence is uncommon and the reported cases present a benign clinical course and a good response to treatment. This study aimed to strengthen the clinical and epidemiological importance of L. (V. naiffiin the Amazon Region (Manaus, state of Amazonas and to report therapeutic failure in patients infected with this species. Thirty Leishmania spp samples isolated from cutaneous lesions were characterised by multilocus enzyme electrophoresis. As expected, the most common species was Leishmania (V. guyanensis (20 cases. However, a relevant number ofL. (V. naiffi patients (8 cases was observed, thus demonstrating that this species is not uncommon in the region. No patient infected withL. (V. naiffievolved to spontaneous cure until the start of treatment, which indicated that this species may not have a self-limiting nature. In addition, two of the patients experienced a poor response to antimonial or pentamidine therapy. Thus, either ATL cases due to L. (V. naifficannot be as uncommon as previously thought or this species is currently expanding in this region.

  9. Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum

    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.

  10. Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection

    Alessandro Costagliola

    2016-01-01

    Full Text Available Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection.

  11. Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection.

    Costagliola, Alessandro; Piegari, Giuseppe; Otrocka-Domagala, Iwona; Ciccarelli, Davide; Iovane, Valentina; Oliva, Gaetano; Russo, Valeria; Rinaldi, Laura; Papparella, Serenella; Paciello, Orlando

    2016-01-01

    Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection.

  12. Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection

    Piegari, Giuseppe; Otrocka-Domagala, Iwona; Ciccarelli, Davide; Iovane, Valentina; Oliva, Gaetano; Russo, Valeria; Rinaldi, Laura; Papparella, Serenella; Paciello, Orlando

    2016-01-01

    Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection. PMID:27413751

  13. Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

    Dhekra Chaara; Anne-Laure Bañuls; Najoua Haouas; Loïc Talignani; Patrick Lami; Habib Mezhoud; Zoubir Harrat; Jean-Pierre Dedet; Hamouda Babba; Francine Pratlong

    2015-01-01

    Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples...

  14. Surveillance for antibodies to Leishmania spp. in dogs from Sri Lanka and India

    The global distribution of leishmaniasis is rapidly expanding into new geographic regions. Dogs are the primary reservoir hosts for human visceral leishmaniasis (VL) caused by infection with Leishmania infantum. Natural infections with other Leishmania species can occur in dogs, but their role as re...

  15. Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

    Martins, V T; Lage, D P; Duarte, M C; Costa, L E; Chávez-Fumagalli, M A; Roatt, B M; Menezes-Souza, D; Tavares, C A P; Coelho, E A F

    2016-02-01

    Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine.

  16. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice.

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2007-06-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.

  17. Inhibition by Dications of in vitro growth of Leishmania major and Leishmania tropica: causative agents of old world cutaneous leishmaniasis.

    Rosypal, Alexa C; Werbovetz, Karl A; Salem, Manar; Stephens, Chad E; Kumar, Arvind; Boykin, David W; Hall, James E; Tidwell, Richard R

    2008-06-01

    Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.

  18. Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania infantum chagasi-infected BALB/c mice

    Samanta Etel Treiger Borborema

    2013-08-01

    Full Text Available Pentavalent antimonials such as meglumine antimoniate (MA are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania infantum chagasi-infected mice. MA (Glucantime(r was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

  19. Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection by Leishmania (Leishmania) infantum

    Medeiros, Fernanda Alvarenga Cardoso; Gomes, Luciana Inácia; de Souza, Carolina Senra Alves; Mourão, Maria Vitória; Cota, Gláucia Fernandes; Marques, Letícia Helena dos Santos; Carneiro, Mariângela

    2017-01-01

    A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions. PMID:28163725

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

    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.

  1. Evidência de transmissão de leishmaniose visceral por Lutzomyia cruzi no município de Jaciara, Estado de Mato Grosso, Brasil

    Nanci Akemi Missawa

    2011-02-01

    Full Text Available INTRODUÇÃO: O município de Jaciara foi classificado em 2003, como área de transmissão de leishmaniose visceral em situação de surto. O trabalho objetivou determinar evidência de transmissão de Leishmania (Leishmania infantum chagasi por Lutzomyia cruzi no município de Jaciara, Estado de Mato Grosso, Brasil. MÉTODOS: O município situa-se a 127km da capital Cuiabá e é um importante ponto de atração para os praticantes de eco-turismo. Fêmeas de Lutzomyia cruzi, capturadas com armadilha de CDC, foram dissecadas para confirmação da espécie e armazenadas a -20ºC em pools de 10 indivíduos para extração de DNA, PCR genérico, RFLP específico e eletroforese em gel de poliacrilamida. RESULTADOS: O levantamento entomológico demonstrou a ocorrência abundante de Lutzomyia cruzi e ausência de Lutzomyia longipalpis, principal vetora da Leishmania (Leishmania infantum chagasi. Uma das três amostras analisadas apresentou banda característica de DNA de Leishmania (120pb em PCR genérico. Para confirmação da espécie de Leishmania, na RFLP utilizaram-se controles positivos de Leishmania (Leishmania amazonensis, Leishmania (Viannia braziliensis e Leishmania (Leishmania infantum chagasi digeridas com enzima de restrição HaeIII. Constatou-se um padrão de bandas semelhante à Leishmania (Leishmania infantum chagasi em uma amostra, confirmando a detecção de infecção natural de Leishmania (Leishmania infantum chagasi em Lutzomyia cruzi. CONCLUSÕES: A ocorrência de casos humanos e cães positivos, a presença da Lutzomyia cruzi e a ausência de Lutzomyia longipalpis, bem como a detecção de infecção natural por Leishmania (Leishmania infantum chagasi, evidenciam a participação de Lutzomyia cruzi na transmissão da leishmaniose visceral em Jaciara, Estado de Mato Grosso, Brasil.

  2. Clinical manifestations and genetic variation of Leishmania infantum and Leishmania tropica in Southern Turkey.

    Eroglu, Fadime; Koltas, Ismail S; Alabaz, Derya; Uzun, Soner; Karakas, Mehmet

    2015-07-01

    L. infantum was isolated from cutaneous leishmaniasis (CL) skin lesions in patients having no signs and symptoms of visceral leishmaniasis (VL). Similarly, L. tropica had previously been isolated from patients with VL in the absence of cutaneous lesions. It was not certain how visceralization occurred. Smears (207) and bone marrow samples (135) were taken from CL and VL-suspected patients, respectively. Microscopic examination, ITS1-PCR, RFLP and DNA sequencing for all samples were analyzed. The microscopic examination of smears was found to be 61.3% (127/207) in CL-suspected cases and bone marrow samples were found to be positive 8.8% (12/135) in VL-suspected cases. L. tropica 48.6% (72/148), L. infantum 35.8% (53/148), L. major 15.6% (23/148) in CL, and L. infantum 56.3% (18/32), L. donovani 31.2% (10/32), L. tropica 12.5% (4/32) in VL were found with PCR-RFLP. In addition, the DNA sequencing revealed a genetic variation in L. infantum (variants 1-3) and L. tropica (variants 1-5). We assume that the increased disease occurrence may have resulted from geographical expansion of disease, changing patterns of international travel, population migrations, non-immune people into endemic regions of infected people into non-endemic regions. In this study, L. infantum (variant 3) only in CL-patients and L. tropica (variant 2) only in VL-patients were identified. We hypothesize that genetic variation might play a role in the causation of CL and VL in southern Turkey and the genetic variants may differ according to the geographical location among Leishmania strains.

  3. Presence of Leishmania amastigotes in peritoneal fluid of a dog with leishmaniasis from Alagoas, Northeast Brazil Presença de formas amastigotas de Leishmania em fluido peritoneal de cão com leishmaniose proveniente de Alagoas, nordeste do Brasil

    Filipe Dantas-Torres

    2006-08-01

    Full Text Available The goal of this short communication is to report the uncommon presence of intracellular amastigotes of Leishmania in peritoneal fluid of a dog with leishmaniasis from Alagoas State, Brazil. Physical examination of an adult male rottweiler suspected to be suffering of leishmaniasis revealed severe loss of weight, ascitis, splenomegaly, moderately enlarged lymph nodes, onychogryphosis, generalized alopecia, skin ulcers on the posterior limbs, and conjunctivitis. Samples of bone marrow, popliteal lymph node, skin ulcer, and peritoneal fluid were collected and smears of each sample were prepared and stained with hematoxylin and eosin. Numerous amastigotes were detected in bone marrow, popliteal lymph node, and skin ulcer smears. Smears of peritoneal fluid revealed the unusual presence of several free and intracellular amastigotes of Leishmania. Future studies are needed to determine whether the cytology of ascitic fluid represents a useful tool for diagnosis Leishmania infection in ascitic dogs, particularly in those living in areas where canine leishmaniasis is enzootic.O objetivo desta comunicação é descrever a presença incomum de formas amastigotas de Leishmania em fluido peritoneal de um cão com leishmaniose proveniente do Estado de Alagoas, nordeste do Brasil. O exame físico de um cão macho adulto da raça rottweiler, apresentando suspeita de leishmaniose, revelou perda de peso severa, esplenomegalia, linfonodos moderadamente aumentados, ascite, onicogrifose, alopecia generalizada, conjuntivite e presença de lesões cutâneas ulceradas localizadas nos membros posteriores. Foram coletadas amostras de medula óssea, linfonodo poplíteo, fluido peritoneal e úlcera cutânea. A partir das amostras, foram elaborados esfregaços, os quais foram corados pela hematoxilina e eosina. Inúmeras formas amastigotas foram detectadas na medula óssea, linfonodo poplíteo e úlcera cutânea. Esfregaços de fluido peritoneal revelaram a presença, n

  4. A Molecular Dynamics Study of Allosteric Transitions in Leishmania mexicana Pyruvate Kinase.

    Naithani, Ankita; Taylor, Paul; Erman, Burak; Walkinshaw, Malcolm D

    2015-09-15

    A comparative molecular dynamics analysis of the pyruvate kinase from Leishmania mexicana is presented in the absence and presence of the allosteric effector fructose 2,6-bisphosphate. Comparisons of the simulations of the large 240 kDa apo and holo tetramers show that binding of fructose 2,6-bisphosphate cools the enzyme and reduces dynamic movement, particularly of the B-domain. The reduced dynamic movement of the holo form traps the pyruvate kinase tetramer in its enzymatically active state with the B-domain acting as a lid to cover the active site. The simulations are also consistent with a transition of the mobile active-site α6' helix, which would adopt a helical conformation in the active R-state and a less structured coil conformation in the inactive T-state. Analysis of the rigid body motions over the trajectory highlights the concerted anticorrelated rigid body rocking motion of the four protomers, which drives the T to R transition. The transitions predicted by these simulations are largely consistent with the Monod-Wyman-Changeux model for allosteric activation but also suggest that rigidification or cooling of the overall structure upon effector binding plays an additional role in enzyme activation.

  5. Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as vectors of Leishmania infantum in Tbilisi, Georgia.

    Ekaterina Giorgobiani

    Full Text Available A survey of potential vector sand flies was conducted in the neighboring suburban communities of Vake and Mtatsminda districts in an active focus of visceral Leishmaniasis (VL in Tbilisi, Georgia. Using light and sticky-paper traps, 1,266 male and 1,179 female sand flies were collected during 2006-2008. Five Phlebotomus species of three subgenera were collected: Phlebotomus balcanicus Theodor and Phlebotomus halepensis Theodor of the subgenus Adlerius; Phlebotomus kandelakii Shchurenkova and Phlebotomus wenyoni Adler and Theodor of the subgenus Larroussius; Phlebotomus sergenti Perfil'ev of the subgenus Paraphlebotomus. Phlebotomus sergenti (35.1% predominated in Vake, followed by P. kandelakii (33.5%, P. balcanicus (18.9%, P. halepensis (12.2%, and P. wenyoni (0.3%. In Mtatsminda, P. kandelakii (76.8% comprised over three fourths of collected sand flies, followed by P. sergenti (12.6%, P. balcanicus (5.8%, P. halepensis (3.7%, and P. wenyoni (1.1%. The sand fly season in Georgia is exceptionally short beginning in early June, peaking in July and August, then declining to zero in early September. Of 659 female sand flies examined for Leishmania, 12 (1.8% specimens without traces of blood were infected including 10 of 535 P. kandelakii (1.9% and two of 40 P. balcanicus (5.0%. Six isolates were successfully cultured and characterized as Leishmania by PCR. Three isolates from P. kandelakii (2 and P. balcanicus (1 were further identified as L. infantum using sequence alignment of the 70 kDa heat-shock protein gene. Importantly, the sand fly isolates showed a high percent identity (99.8%-99.9% to human and dog isolates from the same focus, incriminating the two sand fly species as vectors. Blood meal analysis showed that P. kandelakii preferentially feeds on dogs (76% but also feeds on humans. The abundance, infection rate and feeding behavior of P. kandelakii and the infection rate in P. balcanicus establish these species as vectors in the Tbilisi

  6. Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection.

    Luz, Nívea F; Andrade, Bruno B; Feijó, Daniel F; Araújo-Santos, Théo; Carvalho, Graziele Q; Andrade, Daniela; Abánades, Daniel R; Melo, Enaldo V; Silva, Angela M; Brodskyn, Cláudia I; Barral-Netto, Manoel; Barral, Aldina; Soares, Rodrigo P; Almeida, Roque P; Bozza, Marcelo T; Borges, Valéria M

    2012-05-01

    Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

  7. Serological Evidence of Infection by Leishmania (Leishmania) infantum (Synonym: Leishmania (Leishmania) chagasi) in Free-Ranging Wild Mammals in a Nonendemic Region of the State of São Paulo, Brazil.

    Paiz, Laís Moraes; Fornazari, Felipe; Menozzi, Benedito Donizete; Oliveira, Gabriela Capriogli; Coiro, Carla Janeiro; Teixeira, Carlos Roberto; da Silva, Valdinei Moraes Campanucci; Donalisio, Maria Rita; Langoni, Helio

    2015-11-01

    Concerns about the interface between wildlife, domestic animals, and humans in the transmission of visceral leishmaniasis (VL) have been growing due to natural or anthropogenic environmental changes. In this context, investigations of the infection in wild mammals are important to assess their exposure to the vector and the parasite. A study of anti-Leishmania (Leishmania) infantum antibodies was carried out using the direct agglutination test (DAT) on 528 free-ranging wild mammals of 38 species from the region of Botucatu, state of São Paulo, Brazil, a municipality that has no records of the vector or of human or canine autochthony. Antibodies were detected, with a cutoff of 1:320, in 9/528 (1.7%; 95% confidence interval [CI] 0.6-2.8%) mammals of the species Callithrix jacchus, Lepus europaeus, Sphiggurus villosus, Nasua nasua, Eira barbara, and Galictis cuja, with high titers (≥1280) for the last three. These three are little-studied species, and previous records of the detection of anti-Leishmania spp. antibodies in Brazil exist only for coatis (N. nasua), whereas worldwide, infection by L. (L.) infantum has been confirmed only in hares (Le. europaeus). On the other hand, opossums and canids, the species most commonly reported to be naturally infected by L. (L.) infantum, were not seropositive. Fifty-eight (58/528; 10.9%) mammals were found to have antibody titers ranging from 20 to 160 and were not included among the seropositive animals due to the adopted cutoff. However, the possibility of infection in these animals should not be discarded, because there is no standard cutoff point for the different wild species. Our findings indicate the need for investigations into the exact role of the seropositive species in the epidemiology of VL and for effective epidemiological surveillance to prevent its expansion, because even in regions where there are no records of canine or human autochthonous cases, there may be parasite circulation among wild mammals.

  8. In vitro activity of amphotericin B cochleates against Leishmania chagasi

    Aretha Molina Sesana; Renata Monti-Rocha; Solange Alves Vinhas; Carlos Gustavo Morais; Reynaldo Dietze; Elenice Moreira Lemos

    2011-01-01

    Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB). In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vi...

  9. [Leishmania tropica in Morocco. IV--Intrafocal enzyme diversity].

    Pratlong, F; Rioux, J A; Dereure, J; Mahjour, J; Gallego, M; Guilvard, E; Lanotte, G; Perieres, J; Martini, A; Saddiki, A

    1991-01-01

    Ecoepidemiological analysis of a Moroccan focus of leishmaniasis caused by Leishmania tropica revealed considerable enzymatic diversity. Seven zymodemes belonging to the complex were identified in 149 strains isolated from humans, dogs, and the vector Phlebotomus sergenti. Three distinct subgroups were identifiable, two of which were in turn, composed of three "small variant" zymodemes. The diversity appears to be related to the age of the focus, which may have allowed colonization by zymodemes of different geographic origins. Diversification into "small variants" is apparently the result of recent mutation, possibly associated with genetic exchange.

  10. Aluminium: a natural adjuvant in Leishmania transmission via sand flies?

    Maingon, Rhayza; Khela, Amandeep; Sampson, Christopher; Ward, Richard; Walker, Karen; Exley, Christopher

    2008-11-01

    Genetically identical Leishmania chagasi/infantum parasites cause both atypical cutaneous leishmaniasis and visceral leishmaniasis. In this report we have tested the first part of a hypothesis that states that the form of this disease that is manifested depends upon the adjuvant-like activity of aluminium of dietary origin accumulated in the salivary gland of the sand fly vector. In sand flies fed aluminium-supplemented sucrose we have used histochemistry to qualitatively identify aluminium in their salivary glands and graphite furnace atomic absorption spectrometry to quantify the aluminium content of dissected salivary glands. Aluminium may be acting as a natural adjuvant in some forms of leishmaniasis.

  11. 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

    Yang Bin-Bin

    2013-02-01

    Full Text Available Abstract 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

  12. Phylogenetic position of Leishmania isolates from Khyber Pakhtunkhwa province of Pakistan.

    Khan, Nazma Habib; Messenger, Louisa A; Wahid, Sobia; Sutherland, Colin J

    2016-08-01

    Several species of the genus Leishmania are causative agents of cutaneous leishmaniasis in Pakistan. This study aimed to determine phylogenetic placement of Leishmania species causing cutaneous leishmaniasis in Khyber Pakhtunkhwa province, Pakistan (34 Leishmania tropica, 3 Leishmania infantum), in-relation to species from other geographical areas using gene sequences encoding cytochrome b (cytb) and internal transcribed spacer 2 (its2). Based on cytochrome b sequence analysis, L. tropica strains from Pakistan and other geographical regions were differentiated into two genotype groups, A and B. Within the province, five distinct L. tropica genotypes were recognized; two in group A, three in group B. Two L. infantum isolates from the province were closely associated with both Afro-Eurasian and American species of the Leishmania donovani complex, including Leishmania chagasi, L. infantum and L. donovani from Sudan and Ethiopia; while a third L. infantum isolate could not be differentiated from visceralizing Kenyan and Indian L. donovani. We observed apposite phylogenetic placement of CL-causing L. tropica and L. infantum from Khyber Pakhtunkhwa. Affinities ascribed to Leishmania spp. From the region are valuable in tracing potential importation of leishmaniasis.

  13. First molecular detection of Leishmania tarentolae-like DNA in Sergentomyia minuta in Spain.

    Bravo-Barriga, Daniel; Parreira, Ricardo; Maia, Carla; Blanco-Ciudad, Juan; Afonso, Maria Odete; Frontera, Eva; Campino, Lenea; Pérez-Martín, Juan Enrique; Serrano Aguilera, Francisco Javier; Reina, David

    2016-03-01

    Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

  14. Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis.

    Telleria, Erich L; Sant'Anna, Maurício R V; Ortigão-Farias, João R; Pitaluga, André N; Dillon, Viv M; Bates, Paul A; Traub-Csekö, Yara M; Dillon, Rod J

    2012-04-13

    Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.

  15. Caspar-like Gene Depletion Reduces Leishmania Infection in Sand Fly Host Lutzomyia longipalpis*

    Telleria, Erich L.; Sant'Anna, Maurício R. V.; Ortigão-Farias, João R.; Pitaluga, André N.; Dillon, Viv M.; Bates, Paul A.; Traub-Csekö, Yara M.; Dillon, Rod J.

    2012-01-01

    Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on Gram-negative and Gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly. PMID:22375009

  16. A soro-aglutinação das Leishmanias Agglutination of Leishmanias

    A. M. da Cunha

    1942-01-01

    Full Text Available The first agglutination experiments (Tables 1 and 2 showed that the serum obtained with any one strain of Leishmania, agglutinates all the others even of another species. This finding reveals the existence of a common antigen. However as the titre of agglutination did not permit a sharp differentiation of species we tried the adsorption method. The first adsorption tests made demonstrated differences in antigenic constitution between a strain of. L. donovani on one hand and strains of L. tropica or L. brasiliensis on the other. Further experiments in which L. chagasi was tested against the other species revealed that the former was antigenically different from the others. These tests were performed by adsorbing an anti-chagasi serum with organisms belonging to the other species or, conversely, adsorbing with L. chagasi sera prepared against the other species (See Tables 9 to 24. On the other hand, the adsorption of a serum prepared against one strain of l. chagasi by another of the same species showed that they had identifical antigenie constitution. These findings suggested the possibility of separating different species of Leishmania by this method. However, tests to separate the other species from one to another gave inconclusive results. (See Tables 27 to 35. It was soon observed that all the strains of L. chagasi were of recent isolation while all the others had been maintained in artificial culture media for a long time. We were led to believe that this condition was responsible for the differences in behaviour encountered. Accordingly, recently isolated strains of L. brasiliensis and L. donovani were tested and shown to be antigenically similar to strains of L. chagasi also recently isolated. The conclusion may be drawn that all strains have the same antigenic constitution when freshly isolated. It has been noted that when a serum which has been prepared against a freshly isolated is adsorbed with an old strain, the amount of agglutinins

  17. Complete conservation of an immunogenic gene (lcr1 in Leishmania infantum and Leishmania chagasi isolated from Iran, Spain and Brazi

    H. Mahmoudzadeh-Niknam , F. Abrishami , M. Doroudian , M. Moradi , M.H. Alimohammadian , P. Parvizi

    2010-12-01

    Full Text Available Background & objectives: Kala-azar is the visceral and most severe form of leishmaniasis thatleads to death if untreated. The causative agents of visceral leishmaniasis (VL are members ofLeishmania (L. donovani complex which includes L. chagasi and L. infantum. Genome sequenceshave raised the question whether L. chagasi and L. infantum are synonymous or different. Thisquestion has important implications for clinical and epidemiological studies, evaluation of vaccinesand drugs, and disease control. LCR1 is an immunogenic molecule discovered from L. chagasiwith potential as a component of a Leishmania subunit vaccine. If this protein has potentials forbeing used in a vaccine or diagnostic testing, there should be little variability in this moleculebetween L. infantum isolates from diverse geographic regions. The aim of this study was to determinewhether lcr1 of an Iranian strain of L. infantum was identical to lcr1 of both L. infantum strainfrom a different geographic region (Spain and that of an L. chagasi isolate from Brazil.Methods: L. infantum isolated from an Iranian kala-azar patient was studied. Lcr1 from this isolatewas PCR amplified, cloned, and studied by restriction digest analysis and sequencing.Results: The sequences of lcr1 of the Iranian L. infantum were completely identical at nucleotidelevel to lcr1 sequences of both the Spanish L. infantum and the Brazilian L. chagasi strains.Conclusion: Complete conservation of the DNA sequence encoding for LCR1 molecule betweengeographically distinct Leishmania species adds credibility to the potential for LCR1 as a componentof a subunit vaccine and diagnostic test for kala-azar.

  18. Presence of amastigotes in the central nervous system of hamsters infected with Leishmania sp. Presença de amastigotas em sistema nervoso central de hamster infectado com Leishmania sp.

    Elisangela de Oliveira

    2011-06-01

    Full Text Available Visceral leishmaniasis (VL is a severe chronic disease caused by Leishmania (Leishmania infantum chagasi. Better knowledge on the effects caused by this disease can help develop adequate clinical management and treatment. Parasitological and immunohistochemical studies were performed golden hamsters Mesocricetus auratus infected with bone marrow from individuals with VL in the State of Mato Grosso do Sul, central-west Brazil. The effects of parasitism in the spleen, liver, kidneys, lungs, heart and brain of the animals were examined. Eighteen hamsters were inoculated intraperitoneally, and six healthy animals were used as negative controls. The animals were kept in the animal house and checked for clinical signs. Specimens of each organ were examined for the presence of amastigotes. Immunohistochemical technique was performed in all brain specimens and organs negative on the direct examination of parasites. Direct examination of amastigotes was positive in the spleen and liver of all infected animals; 33.3% showed the parasite in the kidneys and lungs, and 16.7% in the heart. Parasitic forms were seen in 83.3% (15/18 of the brain examined. Immunohistochemistry confirmed the results of the direct examination, except in two specimens of lung tissue and in the brain specimens. Other studies are needed to further clarify the effect of the parasite in the central nervous system.A leishmaniose visceral (LV é uma doença crônica grave, causada pelo parasito Leishmania (Leishmania infantum chagasi. Esclarecer as alterações provocadas pela doença é fundamental para que se adotem condutas clínicas e de tratamento adequadas. Com o objetivo de analisar a infecção experimental em hamsters da linhagem golden, Mesocricetus auratus, infectados com tecido de medula óssea de pacientes com LV no Estado de Mato Grosso do Sul, foram realizados estudos parasitológicos e de imunomarcação. Foi verificada a distribuição do parasitismo no baço, f

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

    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.

  20. The Multiple Forms of Leishmania Major in BALB/C Mice Lung in Iran

    M Jafari

    2012-06-01

    Full Text Available Cutaneous leishmaniasis is one of the most important parasitic diseases, which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days, the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected.

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

    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.

  2. Infecção natural de Lutzomyia (Lutzomyia longipalpis (Diptera: Psychodidae por Leishmania infantum chagasi em flebotomíneos capturados no município de Janaúba, Estado de Minas Gerais, Brasil

    Érika Monteiro Michalsky

    2011-02-01

    Full Text Available INTRODUÇÃO: A leishmaniose visceral tem sido notificada em quase todos os estados do Brasil, e principalmente no norte de Minas Gerais, onde a doença é endêmica. Este estudo visou detectar a infecção natural de Lutzomyia longipalpis e identificar através da técnica de PCR/RFLP a espécie de Leishmania encontrada nos flebotomíneos do município de Janaúba. MÉTODOS: Utilizando-se armadilhas luminosas, foram capturadas 1.550 fêmeas de L. longipalpis, que agrupadas em pool de 10 exemplares foram submetidas à extração e amplificação de DNA, através das técnicas de PCR genérico e cacofonia. RESULTADOS: Dos 155 pools, seis apresentaram-se positivos para Leishmania sp., sendo a taxa de infecção do município de 3,9%. Através da PCR/RFLP determinou-se que o padrão de digestão das amostras positivas foi semelhante ao da cepa referência Leishmania chagasi (MHOM/BR/74/PP75. CONCLUSÕES: A detecção de infecção natural associada a estudos sobre a epidemiologia da LV sugere que L. longipalpis esteja envolvida na transmissão de L. infantum chagasi em Janaúba, principalmente nas áreas de intensa transmissão de LV.

  3. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    Patricia Barrera

    2013-01-01

    Full Text Available 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 the host’s cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc, dehydroleucodine (DhL, psilostachyin (Psi, and, at lesser extent, psilostachyin C (Psi C is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.

  4. Aurapten, a coumarin with growth inhibition against Leishmania major promastigotes

    Napolitano H.B.

    2004-01-01

    Full Text Available Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compund was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg of shows significant growth inhibition with an LD50 of 30 µM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.

  5. Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

    Wanderley J.L.M.

    2005-01-01

    Full Text Available Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS exposure and its recognition by a specific receptor (PSR were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

  6. Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

    Sarah A C Falcão

    2015-03-01

    Full Text Available BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

  7. Natural Leishmania infection of Lutzomyia spp. in Peru.

    Perez, J E; Ogusuku, E; Inga, R; Lopez, M; Monje, J; Paz, L; Nieto, E; Arevalo, J; Guerra, H

    1994-01-01

    Natural infection of Lutzomyia spp. with Leishmania was studied with the aid of the polymerase chain reaction (PCR) in Chaute, Lima, Perú, a locality endemic for Andean cutaneous leishmaniasis (uta). The PCR, with primers specific for the L. braziliensis complex, was applied to sandfly pools. Sandflies were sampled from April 1990 to May 1991 with CDC light traps in homes, and from near homes with a Shannon trap using protected human bait. Lu. verrucarum (4 pools) and Lu. peruenis (2 pools) from the anthropophilic collections, and Lu. verrucarum (2 pools) from indoors were found to be infected with Leishmania. The majority of infected sandflies were recorded mainly in April 1991 (4 pools), coinciding with the highest sandfly densities and the maximum number of new cases of uta (7). Non-infected sandflies were found from May to October 1990 and January to March 1991. Thus, these 2 sandfly species play a role in the spread of leishmaniasis among humans and other animals in Chaute.

  8. Proteinases as virulence factors in Leishmania spp. infection in mammals

    Silva-Almeida Mariana

    2012-08-01

    Full Text Available Abstract Leishmania parasites cause human tegumentary and visceral infections that are commonly referred to as leishmaniasis. Despite the high incidence and prevalence of cases, leishmaniasis has been a neglected disease because it mainly affects developing countries. The data obtained from the analysis of patients’ biological samples and from assays with animal models confirm the involvement of an array of the parasite’s components in its survival inside the mammalian host. These components are classified as virulence factors. In this review, we focus on studies that have explored the role of proteinases as virulence factors that promote parasite survival and immune modulation in the mammalian host. Additionally, the direct involvement of proteinases from the host in lesion evolution is analyzed. The gathered data shows that both parasite and host proteinases are involved in the clinical manifestation of leishmaniasis. It is interesting to note that although the majority of the classes of proteinases are present in Leishmania spp., only cysteine-proteinases, metalloproteinases and, to a lesser scale, serine-proteinases have been adequately studied. Members from these classes have been implicated in tissue invasion, survival in macrophages and immune modulation by parasites. This review reinforces the importance of the parasite proteinases, which are interesting candidates for new chemo or immunotherapies, in the clinical manifestations of leishmaniasis.

  9. Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum.

    Brotherton, Marie-Christine; Bourassa, Sylvie; Légaré, Danielle; Poirier, Guy G; Droit, Arnaud; Ouellette, Marc

    2014-08-01

    Amphotericin B (AmB) in its liposomal form is now considered as either first- or second-line treatment against Leishmania infections in different part of the world. Few cases of AmB resistance have been reported and resistance mechanisms toward AmB are still poorly understood. This paper reports a large-scale comparative proteomic study in the context of AmB resistance. Quantitative proteomics using stable isotope labeling of amino acids in cell culture (SILAC) was used to better characterize cytoplasmic and membrane-enriched (ME) proteomes of the in vitro generated Leishmania infantum AmB resistant mutant AmB1000.1. In total, 97 individual proteins were found as differentially expressed between the mutant and its parental sensitive strain (WT). More than half of these proteins were either metabolic enzymes or involved in transcription or translation processes. Key energetic pathways such as glycolysis and TCA cycle were up-regulated in the mutant. Interestingly, many proteins involved in reactive oxygen species (ROS) scavenging and heat-shock proteins were also up-regulated in the resistant mutant. This work provides a basis for further investigations to understand the roles of proteins differentially expressed in relation with AmB resistance.

  10. Genetic diversity of Leishmania infantum field populations from Brazil

    Marcela Segatto

    2012-02-01

    Full Text Available Leishmania infantum (syn. Leishmania chagasi is the etiological agent of visceral leishmaniasis (VL in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT, random amplification of polymorphic DNA (RAPD and simple sequence repeats-polymerase chain reaction (SSR-PCR, were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

  11. Structure of the SAS-6 cartwheel hub from Leishmania major.

    van Breugel, Mark; Wilcken, Rainer; McLaughlin, Stephen H; Rutherford, Trevor J; Johnson, Christopher M

    2014-01-01

    Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001.

  12. Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

    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.

  13. Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major

    Bisacchi, Davide [Bioinformatics and Structural Proteomics, IST-National Cancer Research Institute, Genova (Italy); Zhou, Yao; Rosen, Barry P.; Mukhopadhyay, Rita [Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan (United States); Bordo, Domenico, E-mail: domenico.bordo@istge.it [Bioinformatics and Structural Proteomics, IST-National Cancer Research Institute, Genova (Italy)

    2006-10-01

    LmACR2 from L. major is the first rhodanese-like enzyme directly involved in the reduction of arsenate and antimonate to be crystallized. Diffraction data have been collected to 1.99 Å resolution using synchrotron X-rays. Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3{sub 1}21/P3{sub 2}21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 Å and a = b = 111.0, c = 175.6 Å, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 Å.

  14. The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan Province, Iran.

    Spotin, Adel; Rouhani, Soheila; Parvizi, Parviz

    2014-01-01

    Cutaneous leishmaniasis has various phenotypic aspects consisting of polymorphic amastigotes with different genetic ranges. Samples were collected from suspected patients of Khuzestan province. Prepared smears were stained, scaled, and measured using ocular micrometer. The Cyt b, ITS-rDNA, and microsatellite genes of Leishmania were amplified and Leishmania species were identified by molecular analyses. Of 150 examined suspected patients, 102 were identified to Leishmania species (90 L. major, nine L. tropica, and three unidentified). The amastigotes of 90 L. major had regular and different irregular shapes within three clinical lesions with no and/or low genetic diversity. Three haplotypes of Cyt b of L. major were found but no variation was observed using ITS-rDNA gene. Interesting findings were that all nine L. tropica had regular amastigote shapes with more genetic variations, also a patient which had coinfection of L. major, L. tropica, and Crithidia. At least two L. major and L. tropica were identified in suspected patients of the regions. Different irregular amastigotes' shapes of L. major can be explained by various reservoir hosts and vectors. In contrast, more molecular variations in L. tropica could be justified by genetic characters. Unidentified Leishmania could be mixed pathogens or nonpathogens with mammals' Leishmania or Crithidia.

  15. Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

    Chaara, Dhekra; Bañuls, Anne-Laure; Haouas, Najoua; Talignani, Loïc; Lami, Patrick; Mezhoud, Habib; Harrat, Zoubir; Dedet, Jean-Pierre; Babba, Hamouda; Pratlong, Francine

    2015-12-01

    Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

  16. The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

    Mahmoudzadeh-Niknam, Hamid; Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-02-01

    Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

  17. Comparison of Leishmania killicki (syn. L. tropica and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

    Dhekra Chaara

    2015-12-01

    Full Text Available Leishmania (L. killicki (syn. L. tropica, which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica (from Tunisia, Algeria and Libya and 113 L. tropica specimens (all from Morocco were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE and MultiLocus Microsatellite Typing (MLMT approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica in space and time. Comparison of the L. tropica (exclusively from Morocco and L. killicki (syn. L. tropica population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

  18. Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused bY Leishmania (Leishmania mexicana

    Erika Ivett Sosa-Bibiano

    2012-06-01

    Full Text Available There is not an experimental model of localized cutaneous leishmaniasis (LCL caused by Leishmania (Leishmania mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L. mexicana. A total of 54 P. yucatanicus (groups of 18 were inoculated with 1x10(6 promastigotes of L. (L. mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18 and in 11.11% (2/18 P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100% biopsies of euthanized P. yucatanicus at three (n = 5 and six (n = 2 months, respectively. These results resembled clinical and histological features caused by L. (L. mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.

  19. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    , identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some...

  20. Actividad in vitro de la mezcla de alcaloides de Ervatamia coronaria (Jacq Staff. Apocynaceae sobre amastigotes de Leishmania braziliensis

    Amanda Moreno Rodríguez

    2008-09-01

    Full Text Available A leishmaniose é considerada uma importante causa de morbidade e mortalidade a nível mundial, principalmente nos países tropicais. As formas cutânea e mucocutânea são causadas, entre outras espécies, por Leishmania braziliensis. Na procura de compostos leishmanicidas de origem natural, foi estudada a atividade da mistura de alcalóides de Ervatamia coronaria (Apocynaceae contra amastigotas de L. braziliensis em 6 concentrações diferentes (1, 10, 20, 25, 50 e 100 µg/mL. Foram tratados macrófagos de ratos da linha J774, infectados com promastigotas de L. braziliensis, com a mistura de alcalóides 1 hora após-infecção e diariamente por 3 dias sem mudança de meio. As experiências de citotoxicidade foram efetuadas sobre os macrófagos com azul tripam. Todos os cultivos foram feitos de forma triplicada e os grupos de controle não foram submetidos à mistura de alcalóides. Foi obtido que o composto adicionado exerce atividade doses/dependente sobre a parasita. No entanto, as concentrações mais altas (50 e 100 µg/mL, adicionado durante 3 dias, mostraram os maiores índices de infecção, provavelmente devido a diminuição no número de macrófagos, sobre os quais não foi observado efeito tóxico do tratamento durante 24 horas DL50/24h = 233,52 µg/mL. Os resultados dessa pesquisa revelaram uma nova atividade farmacológica de alcalóides da espécie Ervatamia coronaria sobre a forma amastigota de Leishmania braziliensis, com IC50 = 2,6 e 12,4 µg/mL sem mostrar toxicidade sobre a célula hospedeira.

  1. ¿ESTÁN LOS CLC DE LEISHMANIA ASOCIADOS CON LA ADAPTACIÓN DEL PARÁSITO A CAMBIOS DE pH Y/O DE OSMOLARIDAD?

    Marcela Camacho Navarro

    2016-03-01

    Full Text Available Leishmania es un protozoario patógeno, que transita durante su ciclo de vida desde el intestino de su vector hasta un compartimientofagolisosomal al interior de su célula hospedera, el macrófago. Durante este recorrido el parásito está expuesto a ambientes quecambian en temperatura, pH y presumiblemente osmolaridad. Para su supervivencia, Leishmania debe hacer ajustes para adaptarsey la expresión de canales de cloruro ha sido implicada en estos. Basándose en los antecedentes de nuestro grupo de investigación,que registró corrientes de cloruro voltaje dependientes luego de la inyección de mRNA de promastigotes de Leishmania en ovocitos deXenopues laevis, se postula que éstas son el resultado de la actividad de proteínas CLC. Se mencionan algunas bases moleculares de laadaptación de este párasito con énfasis en transportadores, regulación de calcio, pH y osmolaridad. Con base en datos del grupose argumenta que la osmolaridad de la vacuola parasitófora es alta. Además se da evidencia de la transcripción en promastigotes detres genes que codificarían CLC putativos, y se postulan funciones potenciales que llevarían a cabo en los dos estadios del parásito. AbstractLeishmania is a protozoan pathogen that transits during its lifecycle from the gut of the vector to a phagolysosomal compartmentwithin its host cell, the macrophage. During this journey, the parasite is exposed to environments that change in temperature, pHand osmolarity. For their survival, Leishmania must make adjustments to adapt and expression of chloride channels has been involved in these. Based on our research group results: reported voltage-dependent chloride currents after mRNA injection of promastigotes of Leishmania in Xenopus laevis oocytes, it is postulated that these are the result of CLC protein activity. Some molecular bases of adaptation of this parasite are mentioned with emphasis on regulation of calcium, pH and osmolarity. Based on data from our

  2. Leishmania Exosomes Deliver Preemptive Strikes to Create an Environment Permissive for Early Infection

    Judith Maxwell Silverman

    2012-01-01

    Full Text Available Herein, we review evidence supporting a role for leishmania exosomes during early infection. We suggest a model in which leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, ; wild-type (WT exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α by leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The leishmania proteins GP63 and elongation factor-1α (EF-1α are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein tyrosine phosphatases (PTPs. Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell

  3. Leishmania exosomes deliver preemptive strikes to create an environment permissive for early infection.

    Silverman, Judith Maxwell; Reiner, Neil E

    2011-01-01

    Herein, we review evidence supporting a role for Leishmania exosomes during early infection. We suggest a model in which Leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for Leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, wild-type (WT) exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) by Leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The Leishmania proteins GP63 and elongation factor-1α (EF-1α) are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein-tyrosine phosphatases (PTPs). Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that Leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell signaling pathways.

  4. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

  5. First case of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Suriname.

    Hu, Ricardo V P F; Kent, Alida D; Adams, Emily R; van der Veer, Charlotte; Sabajo, Leslie O A; Mans, Dennis R A; de Vries, Henry J C; Schallig, Henk D F H; Lai A Fat, Rudy F M

    2012-05-01

    The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.

  6. Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

    Kasra Hassani; Elisabeth Antoniak; Armando Jardim; Martin Olivier

    2011-01-01

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 p...

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

    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...... that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs....

  8. New Record of Phlebotomus Sergenti, the Vector of Leishmania Tropica, in the Southern Nile Valley of Egypt

    2001-01-01

    SCIENTIFIC NOTE NEW RECORD OF PHLEBOTOMUS SERGENTI, THE VECTOR OF LEISHMANIA TROPICA , IN THE SOUTHERN NILE VALLEY OF EGYPT HANAFI A. HANAFI’ GREGORY M...forms of Leishmania tropica , from southern Egypt . Four female and I male P. sergenti were collected from unlit Centers for Disease Control light traps...sergenti (Parrot) is a widely distrib- uted sand fly species that feeds readily on humans and is a known vector of Leishmania tropica (Ash- ford and

  9. Leishmania infantum AS A CAUSATIVE AGENT OF CUTANEOUS LEISHMANIASIS IN THE STATE OF MATO GROSSO DO SUL, BRAZIL

    CASTRO, Ludiele Souza; FRANÇA, Adriana de Oliveira; FERREIRA, Eduardo de Castro; HANS, Günther; HIGA, Minoru German; GONTIJO, Célia Maria Ferreira; PEREIRA, Agnes Antônia Sampaio; DORVAL, Maria Elizabeth Moraes C.

    2016-01-01

    Cutaneous leishmaniasis is caused by different species of theLeishmania genus. Leishmania(Leishmania) infantum, causing cutaneous leishmaniasis, has been described in patients living in areas where visceral leishmaniasis is endemic. In this study, it was possible to characterize this species in seven slides from cutaneous tissue imprints from patients with cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil. PMID:27007566

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

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

    2001-01-01

    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....... Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs....

  11. Leishmanicidal activity of synthetic chalcones in Leishmania (Viannia) braziliensis.

    de Mello, Tatiane F P; Bitencourt, Heriberto R; Pedroso, Raissa B; Aristides, Sandra M A; Lonardoni, Maria V C; Silveira, Thais G V

    2014-01-01

    The treatment of American cutaneous leishmaniasis (ACL) is based on a small group of compounds that were developed decades ago, all of which are highly toxic and have a high rate of treatment failure. The chalcones show leishmanicidal activity, yet few studies have evaluated this activity against Leishmania (Viannia) braziliensis, one of the most important species of Leishmania across Latin America. Four new synthetic chalcones (1-4) were evaluated for inhibitory activity in vitro against promastigotes and intracellular parasites 24h post infection of L. (V.) braziliensis, cytotoxicity for macrophages J774.A1 and red blood cells, and the ability to stimulate nitric oxide production. The results for the inhibitory concentration for 50% of the promastigotes (IC50) (1.38±1.09-6.36±2.04μM), cytotoxic concentration for 50% of the macrophages (CC50) (13.49±3.13-199.43±4.11μM), and selectivity index (SI) (3.76 to 33.94) indicate that all chalcones (1-4) showed an effect on promastigotes of L. (V.) braziliensis; chalcone 2 had the highest SI. The haemolytic assay with chalcones 1 (301.93μM), 2 (534.18μM), 3 (419.46μM) and 4 (381.11μM) showed 0.00%, 2.33%, 0.57% and 1.74% haemolysis, respectively. All chalcones significantly reduced the infection index of macrophages by parasites; for chalcones (1-3) this effect may be dependent on nitric-oxide production by macrophages. The chalcones tested exhibited inhibitory activity for promastigotes and intracellular parasites of L. (V.) braziliensis, with low toxicity for macrophages and red blood cells. The anti-Leishmania activity of chalcones (1-3) may depend on the stimulation of nitric-oxide production in the initial stage of infection. These results show an initially encouraging potential for the use of chalcones (1-4) to treat ACL.

  12. Leishmania donovani HslV does not interact stably with HslU proteins.

    Chrobak, Mareike; Förster, Sabine; Meisel, Sarah; Pfefferkorn, Roxana; Förster, Frank; Clos, Joachim

    2012-04-01

    Genes for HslVU-type peptidases are found in bacteria and in a few select Eukaryota, among those such important pathogens as Plasmodium spp. and Leishmania spp. In this study, we performed replacements of all three HslV/HslU gene homologues and found one of those, HslV, to be essential for Leishmania donovani viability. The Leishmania HslV gene can also partially relieve the thermosensitive phenotype of a combined HslVU/Lon/ClpXP knockout mutant of Escherichia coli, indicating a conserved function. However, we found that the role and function of the two Leishmania HslU genes has diverged since neither of those interacts stably with HslV. The latter forms a dodecameric complex by itself and shows a punctate distribution. We conclude that whilst the basic function of HslV may be conserved in Leishmania, its organisation and interaction with its canonical complex partner HslU is not. Nevertheless, given the absence of HslV from the proteome of mammals and its essential role in Leishmania viability, HslV is a promising target for intervention.

  13. Fatores associados à soropositividade para Babesia, Toxoplasma, Neospora e Leishmania em cães atendidos em nove clínicas veterinárias do município de Lavras, MG Factors associated the seropositivity for Babesia, Toxoplasma, Neospora e Leishmania in dogs attended at nine veterinary clinics in the municipality of Lavras, MG

    Antônio Marcos Guimarães; Christiane M.B.M. Rocha; Oliveira, Trícia M. F. S.; Rosado, Isabel R; Morais,Letícia G.; Raquel R. D. Santos

    2009-01-01

    O objetivo deste estudo foi determinar a frequência e avaliar a influência da idade, sexo e raça na soropositividade anti-Babesia canis, Toxoplasma gondii, Leishmania (L.) chagasi e Neospora caninum, por meio da reação de imunofluorescência indireta (RIFI), em amostras de soros coletadas de cães atendidos em nove clínicas veterinárias particulares do município de Lavras, MG, no período de agosto de 2000 a abril de 2002. De 300 cães, 73,3% foram soropositivos (RIFI > 1:80) para B. canis, e hou...

  14. Leishmania Mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. Mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

    SA Ali

    2011-09-01

    Full Text Available Background: Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical coun­tries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effec­tive in Leishmania vaccination. Leishmania Soluble Antigen (SLA has also recently been used Leishmania vaccination.Methods: The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of den­dritic cells (DCs loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun.Results: Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administra­tion of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection.Conclusion: The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vac­cine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These find­ings would be helpful in opening new windows in Leishmania vaccine research.

  15. Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro

    Gabriel Grimaldi Junior

    1983-06-01

    Full Text Available Unstimulated adherent mouse peritoneal cells were cultured in vitro and infected with equal numbers of a single strain of Leishmania m. mexicana amastigotes (AM, virulent promastigotes (VP, avirulent promastigotes (AVP and fixed promastigotes (FP. Duplicate May-Grünwald-Giemsa stained coverslips were examined at time intervals up to 13 days. By 3 hr post infection, the number of macrophages containing parasites varied between 60.5% (VP and 84% (AM for macrophages exposed to living parasites, compared to 6.5% for macrophages exposed for FP. However, variable numbers of parasites showed degenerative changes by 3 hr, and the number of macrophages containing morphologically intact parasites varied significantly between cells infected with AM (84% and those infected with VP (42% or AVP(40%. The mean number on intacte parasites/macrophage also differed significantly between AM-infected cells and living or fixed promastigotes-infected cells. Quantitation of intact and degenerated parasites indicated parasite multiplication, as well as destruction, in VP-infected cells and parasite survival and multiplication in AM-infecte monolayers; in contrast no evidence of parasite multiplication was seen in AVP-infected cells. Changes in the mono layer itself (cell loss and macrophage vacuolization were also evaluated. These results suggest that crucial events determining the outcome of infection occur in the host-parasite relationship during the fist 24 hours of infection. These events are apparently influenced not only by parasite or host strain but by environmentally induced variation within a given strain.Células peritoneais de camundongos cultivadas in vitro foram infectadas com inóculos idênticos de amastigotas (AM e de promastigotas, respectivamente, virulentas (VP, avirulentas (AVP e fixadas (FP de uma mesma cepa de Leishmania m. mexicana. As monocamadas coradas com May-Grunwald-Giemsa foram examinadas em diferentes intervalos de tempo. Ao nível de 3

  16. Leishmania spp. Epidemiology of Canine Leishmaniasis in the Yucatan Peninsula

    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.

  17. Drought, smallpox, and emergence of Leishmania braziliensis in northeastern Brazil.

    Sousa, Anastácio Q; Pearson, Richard

    2009-06-01

    Cutaneous leishmaniasis caused by Leishmania (Vianna) braziliensis is a major health problem in the state of Ceará in northeastern Brazil. We propose that the disease emerged as a consequence of the displacement of persons from Ceará to the Amazon region following the Great Drought and smallpox epidemic of 1877-1879. As the economic and social situation in Ceará deteriorated, approximately 55,000 residents migrated to the Amazon region to find work, many on rubber plantations. Those that returned likely introduced L. (V.) brazilensis into Ceará, where the first cases of cutaneous leishmaniasis were reported early in the 20th century. The absence of an animal reservoir in Ceará, apart from dogs, supports the hypothesis. The spread of HIV/AIDS into the region and the possibility of concurrent cutaneous leishmaniasis raise the possibility of future problems.

  18. A Pediatric Case of Concomitant Leishmania and Brucella Infection

    Perihan Yasemen Canöz

    2014-08-01

    Full Text Available Visceral leishmaniasis (Kala-azar is a disease caused by protozoan parasites of the Leishmania Genus, and Brucellosis is a zoonotic disease infecting human host by infective animals. A sixteen year-old girl presented to our clinic with complaints of fatigue, myalgia, pallor, stomach ache, leg swelling, and diffuse body rash. Physical examination revealed findings of elevated body temperature, splenomegaly, upper and lower extremity edema, and diffuse erythema. Patients’ brucella agglutination test was positive at titers 1: 640. Since the treatment of Brucellosis was unsuccessful, other disease processes were investigated and extracellular and intracellular amastigots were detected in the bone marrow aspirate preparations. Kala-azar dipstick (rk-39 was also positive. We present a 16 year-old girl who was diagnosed with Kala-azar and Brucellosis together infection and successfully treated.

  19. Leishmania: origin, evolution and future since the Precambrian.

    Tuon, Felipe Francisco; Neto, Vicente Amato; Amato, Valdir Sabbaga

    2008-11-01

    This brief review discusses the history of leishmaniasis, considering its origin from the Paleoartic, Neoartic or Neotropic. We reassess some of the theories of the likely origin of this protozoan since the beginning of life on Earth, passing through the Mesozoic and continuing to the appearance of humans. The relationship between this parasite or its ancestors, possible vectors and hosts with regard to ecological modifications is discussed. Recent molecular techniques have helped to elucidate some of the evolutionary questions regarding Leishmania, but have also brought doubts about the origin and evolution of this human parasite. PCR has been used for studies in the new discipline of paleoparasitology, helping to elucidate some of the remaining evolutionary questions. Understanding of this global condition is fundamental in determining the best approach to use against the parasite, specifically for the development of an efficient vaccine.

  20. Molecular Cloning, Expression and Characterization of Ribokinase of Leishmania major

    Patrick. O.J. OGBUNUDE; Nadia LAMOUR; Michael P. BARRETT

    2007-01-01

    Ribokinase (EC 2.1.7.15) from Leishmania major was cloned, sequenced and overexpressed in Escherichia coli. The gene expressed an active enzyme that had comparable activity to the same enzyme studied in E. coli. It specifically phosphorylated D-ribose. Under defined conditions, the Km for the substrates D-ribose and ATP were 0.3±0.04 mM and 0.2±0.02 mM, respectively. The turnover numbers of the enzyme for the substrates were 10.8 s-1 and 10.2 s-1, respectively. The enzyme product ribose 5-phosphate inhibited the phosphorylation of D-ribose with an apparent Ki of 0.4 mM, which is close to the Km (0.3 mM) of D-ribose, suggesting that it might play a role in regulating flux through the enzyme.

  1. Antimony Resistance in Leishmania, Focusing on Experimental Research

    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.

  2. Development of Cutaneous Leishmaniasis after Leishmania Skin Test

    Machado, Paulo R.; Carvalho, Augusto M.; Machado, Gustavo U.; Dantas, Marina L.; Arruda, Sérgio

    2011-01-01

    Thirty-year-old female with a previous history of a cutaneous ulcer suspicious of leishmaniasis 20 years ago presented with a new complaint of a depressed papular lesion 8 × 7 mm in the right lower extremity. The lesion was of 10-day duration. Because early cutaneous leishmaniasis (CL) lesions may have a non-ulcerated appearance, a Leishmania skin test (LST) was performed on the forearm with a strong positive result (38 × 32 mm). After 8 days, the lesion in the leg, which was diagnosed as folliculitis, completely healed. However, a typical CL ulcer (26 × 24 mm) developed at the LST site. Histopathology of the new lesion did not identifiy parasites, but the findings were consistent with a diagnosis of CL. Further analysis identified amastigotes by immunohistochemical stain. Mononuclear cells harvested from the patient were stimulated with Leishmania antigen and showed high levels of production of both tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ): 2,943 pg/mL and 2,313 pg/mL, respectively. After 40 days of treatment with antimony and pentoxifylline, the ulcer resolved. The development of CL at the LST site suggests a strong Th1 immune response, and it is an in vivo documentation of the role of the host immune response in the pathology of CL. It teaches us that LST should be cautiously, if at all, used in patients with self-healing CL ulcers. PMID:22162702

  3. Allopurinol Resistance in Leishmania infantum from Dogs with Disease Relapse.

    Daniel Yasur-Landau

    2016-01-01

    Full Text Available Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of Leishmania strains from dogs to allopurinol has not been described before in clinical studies.Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group showed an average half maximal inhibitory concentration (IC50 of 996 μg/mL. A significantly lower IC50 (P = 0.01 was found for isolates from ten dogs before treatment (NT group, 200 μg/mL, as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL. Axenic amastigotes produced from isolates of the TR group also showed significantly higher (P = 0.002 IC50 compared to the NT group (1678 and 671 μg/mL, respectively. The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (P = 0.002 was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol.This is the first study to demonstrate allopurinol resistance in L. infantum and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.

  4. Screening and characterization of RAPD markers in viscerotropic Leishmania parasites.

    Imen Mkada-Driss

    Full Text Available Visceral leishmaniasis (VL is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5' end transversions, and presence of inter- and intra- taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers

  5. Leishmania tropica in Stray Dogs in Southeast Iran

    Mehdi BAMOROVAT

    2015-10-01

    Full Text Available  Background: Cutaneous leishmaniasis (CL caused by Leishmania tropica is endemic in Kerman, southeastern Iran. While dogs have long been implicated as the main domestic reservoirs of L. infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL, they can also carry L. tropica infection. The objective of the present study was to determine molecular identity and to evaluate histopathological changes due to CL in dogs in a well-known focus of anthroponotic CL (ACL in Kerman, southeastern Iran.Methods: This study was carried out in three prospective series from 1994 to 2013 on dogs. Tissue samples were taken from 471 stray dogs. Pathological specimens including skin, spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning and staining for further histopathological examination. PCR amplification of kDNA was performed to identify the causative agent and sequencing. Overall, two out of 471 stray dogs were infected with L. tropica. Hyperplasia of red pulp by the proliferation of histiocytes, lymphocytes, plasma cells and cytoplasm of histiocytes collection of amastigotes was noted.Results: Based on the results of PCR products and sequencing analysis, the parasites isolated from the lesions of two dogs were characterized as L. tropica, corresponding to a band of 830 bp Conclusion: This finding revealed infection with L. tropica in stray dogs in the city and suburbs of Kerman. This information is essential for public health concerns and planning effective future control programs. The role of dogs as potentional reservoir in the epidemiology of ACL needs further investigation. Keywords: Leishmania tropica, Dog, Histopathology, Molecular, Epidemiology, Iran

  6. Antimony quantification in Leishmania by electrothermal atomic absorption spectroscopy.

    Roberts, W L; Rainey, P M

    1993-05-15

    Tri- and pentavalent antimony were quantified in Leishmania mexicana pifanoi amastigotes and promastigotes by atomic absorption spectroscopy with electrothermal atomization. Leishmania grown in axenic culture were treated with either potassium antimony tartrate [Sb(III)] or sodium stibogluconate [Sb(V)]. The parasites were collected, digested with nitric acid, and subjected to atomic absorption spectroscopy. The method was linear from 0 to 7 ng of antimony. The interassay coefficients of variation were 9.6 and 5.7% (N = 5) for 0.52 and 3.7-ng samples of leishmanial antimony, respectively. The limit of detection was 95 pg of antimony. The assay was used to characterize Sb(III) and Sb(V) influx and efflux kinetics. Influx rates were determined at antimony concentrations that produced a 50% inhibition of growth (IC50). The influx rates of Sb(V) into amastigotes and promastigotes were 4.8 and 12 pg/million cells/h, respectively, at 200 micrograms antimony/ml. The influx rate of Sb(III) into amastigotes was 41 pg/million cells/h at 20 micrograms antimony/ml. Influx of Sb(III) into promastigotes at 1 microgram antimony/ml was rapid and reached a plateau of 175 pg/million cells in 2 h. Efflux of Sb(III) and Sb(V) from amastigotes and promastigotes exhibited biphasic kinetics. The initial (alpha) half-life of Sb(V) efflux was less than 4 min and that of Sb(III) was 1-2 h. The apparent terminal (beta) half-lives ranged from 7 to 14 h.

  7. Development of cutaneous leishmaniasis after leishmania skin test.

    Machado, Paulo R; Carvalho, Augusto M; Machado, Gustavo U; Dantas, Marina L; Arruda, Sérgio

    2011-01-01

    Thirty-year-old female with a previous history of a cutaneous ulcer suspicious of leishmaniasis 20 years ago presented with a new complaint of a depressed papular lesion 8 × 7 mm in the right lower extremity. The lesion was of 10-day duration. Because early cutaneous leishmaniasis (CL) lesions may have a non-ulcerated appearance, a Leishmania skin test (LST) was performed on the forearm with a strong positive result (38 × 32 mm). After 8 days, the lesion in the leg, which was diagnosed as folliculitis, completely healed. However, a typical CL ulcer (26 × 24 mm) developed at the LST site. Histopathology of the new lesion did not identifiy parasites, but the findings were consistent with a diagnosis of CL. Further analysis identified amastigotes by immunohistochemical stain. Mononuclear cells harvested from the patient were stimulated with Leishmania antigen and showed high levels of production of both tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ): 2,943 pg/mL and 2,313 pg/mL, respectively. After 40 days of treatment with antimony and pentoxifylline, the ulcer resolved. The development of CL at the LST site suggests a strong Th1 immune response, and it is an in vivo documentation of the role of the host immune response in the pathology of CL. It teaches us that LST should be cautiously, if at all, used in patients with self-healing CL ulcers.

  8. Natural infection of Algerian hedgehog, Atelerix algirus (Lereboullet 1842) with Leishmania parasites in Tunisia.

    Chemkhi, Jomaa; Souguir, Hejer; Ali, Insaf Bel Hadj; Driss, Mehdi; Guizani, Ikram; Guerbouj, Souheila

    2015-10-01

    In Tunisia, Leishmania parasites are responsible of visceral leishmaniasis, caused by Leishmania infantum species while three cutaneous disease forms are documented: chronic cutaneous leishmaniasis due to Leishmania killicki, sporadic cutaneous form (SCL) caused by L. infantum and the predominant zoonotic cutaneous leishmanaisis (ZCL) due to Leishmania major. ZCL reservoirs are rodents of the Psammomys and Meriones genera, while for SCL the dog is supposed to be a reservoir. Ctenodactylus gundii is involved in the transmission of L. killicki. However, other mammals could constitute potential reservoir hosts in Tunisia and other North African countries. In order to explore the role of hedgehogs as potential reservoirs of leishmaniasis, specimens (N=6) were captured during July-November period in 2011-2013 in an SCL endemic area in El Kef region, North-Western Tunisia. Using morphological characteristics, all specimens were described and measured. Biopsies from liver, heart, kidney and spleen of each animal were used to extract genomic DNA, which was further used in PCR assays to assess the presence of Leishmania parasites. Different PCRs targeting kinetoplast minicircles, ITS1, mini-exon genes and a repetitive Leishmania- specific sequence, were applied. To further identify Leishmania species involved, RFLP analysis of amplified fragments was performed with appropriate restriction enzymes. Using morphological characters, animals were identified as North African hedgehogs, also called Algerian hedgehogs, that belong to the Erinaceidae family, genus Atelerix Pomel 1848, and species algirus (Lereboullet, 1842). PCR results showed in total that all specimens were Leishmania infected, with different organs incriminated, mainly liver and spleen. Results were confirmed by direct sequencing of amplified fragments. Species identification showed that all specimens were infected with L. major, three of which were additionally co-infected with L. infantum. The present study

  9. In vivo antileishmanial action of Ir-(COD-pentamidine tetraphenylborate on Leishmania donovani and Leishmania major mouse models

    Loiseau P.M.

    2000-06-01

    Full Text Available lr-(COD-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 μM. In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9 /Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 μmoles/ kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23 % of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 μmoles/ kg/day x 5 (54 % of parasite suppression, the iridium complex exhibited 32 % of parasite suppression after a treatment at 76 μmoles/ kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

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

    Matthew B Rogers

    2014-01-01

    Full Text Available 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.

  11. Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population

    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. PMID:24453988

  12. Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

    Ranasinghe, Shalindra; Wickremasinghe, Renu; Hulangamuwa, Sanjeeva; Sirimanna, Ganga; Opathella, Nandimithra; Maingon, Rhaiza D C; Chandrasekharan, Vishvanath

    2015-12-01

    Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

  13. Ocurrence of co-infection by Leishmania (Leishmania chagasi and Trypanosoma (Trypanozoon evansi in a dog in the state of Mato Grosso do Sul, Brazil

    Elisa San Martin Mouriz Savani

    2005-11-01

    Full Text Available A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L. chagasi, the natural transmission of T. (T. evansi occurs in the area under study.

  14. 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

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history...

  15. Synthesis, Leishmanicidal Activity and Theoretical Evaluations of a Series of Substituted bis-2-Hydroxy-1,4-Naphthoquinones

    Morgana V. de Araújo

    2014-09-01

    Full Text Available A series of eight substituted bis-2-hydroxy-1,4-naphthoquinone derivatives was synthesized through lawsone condensation with various aromatic and aliphatic aldehydes under mild acidic conditions. The title compounds were evaluated for antileishmanial activity in vitro against Leishmania amazonensis and Leishmania braziliensis promastigotes; six compounds showed good activity without significant toxic effects. The compound with the highest activity was used for an in vivo assay with Leishmania amazonensis.

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

    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...

  17. Serological survey of Leishmania infantum and Trypanosoma cruzi in dogs from urban areas of Brazil and Colombia

    Leishmania infantum and Trypanosoma cruzi are zoonotic parasites that are endemic throughout many parts of Latin America. Infected dogs play an important role in transmission of both parasites to humans. A serological survey of Leishmania and Trypanosoma infection was conducted on 365 dogs from São ...

  18. Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.

    Kumar, Pranav; Lodge, Robert; Raymond, Frédéric; Ritt, Jean-François; Jalaguier, Pascal; Corbeil, Jacques; Ouellette, Marc; Tremblay, Michel J

    2013-08-01

    Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.

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

    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.

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

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

    2016-01-01

    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. PMID:27548150

  1. Naloxonazine, an Amastigote-Specific Compound, Affects Leishmania Parasites through Modulation of Host-Encoded Functions

    Vanhollebeke, Benoit; Caljon, Guy; Wolfe, Alan R.; McKerrow, James; Dujardin, Jean-Claude

    2016-01-01

    Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy. PMID:28036391

  2. Deciphering the Leishmania exoproteome: what we know and what we can learn.

    Corrales, Rosa Milagros; Sereno, Denis; Mathieu-Daudé, Françoise

    2010-02-01

    Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis. Survival and transmission of these parasites in their different hosts require membrane-bound or extracellular factors to interact with and modify their host environments. Over the last decade, several approaches have been applied to study all the extracellular proteins exported by an organism at a particular time or stage in its life cycle and under defined conditions, collectively termed the secretome or the exoproteome. In this review, we focus on emerging data shedding light on the secretion mechanisms involved in the production of the Leishmania exoproteome. We also describe other methodologies currently available that could be used to analyse the Leishmania exoproteome. Understanding the complexity of the Leishmania exoproteome is a key component to elucidating the mechanisms used by these parasites for exporting proteins to the extracellular space during its life cycle. Given the importance of extracellular factors, a detailed knowledge of the Leishmania exoproteome may provide novel targets for rational drug design and/or a source of antigens for vaccine development.

  3. The effect of temperature on Leishmania (Kinetoplastida: Trypanosomatidae) development in sand flies.

    Hlavacova, J; Votypka, J; Volf, P

    2013-09-01

    The spread of leishmaniasis to areas where it was previously considered nonendemic has been recently found in the New and Old Worlds, and climate changes are suspected as a crucial factor responsible for this spread. Ambient temperature is known to significantly affect the metabolism of sand flies and their developmental times, but little is known about the effect of temperature on the Leishmania life cycle in vectors. This study assesses the effect of temperature on the development of two closely related New World Viannia species, Leishmania braziliensis and Leishmania peruviana, in the permissive vector Lutzomyia longipalpis, and on the development of New and Old World Leishmania infantum in its natural vectors Lu. longipalpis and Phlebotomus perniciosus, respectively. The mountain species L. peruviana developed well in sand fly females kept at 20 degrees C, whereas at 26 degrees C, most infections were lost during the defecation ofbloodmeal remains; this suggests an adaptation to the slower metabolism of sand flies living at lower ambient temperature. On the contrary, L. infantum and L. braziliensis developed well at both temperatures tested; heavy late-stage infections were observed in a majority of sand fly females maintained at 20 degrees C as well 26 degrees C. Frequent fully developed infections of L. infantum and L. braziliensis at 20 degrees C suggest a certain risk of the spread of these two Leishmania species to higher latitudes and altitudes.

  4. Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil.

    Coelho, Willian Marinho Dourado; Richini-Pereira, Virgínia Bodelão; Langoni, Helio; Bresciani, Katia Denise Saraiva

    2011-03-10

    The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, São Paulo State.

  5. Avaliação da atividade leishmanicida in vitro de plantas medicinais Evaluation of the in vitro leishmanicidal activity of medicinal plants

    Jeamile L. Bezerra

    2006-12-01

    Full Text Available Na busca de novos agentes leishmanicidas, avaliamos em culturas de promastigotas de Leishmania amazonensis o efeito dos extratos hidroalcoólicos das folhas de Tephrosia cinerea (L. Pers. (Fabaceae, Dichorisandra sp (Commelinaceae, Syzygium jambolanum DC. (Myrtaceae, Julocroton triqueter (Lam. Didr. var. triqueter (Euphorbiaceae, Passiflora edulis Sims(Passifloraceae, Cecropia sp (Cecropiaceae, Chenopodium ambrosioides L.(Chenopodiaceae, Pedilanhus tithymaloides (L. Poit (Euphorbiaceae, Peristrophe angustifolia Nees(Acanthaceae e o extrato aquoso do mesocarpo de Orbignya phalerata Mart. (Arecaceae. As promastigotas de Leishmania amazonensis foram cultivadas em presença de 31,3; 63,5; 125,0; 250,0 e 500,0 µg/mL dos extratos por 24 horas. Ao final desse período foi calculada a concentração inibitória do crescimento (CI50 em relação às culturas não tratadas com os extratos. Os extratos das espécies J. triqueter, Dichorisandra sp e T. cinerea apresentaram maior eficácia em induzir a morte das promastigotas, com CI50 de 29,5; 32,9 e 43,6 µg/mL, respectivamente. P. edulis, C. ambrosioides e S. jambolanum apresentaram eficácia moderada com CI50 de 150,1; 151,9 e 166,6 µg/mL, respectivamente. P. tithymaloides e O. phalerata apresentaram baixo efeito leishmanicida com CI50 >500 µg/mL, enquanto Peristrophe angustifolia e Cecropia spnão apresentaram efeito. Dessa forma, dos dez extratos testados, três apresentaram uma expressiva atividade leishmanicida in vitro.Searching for new leishmanicidal agents, promastigotes forms of L. amazonensis were cultured with the hydroalcoholic extracts obtained from the Tephrosia cinerea (L. Pers. (Fabaceae, Dichorisandra sp (Commelinaceae, Syzygium jambolanum DC. (Myrtaceae, Julocroton triqueter (Lam. Didr. var. triqueter (Euphorbiaceae, Passiflora edulis Sims(Passifloraceae, Cecropia sp (Cecropiaceae, Chenopodium ambrosioides L.(Chenopodiaceae, Pedilanhus tithymaloides (L. Poit (Euphorbiaceae

  6. Notas e descrições em Hesperophanini, Eburiini, Piezocerini e Trachyderini (Coleoptera, Cerambycidae, Cerambycinae do Brasil e da Bolívia

    Ubirajara R. Martins

    2010-01-01

    Full Text Available Novos táxons descritos - Hesperophanini: Paraliostola nigramacula sp. nov., do Brasil (Rondônia; Eburiini: Simplexeburia gen. nov., e sua espécie-tipo, S. divisa sp. nov. do Brasil (Amazonas; Piezocerini: Gorybia amazonensis sp. nov. and G. sulcata sp. nov., ambas do Brasil (Amazonas; Trachyderini: Galissus rubiventris sp. nov., da Bolívia (Santa Cruz. Notas e novos registros são apresentados para Liostola nitida Zajciw, 1962 e Ochrus chapadense Napp & Martins, 1982 (Hesperophanini; Uncieburia rogersi (Bates, 1870 e Quiacaua taguaiba Martins, 1970 (Eburiini.Notes and descriptions on Hesperophanini, Eburiini, Piezocerini and Trachyderini (Coleoptera, Cerambycidae, Cerambycinae from Brazil and Bolivia. New taxa described - Hesperophanini: Paraliostola nigramacula sp. nov. from Brazil (Rondônia; Eburiini: Simplexeburia gen. nov., and its type species, S. divisa sp. nov. from Brazil (Amazonas; Piezocerini: Gorybia amazonensis sp. nov. and G. sulcata sp. nov. both from Brazil (Amazonas; Trachyderini: Galissus rubiventris sp. nov. from Bolivia (Santa Cruz de la Sierra. Notes and records are presented for Liostola nitida Zajciw, 1962 and Ochrus chapadense Napp & Martins, 1982 (Hesperophanini; Uncieburia rogersi (Bates, 1870 and Quiacaua taguaiba Martins, 1997 (Eburiini.

  7. 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

    C.A. Cuba Cuba

    1996-12-01

    pacientes humanos com lesões cutâneas de leishmaniose na Província de Santiago del Estero, no Norte da Argentina. Os procedimentos de diagnóstico foram: biópsias de lesões para utilização em esfregaços e inoculação em hamster; aspiração (com agulha de úlceras, para cultura "in vitro". As técnicas imunodiagnósticas empregadas foram a IFAT-IgG e o teste intradérmico de Montenegro. Oito cepas de parasitas foram isoladas, sendo estas obtidas de pacientes com lesões ativas. Todas as cepas foram inicialmente caracterizadas biologicamente por seu comportamento na infecção experimental do hamster, mensuração dos amastigotas e promastigotas e crescimento "in vitro". As mesmas oito cepas foram logo identificadas e caracterizadas a nível de espécie, devido a sua reatividade frente a um painel de anticorpos monoclonais subgênero e espécie-específicos. Isso foi realizado utilizando o teste de Imunofluorescência Indireta (IFAT/MAbs e de um procedimento de Dot-ELISA. Nós concluímos a partir da análise de serodema dos isolados argentinos que: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 e SE-36 são Leishmania (Viannia braziliensis. Entretanto, três dos isolados de Leishmania (SE-1; SE-2 e SE-30 não foram reconhecidos quando testados com um anticorpo monoclonal de reconhecida alta espécie-especificidade (clone B-18, marcador consagrado de Leishmania (Viannia braziliensis, revelando a existência de dois tipos de serodemas entre as cepas estudadas. Cinco dos oito extratos solúveis de Leishmania foram submetidos à eletroforese em gel de amido de camada fina e subseqüentemente examinadas a fim de constatar a atividade das enzimas MPI, MDH, 6PGD, NH, NH-D, SOD, GPI e ES. Fundamentados nos estudos dos corridos eletroforéticos obtidos nos ensaios isoenzimáticos chegamos à conclusão que as cepas MHOM/AR/92 SE-1; SE-2; SE-4; SE-8 e SE-8-I são Leishmania (Viannia braziliensis. É necessário analisar mais enzimas antes de enquadrá-los nos

  8. Disseminated Leishmaniasis Caused by Leishmania Tropica in a Puppy from Karaj, Central Iran

    M Mohebali

    2011-06-01

    Full Text Available A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was exam­ined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large num­ber of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT. DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.

  9. Geographical Distribution of Leishmania Species of Human Cutaneous Leishmaniasis in Fars Province, Southern Iran

    M Akhoundi

    2013-03-01

    Full Text Available Background: The goal of this study was to know the identity of Leishmania species responsible of cutaneous leishmaniasi