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Sample records for leishmania donovani antigen

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

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    Michael Forgber

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

  2. Induction of cellular and humoral responses by autoclaved and heat-killed antigen of Leishmania donovani in experimental visceral leishmaniasis.

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    Nagill, Rajeev; Mahajan, Raman; Sharma, Meenakshi; Kaur, Sukhbir

    2009-12-01

    The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated and humoral response has been evaluated in the present study. The vaccines were delivered thrice subcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challenged with 10(7) stationary phase promastigotes of L. donovani. Significant protection was achieved in immunized mice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver and spleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH) response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune response was assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody response was predominantly of IgG type with increased IgG2a production and lesser amount of IgM. The immunization preferentially stimulates the production of IFN-gamma and IL-2 in splenocytes which suggests a Th1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potential for the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.

  3. Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

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    Lynette Beattie

    2010-03-01

    Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

  4. Studies on the protective efficacy of freeze thawed promastigote antigen of Leishmania donovani along with various adjuvants against visceral leishmaniasis infection in mice.

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    Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

    2015-09-01

    Visceral leishmaniasis (VL) caused by Leishmania donovani persists as a major public health issue in tropical and subtropical areas of the world. Current treatment of this disease relies on use of drugs. It is doubtful that chemotherapy can alone eradicate the disease, so there is a need for an effective vaccine. Killed antigen candidates remain a good prospect considering their ease of formulation, stability, low cost and safety. To enhance the efficacy of killed vaccines suitable adjuvant and delivery system are needed. Therefore, the current study was conducted to determine the protective efficacy of freeze-thawed L. donovani antigen in combination with different adjuvants against experimental infection of VL. For this, BALB/c mice were immunized thrice at an interval of two weeks. Challenge infection was given two weeks after last immunization. Mice were sacrificed after last immunization and on different post challenge/infection days. Immunized mice showed significant reduction in parasite burden, enhanced DTH responses with increased levels of Th1 cytokines and lower levels of Th2 cytokines, thus indicating the development of a protective Th1 response. Maximum protection was achieved with liposome encapsulated freeze thawed promastigote (FTP) antigen of L. donovani and it was followed by group immunized with FTP+MPL-A, FTP+saponin, FTP+alum and FTP antigen (alone). The present study highlights greater efficacy of freeze thawed promastigote antigen as a potential vaccine candidate along with effective adjuvant formulations against experimental VL infection.

  5. Interferon-¿ and interleukin-4 production by human T cells recognizing Leishmania donovani antigens separated by SDS-PAGE

    DEFF Research Database (Denmark)

    Bahrenscheer, J; Kemp, M; Kurtzhals, J A

    1995-01-01

    of proliferation and interferon-gamma (IFN-gamma) production in cultures of peripheral blood mononuclear cells (PBMC) from individuals who had recovered from visceral leishmaniasis caused by L. donovani. The release of interleukin-4 (IL-4) by PBMC stimulated with the isolated L. donovani antigen fractions...... was measured after treatment with phorbol-myristate-acetate and ionomycin. The cells proliferated in response to all protein fractions with molecular weights in the range 12 kDa to 85 kDa. In general, IFN-gamma was secreted in response to stimulation with all the protein fractions, whereas IL-4 production...

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

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    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.

  7. Humoral and cellular immune responses to glucose regulated protein 78 - a novel Leishmania donovani antigen

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Ismail, Ahmed; Gaafar, Ameera

    2002-01-01

    evaluation of plasma samples obtained in Sudan revealed that 89% of patients with visceral leishmaniasis (VL), 78% with post kala-azar dermal leishmaniasis (PKDL), and 85% with cutaneous leishmaniasis (CL) had antibody reactivity to this Leishmania antigen. Plasma from healthy Sudanese individuals living...... in an area endemic for malaria but free of leishmaniasis and plasma from healthy Danes was negative in the assay. GRP78 antibody was detected in 10% and 5% of plasma samples from Sudanese and Ghanaian malaria patients, respectively, whereas 35% of plasma samples from otherwise healthy Sudanese individuals...

  8. Haemolytic activities of plant saponins and adjuvants. Effect of Periandra mediterranea saponin on the humoral response to the FML antigen of Leishmania donovani.

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    Santos, W R; Bernardo, R R; Peçanha, L M; Palatnik, M; Parente, J P; Palatnik de Sousa, C B

    1997-06-01

    An 87.7% (P saponin combined to the fucose-mannose ligand of Leishmania donovani (FML). However, an undesirable haemolytic effect was described for several saponins. Aiming to improve the formulation with FML/saponin, we comparatively analysed the haemolytic potential of recently characterized plant saponins and currently used adjuvants. The haemolytic activity of steroidic saponins from Agave sisalana; Smilax officinalis as well as commercial saponin (Riedel De Haën's), was higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra mediterranea) and the Freund's complete adjuvant. The concentration resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum hydroxide. The low haemolytic effect of P. mediterranea saponin was abolished by removal of its glycidic moiety and its sapogenin fraction as well as the Freund's Incomplete Adjuvant were non-haemolytic within this range. Furthermore, the adjuvant effect of three doses of P. mediterranea saponin injected with the FML antigen of L. donovani, was assayed in mice, either by the intraperitoneal (i.p.) or the subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased and detectable levels were observed up to 3 months in the s.c. group. The response was expanded in both groups after an injection with a fourth vaccine dose. The IgG response showed increased levels of IgG2a only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies were higher than controls in both groups. In conclusion, the results suggest that the recently described triterpenoid fractions of P. mediterranea can be safely used as adjuvant with low or non-haemolytic effect.

  9. Antigen pulsed CpG-ODN activated dendritic cells induce host-protective immune response by regulating the T regulatory cell functioning in Leishmania donovani-infected mice: critical role of CXCL10

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    Saikat eMajumder

    2014-06-01

    Full Text Available Visceral leishmaniasis (VL, caused by Leishmania donovani, is a systemic infection of reticulo-endothelial system. There is currently no protective vaccine against VL and chemotherapy is increasingly limited due to appearance of drug resistance to first line drugs such as antimonials and amphotericin B. In the present study, by using a murine model of leishmaniasis we evaluated the function played by soluble leishmanial antigen (SLA pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs in restricting the intracellular parasitic growth. We establish that a single dose of SLA-CpG-DCs vaccination is sufficient in rendering complete protection against Leishmania donovani infection. In probing the possible mechanism, we observed that SLA-CpG-DCs vaccination results in the significant decrease in Foxp3+GITR+CTLA4+CD4+CD25+ Treg cell population in Leishmania-infected mice. Vaccination with these antigen stimulated dendritic cells results in the decrease in the secretion of TGF-β by these Treg cells by possible regulation of the SMAD signalling. Moreover, we demonstrated that a CXC chemokine, IFN-γ-inducible protein 10 (IP-10, has a direct role in the regulation of CD4+CD25+ Treg cells in SLA-CpG-DCs vaccinated parasitized mice as Treg cells isolated from IP-10 depleted vaccinated mice showed significantly increased TGF-β production and suppressive activity.

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

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    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.

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

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    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...

  12. Patchy Parasitized Skin Governs Leishmania donovani Transmission to Sand Flies.

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    Kamhawi, Shaden; Serafim, Tiago D

    2017-08-31

    Doehl et al. have combined empirical data with computer simulation to demonstrate that RAG-2 mice intravenously infected with Leishmania donovani form heterogeneous skin parasite patches that govern infectiousness to sand flies. This model provides a much-needed tool to explore the relevance of asymptomatic and symptomatic visceral leishmaniasis patients as infection reservoirs. Published by Elsevier Ltd.

  13. Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

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    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

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

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    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.

  15. In vivo activities of farnesyl pyrophosphate synthase inhibitors against Leishmania donovani and Toxoplasma gondii.

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    Yardley, Vanessa; Khan, Anis A; Martin, Michael B; Slifer, Teri R; Araujo, Fausto G; Moreno, Silvia N J; Docampo, Roberto; Croft, Simon L; Oldfield, Eric

    2002-03-01

    The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.

  16. In vitro activity of an essential oil against Leishmania donovani.

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    Monzote, L; García, M; Montalvo, A M; Scull, R; Miranda, M; Abreu, J

    2007-11-01

    The in vitro antileishmanial effect of the essential oil from Chenopodium ambrosioides against Leishmania donovani was investigated. The product showed significant activity against promastigotes and amastigotes, with a 50% effective concentration of 4.45 and 5.1 microg/mL, respectively. The essential oil caused an irreversible inhibition of the growth of promastigotes after a treatment with 100 or 10 microg/mL for 1 or 24 h, respectively. The phagocytic activity of the macrophages was preserved at a concentration toxic to the parasite. The essential oil from C. ambrosioides may be a potential candidate drug to development a new agent to combat this parasitic disease. Copyright (c) 2007 John Wiley & Sons, Ltd.

  17. Genetically Engineered Ascorbic acid-deficient Live Mutants of Leishmania donovani induce long lasting Protective Immunity against Visceral Leishmaniasis.

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    Anand, Sneha; Madhubala, Rentala

    2015-06-02

    Visceral leishmaniasis caused by Leishmania donovani is the most severe systemic form of the disease. There are still no vaccines available for humans and there are limitations associated with the current therapeutic regimens for leishmaniasis. Recently, we reported functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from L. donovani involved in ascorbate biosynthesis pathway. In this study, we have shown that ΔALO parasites do not affect the ability of null mutants to invade visceral organs but severely impair parasite persistence beyond 16 week in BALB/c mice and hence are safe as an immunogen. Both short term (5 week) and long term (20 week) immunization with ΔALO parasites conferred sustained protection against virulent challenge in BALB/c mice, activated splenocytes and resulted in induction of pro-inflammatory cytokine response. Protection in immunized mice after challenge correlated with the stimulation of IFN-γ producing CD4(+) and CD8(+) T cells. Antigen-mediated cell immunity correlated with robust nitrite and superoxide generation, macrophage-derived oxidants critical in controlling Leishmania infection. Our data shows that live attenuated ΔALO parasites are safe, induce protective immunity and can provide sustained protection against Leishmania donovani. We further conclude that the parasites attenuated in their anti-oxidative defence mechanism can be exploited as vaccine candidates.

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

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    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.

  19. Pharmacological activities of cilantro's aliphatic aldehydes against Leishmania donovani.

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    Donega, Mateus A; Mello, Simone C; Moraes, Rita M; Jain, Surendra K; Tekwani, Babu L; Cantrell, Charles L

    2014-12-01

    Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as a potential source of new drugs against leishmaniasis. In this study, we investigated the antileishmanial activity of cilantro (Coriandrum sativum) essential oil and its main components: (E)-2-undecenal, (E)-2-decenal, (E)-2-dodecenal, decanal, dodecanal, and tetradecanal. The essential oil of C. sativum leaves inhibits growth of Leishmani donovani promastigotes in culture with an IC50 of 26.58 ± 6.11 µg/mL. The aliphatic aldehydes (E)-2-decenal (7.85 ± 0.28 µg/mL), (E)-2-undecenal (2.81 ± 0.21 µg/mL), and (E)-2-dodecenal (4.35 ± 0.15 µg/mL), all isolated from C. sativum essential oil, are effective inhibitors of in vitro cultures of L. donovani promastigotes. Aldehydes (E)-2-decenal, (E)-2-undecenal, and (E)-2-dodecenal were also evaluated against axenic amastigotes and IC50 values were determined to be 2.47 ± 0.25 µg/mL, 1.25 ± 0.11 µg/mL, and 4.78 ± 1.12 µg/mL, respectively. (E)-2-Undecenal and (E)-2-dodecenal demonstrated IC50 values of 5.65 ± 0.19 µg/mL and 9.60 ± 0.89 µg/mL, respectively, against macrophage amastigotes. These cilantro compounds showed no cytotoxicity against THP-1 macrophages.

  20. Cathepsin B gene disruption induced Leishmania donovani proteome remodeling implies cathepsin B role in secretome regulation.

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    Teklu Kuru Gerbaba

    Full Text Available Leishmania cysteine proteases are potential vaccine candidates and drug targets. To study the role of cathepsin B cysteine protease, we have generated and characterized cathepsin B null mutant L. donovani parasites. L. donovani cathepsin B null mutants grow normally in culture, but they show significantly attenuated virulence inside macrophages. Quantitative proteome profiling of wild type and null mutant parasites indicates cathepsin B disruption induced remodeling of L. donovani proteome. We identified 83 modulated proteins, of which 65 are decreased and 18 are increased in the null mutant parasites, and 66% (55/83 of the modulated proteins are L. donovani secreted proteins. Proteins involved in oxidation-reduction (trypanothione reductase, peroxidoxins, tryparedoxin, cytochromes and translation (ribosomal proteins are among those decreased in the null mutant parasites, and most of these proteins belong to the same complex network of proteins. Our results imply virulence role of cathepsin B via regulation of Leishmania secreted proteins.

  1. Studies on Stibanate unresponsive isolates of Leishmania donovani

    Indian Academy of Sciences (India)

    Anindita Bhattacharyya; Mandira Mukherjee; Swadesh Duttagupta

    2002-09-01

    Visceral leishmaniasis, also known as kala-azar (KA) is generally caused by Leishmania donovani. Organic pentavalent antimonials (SbV) is the first line of treatment for KA. However, the number of KA patients unresponsive to treatment with Sb(V) is steadily increasing in India and elsewhere. The primary objective of this work is to determine the factor(s) associated with the rise of unresponsiveness. Analysis of the clonal population of parasites clearly indicated that wild type parasites isolated from KA patients who were clinically cured after treatment with Sb(V), were a mixture of resistant and sensitive cells. The resistant promastigotes were also resistant as amastigotes in vivo. It was further observed that Stibanate sensitive parasites can be made resistant to the drug by repeated passages in experimental animals followed by incomplete treatment with suboptimal doses of the drug. These results suggest that the steady rise in Sb(V) unresponsiveness of KA patients in India is due to infection with resistant parasites, generated as a result of irregular and often incomplete treatment of the patients.

  2. Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans.

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    Hide, M; Bras-Gonçalves, R; Bañuls, A L

    2007-03-01

    Leishmania infantum and Leishmania donovani both pertain to the L. (L.) donovani complex and are responsible for visceral leishmaniasis. To explore the L. donovani complex, we focused our study on cysteine protease B (cpb) and especially on 2 cpb copies: cpbE and cpbF. We selected cpb genes because of their phylogenetic interest and host-parasite interaction involvement. Sequencing these 2 copies revealed (i) that cpbE is specific to L. infantum and cpbF is specific to L. donovani and (ii) that these 2 copies are different in length and sequence. Phylogenetic analysis and protein predictions were carried out in order to compare these copies (i) with other trypanosomatid cpb, especially L. mexicana, and (ii) within the L. donovani complex. Our results revealed patterns specific to the L. donovani complex such as the COOH-terminal extension, potential epitopes and N-glycosylation sites. Moreover, phylogenetic analysis revealed different levels of polymorphism between L. infantum and L. donovani and confirmed the ancestral status of the latter. L. infantum has a shorter sequence and a deleted sequence responsible for modifications in protein conformation and catalytic triad. Considering the clinical aspect, L. infantum dermotropic strains appeared more polymorphic than L. infantum viscerotropic strains.

  3. Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Ismail, A

    1998-01-01

    Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11...... as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63...... Sudanese visceral leishmaniasis patients (VL) and the percentage of patients with anti-KMP-11 antibodies in ELISA were 37% (KMP-11-1), 30% (KMP-11-2) and 58% (KMP-11-3). The fraction of VL patients with measurable antibody reactivity in one or more of the three ELISAs was 79%. Cross-reactivity to the KMP...

  4. Natural infection of the opossum Didelphis albiventris (Marsupialia, Didelphidae with Leishmania donovani, in Brazil

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    Ítalo A. Sherlock

    1984-12-01

    Full Text Available An opossum, Didelphis albiventris, from Jacobina, bahia State, was found naturally infected with Leishmania donovani, being the first non-canid wild mammal to be detected with agent of kala-azar in the New World.Um gambá, Didelphis albiventris, de Jacobina, Bahia, foi encontrado com infecção natural pela Leishmania donovani, sendo o primeiro mamífero silvestre não-canídeo a ser achado com o agente do calazar nas Américas.

  5. Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from Leishmania donovani.

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    Saurabh Pratap Singh

    Full Text Available In addition to the S-adenosylmethionine decarboxylase (AD present in all organisms, trypanosomatids including Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL. Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.

  6. Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

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    Samant, Mukesh; Gupta, Reema; Kumari, Shraddha; Misra, Pragya; Khare, Prashant; Kushawaha, Pramod Kumar; Sahasrabuddhe, Amogh Anant; Dube, Anuradha

    2009-07-01

    Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

  7. Interferon-¿ and interleukin-4 in human Leishmania donovani infections

    DEFF Research Database (Denmark)

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

    1993-01-01

    can direct the T cell response into Th1 or Th2 type. If similar mechanisms operate in humans, the outcome of L. donovani infections may depend on the local cytokine environment in which early activation of Leishmania specific T cells takes place. Cytokines secreted by cross-reactive memory T cells...

  8. Diagnostic Antigens of Leishmania.

    Science.gov (United States)

    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

  9. Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

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    Laura-Isobel McCall

    2015-03-01

    Full Text Available Leishmania protozoan parasites (Trypanosomatidae family are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51 in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.

  10. Bioactivity guided fractionation of Moringa oleifera Lam. flower targeting Leishmania donovani.

    Science.gov (United States)

    Singh, Manoj Kumar; Paul, Joydeep; De, Tripti; Chakraborti, Tapati

    2015-11-01

    Leishmaniases is a group of diseases caused by the protozoan parasite belonging to the genus Leishmania. At least 20 species of Leishmania are known to infect humans transmitted by female sandflies, Phlebotomus spp. Leishmania donovani causes visceral leishmaniasis, considered most lethal among the common three forms of leishmaniasis. Lack of appropriate vaccines, emergence of drug resistance and side effects of currently used drugs stress the need for better alternative drugs, particularly from natural sources. Here, we conducted in vitro and in vivo experiments to study the efficacy of different parts of Moringa oleifera Lam. against Leishmania donovani promastigotes. The flower extract of M. oliefera (MoF) was found to be the most potent antileishmanial agent when compared to other parts of the plant like leaf, root, bark and stem. It imparted significant reduction in parasite number in infected macrophages. The bioactivity guided fractionation of MoF showed ethyl acetate fraction (MoE) as the most active and gave significant parasite reduction in the infected macrophages. Further, growth kinetics studies revealed loss of L. donovani promastigotes viability in the presence of MoE in both time and dose dependent manner. In vivo experiment in Balb/c mouse model of leishmaniasis supported the in vitro findings with a remarkable reduction of the parasite burden in both liver and spleen.

  11. Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes

    Science.gov (United States)

    Boitz, Jan M.; Gilroy, Caslin A.; Olenyik, Tamara D.; Paradis, Dustin; Perdeh, Jasmine; Dearman, Kristie; Davis, Madison J.; Yates, Phillip A.; Li, Yuexin; Riscoe, Michael K.; Ullman, Buddy

    2016-01-01

    ABSTRACT Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite. PMID:27795357

  12. Leishmania Donovani Cell Surface Sialoglycans Regulate Susceptibility for Siglec Mediated Macrophage Invasion and Parasite Survival

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    Tripti De

    2012-02-01

    Full Text Available Glycoconjugates play a pivotal role in the survival of Leishmania parasites in destructive surroundings. An important constituent present on many glycoconjugates is sialic acid. By virtue of their peripheral position on oligosaccharide chains of glycoconjugates, sialic acids are well suited as molecular determinants of specific biological processes, including the interaction of pathogenic microorganisms with sialylated cellular receptors. Differences in a2,3- and a2,6-sialoglycan patterns detected in clonal virulent Leishmania donovani promastigotes, correlated with the level of a2,3- and a2,6-sialyltransferase activity present in these parasites. The role of macrophage sialic acid-receptors in uptake and survival of L.donovani was studied in the murine macrophage cell line raw 264.7. Macrophage invasion was dependent on the binding to Siglec-1, while suppression of MAPK signaling was mediated through Siglec-5. Sialic acid removal by neuraminidase treatment reduced parasite infectivity. The presence of trypsin resistant sialic acid residues in the neuraminidase treated parasites grown in a serum free medium in presence of sialoglycoconjugates indicated that the parasites could salvage sialic acid from exogenous sialoglycans and reutilize it for de novo glycoprotein sialylation in L.donovani parasites. Thus, our results demonstrate the involvement of sialoglycans in the invasion as well as the survival process of L.donovani parasites.

  13. Linking in vitro and in vivo survival of clinical Leishmania donovani strains.

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    Manu Vanaerschot

    Full Text Available BACKGROUND: Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL, and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these 'hostile' environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG was used for over 70 years in the Indian subcontinent. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain. CONCLUSIONS/SIGNIFICANCE: The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.

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

    Science.gov (United States)

    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

  15. Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

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    William P Lafuse

    Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

  16. Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties.

    Science.gov (United States)

    Hudspeth, Elissa M; Wang, Qian; Seid, Christopher A; Hammond, Molly; Wei, Junfei; Liu, Zhuyun; Zhan, Bin; Pollet, Jeroen; Heffernan, Michael J; McAtee, C Patrick; Engler, David A; Matsunami, Risë K; Strych, Ulrich; Asojo, Oluwatoyin A; Hotez, Peter J; Bottazzi, Maria Elena

    2016-07-02

    Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.

  17. Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management.

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    Abul Hasan Sardar

    2016-03-01

    Full Text Available Reactive oxygen and nitrogen species (ROS and RNS produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL. There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed

  18. Leishmania donovani HslV does not interact stably with HslU proteins.

    Science.gov (United States)

    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.

  19. CYP5122A1, a novel cytochrome P450 is essential for survival of Leishmania donovani.

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    Smriti Verma

    Full Text Available BACKGROUND: Cytochrome P450s (CYP450s are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: A CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca(2+ and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol

  20. Functional and phenotypic changes in human lymphocytes after coincubation with Leishmania donovani in vitro

    DEFF Research Database (Denmark)

    Hviid, L; Sørensen, A L; Kharazmi, A

    1990-01-01

    that the inhibition of the proliferative response to PHA by live L. donovani in vitro is associated with early processes in lymphocyte activation. Further studies on the inhibitory phenomena described may be of potential significance in the investigation of the suppressive mechanisms in human visceral leishmaniasis.......In this paper we describe functional and phenotypic changes in T cells after in vitro coincubation of peripheral blood mononuclear cells (PBMC) and Leishmania donovani parasites at different parasite/peripheral blood mononuclear cell ratios. The phytohemagglutinin (PHA)-induced lymphoproliferative...... response was reduced by the coincubation, and at the maximal parasite/peripheral blood mononuclear cell ratio used (7.5:1), the average response was less than 40% of the response in the absence of parasites. The cause of the reduction in lymphoproliferation is not clear, but it requires live parasites...

  1. Imipramine is an orally active drug against both antimony sensitive and resistant Leishmania donovani clinical isolates in experimental infection.

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    Sandip Mukherjee

    Full Text Available BACKGROUND: In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF β level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity. CONCLUSIONS: This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.

  2. Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents.

    Science.gov (United States)

    Kassahun, Aysheshm; Sadlova, Jovana; Dvorak, Vit; Kostalova, Tatiana; Rohousova, Iva; Frynta, Daniel; Aghova, Tatiana; Yasur-Landau, Daniel; Lemma, Wessenseged; Hailu, Asrat; Baneth, Gad; Warburg, Alon; Volf, Petr; Votypka, Jan

    2015-05-01

    Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia

  3. Latent infection with Leishmania donovani in highly endemic villages in Bihar, India.

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    Epco Hasker

    Full Text Available INTRODUCTION: Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India. METHODS: We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants. RESULTS: We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30. Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years. DISCUSSION: Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

  4. The epidemiology of Leishmania donovani infection in high transmission foci in India

    DEFF Research Database (Denmark)

    Singh, Shri P; Picado, Albert; Boelaert, Marleen

    2010-01-01

    OBJECTIVE: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission...... the prevalence of serologically positive individuals taking into account the clustering at household and cluster levels. RESULTS: Of study subjects 18% were DAT positive, and this proportion increased with age. Women had a significantly lower prevalence than men >14 years old. Owning domestic animals (cows...

  5. Activity of dihydroartemisinin against Leishmania donovani both in vitro and vivo

    Institute of Scientific and Technical Information of China (English)

    马莹; 芦殿梅; 陆小军; 廖琳; 胡孝素

    2004-01-01

    @@ Visceral leishmaniasis (VL), also known as kala-azar, is a vector-borne disease caused by protozoa of the Leishmania donovani complex. It is usually fatal if untreated. Pentavalent antimonials have been the mainstay of treatment for more than 60 years, but these drugs have serious side effects and progressive antimonial resistance.1 Other proven therapeutics such as amphotericin B, pentamidine, and paromomycin are costly without oral formulation and unpleasant side effects.2 Thus, new chemotherapeutic drugs are required to supplement or replace currently available drugs.

  6. Comparative study of structural models of Leishmania donovani and human GDP-mannose pyrophosphorylases.

    Science.gov (United States)

    Daligaux, Pierre; Bernadat, Guillaume; Tran, Linh; Cavé, Christian; Loiseau, Philippe M; Pomel, Sébastien; Ha-Duong, Tâp

    2016-01-01

    Leishmania is the parasite responsible for the neglected disease leishmaniasis. Its virulence and survival require biosynthesis of glycoconjugates, whose guanosine diphospho-d-mannose pyrophosphorylase (GDP-MP) is a key player. However, experimentally resolved structures of this enzyme are still lacking. We herein propose structural models of the GDP-MP from human and Leishmania donovani. Based on a multiple sequences alignment, the models were built with MODELLER and then carefully refined with all atom molecular dynamics simulations in explicit solvent. Their quality was evaluated against several standard criteria, including their ability to bind GDP-mannose assessed by redocking calculations. Special attention was given in this study to interactions of the catalytic site residues with the enzyme substrate and competitive inhibitors, opening the perspective of medicinal chemistry developments. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  7. Phytochemical screening of the exudate of Aloe otallensis and its effect on Leishmania donovani

    Institute of Scientific and Technical Information of China (English)

    Zerihun Tesfaye Nigusse; WoldeaAbebe Wondifraw; Sefinew MigbaruAbate

    2016-01-01

    Objective: To evaluate the antileishmanial activity of methanolic extract of Aloe otallensis (A. otallensis) on the promastigote stage of Leishmania donovani (L. donovani) as compared to standard drugs and to screen its phytochemical constituents. Methods: Phytochemical screening was done by using the method mentioned by Evans and Trease on methanolic extract of the exudates of Aloe otallensis leaves. The extract was also evaluated for in vitro antileishmanial activity against L. donavani which is found from the Parasitology Unit of Black Lion Hospital. The result was compared to standard drugs of sodium stibogluconate, milfostin and paramomycin. Results: The extract has a good antileishmanial activity with an IC50 of 0.1230 μg/mL on L. donovani (AM 563). The experimental data showed that relatively it had better activity than paramomycin and milfostin but less activity than sodium stibogluconate. The data analyses were done by GraphPad Prism version 5 software after it was read by ELISA reader at the wave length of 650 nm. The phytochemical screening of the exudates of A. otallensis showed the presence of phenol, alkaloid and saponin. Conclusions: The methanol extract of the exudates of A. otallensis has a good anti-leishmaniasis activity and this may be attributed to phenol, alkaloid and saponin present in the plant. But it needs further analysis for the conformation of which constituent presents in high concentration to know which one has the strongest effect.

  8. Induction of Mitochondrial Dysfunction and Oxidative Stress in Leishmania donovani by Orally Active Clerodane Diterpene

    Science.gov (United States)

    Kathuria, Manoj; Bhattacharjee, Arindam; Sashidhara, Koneni V.; Singh, Suriya Pratap

    2014-01-01

    This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL. PMID:25070112

  9. Phytochemical screening of the exudate of Aloe otallensis and its effect on Leishmania donovani

    Directory of Open Access Journals (Sweden)

    Zerihun Tesfaye Nigusse

    2016-06-01

    Full Text Available Objective: To evaluate the antileishmanial activity of methanolic extract of Aloe otallensis (A. otallensis on the promastigote stage of Leishmania donovani (L. donovani as compared to standard drugs and to screen its phytochemical constituents. Methods: Phytochemical screening was done by using the method mentioned by Evans and Trease on methanolic extract of the exudates of Aloe otallensis leaves. The extract was also evaluated for in vitro antileishmanial activity against L. donavani which is found from the Parasitology Unit of Black Lion Hospital. The result was compared to standard drugs of sodium stibogluconate, milfostin and paramomycin. Results: The extract has a good antileishmanial activity with an IC50 of 0.1230 μg/mL on L. donovani (AM 563. The experimental data showed that relatively it had better activity than paramomycin and milfostin but less activity than sodium stibogluconate. The data analyses were done by GraphPad Prism version 5 software after it was read by ELISA reader at the wave length of 650 nm. The phytochemical screening of the exudates of A. otallensis showed the presence of phenol, alkaloid and saponin. Conclusions: The methanol extract of the exudates of A. otallensis has a good antileishmaniasis activity and this may be attributed to phenol, alkaloid and saponin present in the plant. But it needs further analysis for the conformation of which constituent presents in high concentration to know which one has the strongest effect.

  10. H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

    Energy Technology Data Exchange (ETDEWEB)

    Blackwell, J.M.; Hale, C.; Roberts, M.B.; Ulczak, O.M.; Liew, F.Y.; Howard, J.G.

    1985-01-01

    The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncure phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.

  11. Leishmanization revisited: immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis.

    Science.gov (United States)

    McCall, Laura-Isobel; Zhang, Wen-Wei; Ranasinghe, Shanlindra; Matlashewski, Greg

    2013-02-27

    Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa and associated with three main clinical presentations: cutaneous, mucocutaneous and visceral leishmaniasis. Visceral leishmaniasis is the second most lethal parasitic disease after malaria and there is so far no human vaccine. Leishmania donovani is a causative agent of visceral leishmaniasis in South East Asia and Eastern Africa. However, in Sri Lanka, L. donovani causes mainly cutaneous leishmaniasis, while visceral leishmaniasis is rare. We investigate here the possibility that the cutaneous form of L. donovani can provide immunological protection against the visceral form of the disease, as a potential explanation for why visceral leishmaniasis is rare in Sri Lanka. Subcutaneous immunization with a cutaneous clinical isolate from Sri Lanka was significantly protective against visceral leishmaniasis in BALB/c mice. Protection was associated with a mixed Th1/Th2 response. These results provide a possible rationale for the scarcity of visceral leishmaniasis in Sri Lanka and could guide leishmaniasis vaccine development efforts.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy.

    Directory of Open Access Journals (Sweden)

    Koushik Roy

    2016-05-01

    Full Text Available Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ of Leishmania donovani (LD infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation.MΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ. Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5-2 was conformation specific, whereas the other one (10.2.16 was not. Under parasitized condition, the binding of 11.5-2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5-2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ.The decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of the peptide. Furthermore, liposomal delivery of

  14. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L....... donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL...... for malaria but free of leishmaniasis was negative in both assays....

  15. Identification and functional characterization of Leishmania donovani secretory peroxidase: delineating its role in NRAMP1 regulation.

    Directory of Open Access Journals (Sweden)

    Nisha Singh

    Full Text Available Leishmania silently evades host immune system and establish in the hostile environment of host macrophage phagolysosomes. For differentiation, growth and division parasite acquires divalent cations especially iron from the host nutritive pool. Natural resistance associated with macrophage protein1 (NRAMP1, a cation transporter that effluxes out divalent cations specifically iron from phagosomal milieu to the cytosol, to create ions deprived status for pathogenic microorganisms. The mechanisms of NRAMP1 regulation are largely unknown in leishmanial infections. In the present study, we identified a secretory Leishmania donovani peroxidase (Prx that showed peroxidoxin like peroxidase activity and significantly reduced H(2O(2, O(2.(- and NO levels in LPS activated macrophages. Further, we also observed down regulated Nramp1 expression and concomitantly declined labile iron pool in activated macrophages treated with identified peroxidase. Prx also decreased levels of TNF-α, IFN-γ and IL-12 in LPS activated macrophages. These observations indicate a bifunctional protective role of secretory Prx; first it reduces redox activation of macrophages, and secondly it allows iron access to Leishmania by down regulating NRAMP1 expression.

  16. Involvement of Leishmania donovani major surface glycoprotein gp63 in promastigote multiplication

    Indian Academy of Sciences (India)

    Sanjeev Pandey; Phuljhuri Chakraborti; Rakhi Sharma; Santu Bandyopadhyay; Dwijen Sarkar; Samit Adhya

    2004-03-01

    The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process.

  17. Epidemiology of Leishmania donovani infection in high-transmission foci in Nepal

    DEFF Research Database (Denmark)

    Rijal, Suman; Uranw, Surendra; Chappuis, François

    2010-01-01

    Nepal. METHODS: Ten clusters with highest VL incidence rates were purposefully selected in Nepal. All households were mapped and socio-demographic data and data on past VL incidence were collected. An exhaustive serological survey was performed of individuals aged >2 years, by collecting finger prick......OBJECTIVE: Nepal reports a visceral leishmaniasis (VL) incidence of 5 per 10 000 per year on the basis of notification by health facilities, but little community-based epidemiological information exists. We report data on prevalence rates of Leishmania donovani infection in ten communities in East...... blood on filter paper in November-December 2006. The samples were tested by direct agglutination, and a titre >or=1:1600 was taken as marker of infection. A generalized estimating equation (GEE) model was used to assess risk factors for Direct Agglutination Test (DAT) positivity taking into account...

  18. Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

    Science.gov (United States)

    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.

  19. Transfer of innate resistance and susceptibility to Leishmania donovani infection in mouse radiation bone marrow chimaeras

    Energy Technology Data Exchange (ETDEWEB)

    Crocker, P.R.; Blackwell, J.M.; Bradley, D.J. (London School of Hygiene and Tropical Medicine (UK))

    1984-07-01

    Reciprocal radiation bone marrow chimaeras were made between H-2-compatible strains of mice innately resistant or susceptible to visceral leishmaniasis. In initial experiments, susceptibility but not resistance to Leishmania donovani could be transferred with donor bone marrow into irradiated recipients. In subsequent experiments it was possible to transfer both resistance and susceptibility. This was achieved either by selecting more radiosensitive mouse strains as susceptible recipients, or alternatively by increasing the irradiation dose for the susceptible recipients used in the initial experiments. Using the higher irradiation dose, successful transfer of resistance and susceptibility between congenic mice carrying the Lshsup(r) and Lshsup(s) alleles on the more radioresistant B10 genetic background provided firm evidence that the results obtained in this study were specifically related to expression of the Lsh gene. It is concluded that Lsh gene-controlled resistance and susceptibility to L. donovani is determined by bone marrow-derived cells. The cell type(s) involved is likely to be of the macrophage lineage.

  20. CD4+ Recent Thymic Emigrants Are Recruited into Granulomas during Leishmania donovani Infection but Have Limited Capacity for Cytokine Production

    Science.gov (United States)

    Moore, John W. J.; Beattie, Lynette; Osman, Mohamed; Owens, Benjamin M. J.; Brown, Najmeeyah; Dalton, Jane E.; Maroof, Asher

    2016-01-01

    Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4+ recent thymic emigrants emerging into the periphery of mice with ongoing Leishmania donovani infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4+ RTEs displayed severely restricted differentiation either into IFNγ+ or IFNγ+TNFα+ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4+ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment. PMID:27658046

  1. Single locus genotyping to track Leishmania donovani in the Indian subcontinent: Application in Nepal.

    Science.gov (United States)

    Rai, Keshav; Bhattarai, Narayan Raj; Vanaerschot, Manu; Imamura, Hideo; Gebru, Gebreyohans; Khanal, Basudha; Rijal, Suman; Boelaert, Marleen; Pal, Chiranjib; Karki, Prahlad; Dujardin, Jean-Claude; Van der Auwera, Gert

    2017-03-01

    We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010. As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002-2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution. Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region.

  2. Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

    OpenAIRE

    Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

    2017-01-01

    Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, res...

  3. Leishmania donovani lacking the Golgi GDP-Man transporter LPG2 exhibit attenuated virulence in mammalian hosts

    Science.gov (United States)

    Gaur, Upasna; Showalter, Melissa; Hickerson, Suzanne; Dalvi, Rahul; Turco, Salvatore J.; Wilson, Mary E.; Beverley, Stephen M.

    2009-01-01

    Surface phosophoglycans such as lipophosphoglycan (LPG) or proteophosphoglycan (PPG) and glycosylinositol phospholipids (GIPLs) modulate essential interactions between Leishmania and mammalian macrophages. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by LPG2. LPG2-null (lpg2−) L. major cannot establish macrophage infections or induce acute pathology, whereas lpg2− L. mexicana retain virulence. lpg2− L. donovani has been reported to survive poorly in cultured macrophages but in vivo survival has not been explored. Herein we discovered that, similar to lpg2− L. major, lpg2− L. donovani promastigotes exhibited diminished virulence in mice, but persisted at consistently low levels. lpg2− L. donovani promastigotes could not establish infections macrophages and could not transiently inhibit phagolysosomal fusion. Furthermore, lpg2− promastigotes of L. major, L. donovani and L. mexicana were highly susceptible to complement mediated lysis. We conclude that phosphoglycan assembly and expression mediated by L. donovani LPG2 are important for promastigote and amastigote virulence, unlike L. mexicana but similar to L. major. PMID:19328787

  4. Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

    Science.gov (United States)

    Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

    2016-05-01

    A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

  5. Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

    Science.gov (United States)

    Verma, Sudha; Das, Sushmita; Mandal, Abhishek; Ansari, Md Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, Ruby; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

    2017-06-23

    In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to

  6. In vivo antileishmanial action of Ir-(COD-pentamidine tetraphenylborate on Leishmania donovani and Leishmania major mouse models

    Directory of Open Access Journals (Sweden)

    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.

  7. Activation of Phosphotyrosine Phosphatase Activity Attenuates Mitogen-Activated Protein Kinase Signaling and Inhibits c-FOS and Nitric Oxide Synthase Expression in Macrophages Infected with Leishmania donovani

    OpenAIRE

    Nandan, Devki; Lo, Raymond; Reiner, Neil E

    1999-01-01

    Intracellular protozoan parasites of the genus Leishmania antagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact of Leishmania donovani on mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PM...

  8. Intracellular replication-deficient Leishmania donovani induces long lasting protective immunity against visceral leishmaniasis.

    Science.gov (United States)

    Selvapandiyan, Angamuthu; Dey, Ranadhir; Nylen, Susanne; Duncan, Robert; Sacks, David; Nakhasi, Hira L

    2009-08-01

    No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a live attenuated centrin gene-deleted L. donovani (LdCen1(-/-)) parasite can persist and be both safe and protective in animals. LdCen1(-/-) has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10 wk after infection. Mice immunized with LdCen1(-/-) showed early clearance of virulent parasite challenge not seen in mice immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4(+) T cell population a significant increase of single and multiple cytokine (IFN-gamma, IL-2, and TNF) producing cells and IFN-gamma/IL10 ratio. Immunized mice also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1-type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters. Immunization with LdCen1(-/-) also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leishmaniasis. Results indicate that LdCen1(-/-) can be a safe and effective vaccine candidate against VL as well as mucocutaneous leishmaniasis causing parasites.

  9. Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

    Directory of Open Access Journals (Sweden)

    Moumita Ghosh

    2014-12-01

    Full Text Available The protozoan parasite Leishmania donovani (LD reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2 on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting.To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.

  10. Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

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    Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

    2014-02-27

    The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

  11. Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani.

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    Jessica L Martin

    2014-02-01

    Full Text Available The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

  12. Abietane-Type Diterpenoid Amides with Highly Potent and Selective Activity against Leishmania donovani and Trypanosoma cruzi.

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    Pirttimaa, Minni; Nasereddin, Abedelmajeed; Kopelyanskiy, Dmitry; Kaiser, Marcel; Yli-Kauhaluoma, Jari; Oksman-Caldentey, Kirsi-Marja; Brun, Reto; Jaffe, Charles L; Moreira, Vânia M; Alakurtti, Sami

    2016-02-26

    Dehydroabietylamine (1) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, Leishmania donovani and Trypanosoma cruzi, were assayed. The most potent compound, 10, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC50 value of 0.37 μM against L. donovani axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound 10 fully inhibited the growth of intracellular amastigotes in Leishmania donovani-infected human macrophages with a low IC50 value of 0.06 μM. This compound was also highly effective against T. cruzi amastigotes residing in L6 cells with an IC50 value of 0.6 μM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease.

  13. The neurotrophic receptor Ntrk2 directs lymphoid tissue neovascularization during Leishmania donovani infection.

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    Jane E Dalton

    2015-02-01

    Full Text Available The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB and its ligands brain derived neurotrophic factor (Bdnf, neurotrophin-4 (NT-4/5, and neurotrophin-3 (NT-3 are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hiCD11b(loCD11c(+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.

  14. Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

    Science.gov (United States)

    McAtee, C Patrick; Seid, Christopher A; Hammond, Molly; Hudspeth, Elissa; Keegan, Brian P; Liu, Zhuyun; Wei, Junfei; Zhan, Bin; Arjona-Sabido, Raul; Cruz-Chan, Vladimir; Dumonteil, Eric; Hotez, Peter J; Bottazzi, Maria Elena

    2017-02-01

    The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT).

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    Kima, Peter E; Bonilla, J Alfredo; Cho, Eumin; Ndjamen, Blaise; Canton, Johnathan; Leal, Nicole; Handfield, Martin

    2010-10-05

    Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.

  16. Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT.

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    Peter E Kima

    2010-10-01

    Full Text Available Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.

  17. Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani.

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    Bhavna Chawla

    Full Text Available Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.

  18. Concomitant Infection with Leishmania donovani and L. major in Single Ulcers of Cutaneous Leishmaniasis Patients from Sudan

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    A. M. Babiker

    2014-01-01

    Full Text Available In Sudan human leishmaniasis occurs in different clinical forms, that is, visceral (VL, cutaneous (CL, mucocutaneous (ML, and post-kala-azar dermal leishmaniasis (PKDL. Clinical samples from 69 Sudanese patients with different clinical manifestations were subjected to a PCR targeting the cytochrome oxidase II (COII gene for Leishmania species identification. Mixed infections were suspected due to multiple overlapping peaks presented in some sequences of the COII amplicons. Cloning these amplicons and alignment of sequences from randomly selected clones confirmed the presence of two different Leishmania species, L. donovani and L. major, in three out of five CL patients. Findings were further confirmed by cloning the ITS gene. Regarding other samples no significant genetic variations were found in patients with VL (62 patients, PKDL (one patient, or ML (one patient. The sequences clustered in a single homogeneous group within L. donovani genetic group, with the exception of one sequence clustering with L. infantum genetic group. Findings of this study open discussion on the synergetic/antagonistic interaction between divergent Leishmania species both in mammalian and vector hosts, their clinical implications with respect to parasite fitness and response to treatment, and the route of transmission with respect to vector distribution and or adaptation.

  19. Infecção experimental de Calomys callosus (Rodentia-Cricetidae) com Leishmania donovani chagasi (Laison, 1982) Experimental infection of Calomys callosus (Rodentia-Cricetidae) with Leishmania donovani

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    Mello,Dalva A.; Maria Lucia Teixeira

    1984-01-01

    Foi descrita a infecção experimental em Calomys callosus com uma cepa de Leishmania donovani chagasi de caso humano. Um grupo de 22 roedores foi inoculado por via intraperitoneal com 0,1 ml de um macerado de baço em salina, rico em amastigotas. Esses animais foram sacrificados três meses após as inoculações, tendo sido realizado: cultura "in vitro" em meio acelular (LIT e NNN) e esfregaços, corados pelo Giemsa, de fígado, baço, medula óssea e sangue; cortes histológicos corados com hematoxili...

  20. Identification of R2TP complex of Leishmania donovani and Plasmodium falciparum using genome wide in-silico analysis.

    Science.gov (United States)

    Ahmad, Moaz; Afrin, Farhat; Tuteja, Renu

    2013-11-01

    Recently discovered R2TP complex is an important multiprotein complex involved in multiple cellular process like snoRNP biogenesis, PIKK signaling, RNA polymerase II assembly and apoptosis. Within R2TP complex, Pih1 tightly interacts with Rvb1/Rvb2 and with Tah1 to form R2TP macromolecular complex. R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex, which has been found critical in many cellular process. The genome wide screening of Leishmania donovani and Plasmodium falciparum led to the identification of RuvB like1, RuvB like 2, Pih1, and Tah1. Therefore, we speculate that this complex is also important for these parasites as in the yeast. The detailed analysis of crucial components of R2TP complex, Ld-RuvB like 1, and Ld-RuvB like 2, revealed the presence of characteristic motifs like DNA binding motif and ATPase motifs. Hsp90 is also reported from Leishmania donovani and Plasmodium falciparum suggesting that the R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex. Recently it has been discovered that RuvB like proteins are overexpressed in many cancers and their ATPase activity is crucial for cancer cell proliferation and the human RuvBs have been proposed as suitable drug target for cancer. Similarly one of the Plasmodium falciparum RuvB like protein (PfRuvB3) has been found to be specific to the stage where nuclear division led multiplication of parasite take place. Considering all these it seems that the R2TP complex may be playing some critical role both in the cancer cell proliferation in human and rapid multiplication of the parasites Leishmania donovani and Plasmodium falciparum.

  1. Interaccion de promastigotes de Leishmania donovani con celulas de exudado peritonial de ratones mediante la influencia de la quimotripsina

    OpenAIRE

    Arcay,Lucila; Bruzual,Elizabeth

    1986-01-01

    Se hace un estudio de la interacción de promastigotes de Leishmania donovani con células de exudado peritoneal de ratón (c e p) mediante la influencia de la quimotripsina. La adhesión de los promastigotes a las c e p fue terminal y marginal, y en observaciones hechas a partir de los 10 minutos de enfrentamiento, esta adhesión fue nula hasta los 30 minutos en el grupo tratado, y sólo a las das horas hubo un pequeño incremento (2.4%) con respecto al control. Se observa marcada disminución en to...

  2. Anti-citrullinated peptide antibodies in Sudanese patients with Leishmania donovani infection exhibit reactivity not dependent on citrullination.

    Science.gov (United States)

    Åhlin, E; Elshafie, A I; Nur, M A M; Rönnelid, J

    2015-03-01

    African patients with Leishmania donovani infections have signs of strong systemic inflammation and high levels of circulating immune complexes (IC) and rheumatoid factor (RF), all serologic markers of rheumatic disease. As inflammation in general is associated with citrullination, we sought to investigate ACPA responses in Sudanese Leishmania patients. Serum samples were collected from Sudanese patients with visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) as well as from ACPA-positive Sudanese rheumatoid arthritis patients and compared to healthy Sudanese controls. Levels of circulating C1q-binding IC and anticyclic citrullinated peptide 2(CCP2) were investigated using ELISA, and RF was measured with nephelometry. C1q adsorption was carried out to investigate anti-CCP2 content in IC. Citrulline specificity was evaluated with control plates with cyclic arginine-containing control peptides. Leishmania-infected patients had elevated levels of RF and circulating IC but also a significant increase in anti-CCP2 (12%) as compared to healthy controls. Anti-CCP2-positive Leishmania patients displayed lower anti-CCP2 levels than Sudanese patients with rheumatoid arthritis (RA), and anti-CCP2 levels in Leishmania patients showed a continuum not resembling the dichotomous pattern seen in patients with RA. Whereas the anti-CCP reactivity of Sudanese RA sera was strictly citrulline dependent, anti-CCP2-positive Leishmania sera reacted equally well with ELISA plates containing arginine control peptides. There was a strong correlation between anti-CCP2 and circulating IC among the Leishmania patients, but IC depletion only marginally diminished anti-CCP2 levels. Our findings stress the importance to interpret a positive CCP test carefully when evaluated in non-rheumatic conditions associated with macrophage activation. © 2015 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of Scandanavian Society of

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

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    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.

  4. Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

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    Venkateswara Reddy Gogulamudi

    2015-10-01

    Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

  5. Cutaneous leishmaniasis caused by Leishmania donovani in the tribal population of the Agasthyamala Biosphere Reserve forest, Western Ghats, Kerala, India.

    Science.gov (United States)

    Kumar, N Pradeep; Srinivasan, R; Anish, T S; Nandakumar, G; Jambulingam, P

    2015-02-01

    Cutaneous leishmaniasis (CL), a neglected tropical disease, is reported to be prevalent in tribal villages located in the Agasthyamala Biosphere Reserve forests of Western Ghats, Kerala state, India. We carried out an investigation to characterize the species of Leishmania parasites involved in these infections prevalent among one of the oldest human tribal populations in India. Skin aspirates collected from 13 clinically diagnosed cases were subjected to histopathological investigations, serological rapid tests using 'rk39' and molecular diagnostics. Clinical manifestations recorded among the patients were hypo-pigmented erythematous nodules/papules on limbs and other parts of the body. Histopathological investigations of these skin lesions among patients showed Leishman-Donovan bodies in macrophages. None of the patients were found to be positive for rk39 tests, which detect active visceral leishmaniasis. Using three different genetic markers [kinetoplast minicircle DNA, 3' UTR region of heat-shock protein 70 (Hsp70) and Hsp70 gene] we identified the parasite species involved in these infections to be Leishmania donovani. The 6-phosphogluconate (6-PGDH) gene sequences of the parasite isolates from Western Ghats indicated close genetic relatedness to L. donovani isolates reported from Sri Lanka, also causing CL. This could be cited as another instance of 'local endemism' of organisms in this single 'bio-geographic unit'.

  6. Lipid Isolated from a Leishmania donovani Strain Reduces Escherichia coli Induced Sepsis in Mice through Inhibition of Inflammatory Responses

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    Subhadip Das

    2014-01-01

    Full Text Available Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of gram-negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of leishmanial total lipid (LTL isolated from Leishmania donovani (MHO/IN/1978/UR6 for amelioration of the inflammatory mediators and transcriptional factor with suppression of TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s of lung injury caused by sepsis, namely, edema, vascular permeability, and pathophysiology, and the status of different cytokine-chemokine(s and adhesion molecule(s. Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against gram-negative bacterial sepsis with pulmonary impairment.

  7. Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis

    Science.gov (United States)

    Barbosa Santos, Micheli Luize; Nico, Dirlei; de Oliveira, Fabrícia Alvisi; Barreto, Aline Silva; Palatnik-de-Sousa, Iam; Carrillo, Eugenia; Moreno, Javier; de Luca, Paula Mello; Morrot, Alexandre; Rosa, Daniela Santoro; Palatnik, Marcos; Bani-Corrêa, Cristiane; de Almeida, Roque Pacheco; Palatnik-de-Sousa, Clarisa Beatriz

    2017-01-01

    Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α−IFN-γ−, CD3+CD4+IL-2+TNF-α+IFN-γ−, CD3+CD4+IL-2+TNF-α−IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = −0.428, P = 0.05) and liver sizes (R = −0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = −0.595, P = 0.005) and F2 (R = −0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α−IFN-γ−, CD3+CD8+IL-2+TNF-α+IFN-γ−, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1

  8. Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection.

    Science.gov (United States)

    Mukherjee, Madhuchhanda; Basu Ball, Writoban; Das, Pijush K

    2014-10-01

    Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3β to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent transcriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.

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

    Directory of Open Access Journals (Sweden)

    Abhishek Mandal

    2016-01-01

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

  10. The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V.

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    Adrien F Vinet

    2009-10-01

    Full Text Available We recently showed that the exocytosis regulator Synaptotagmin (Syt V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galbeta1,4Manalpha1-PO(4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.

  11. Stage-dependent expression and up-regulation of trypanothione synthetase in amphotericin B resistant Leishmania donovani.

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    Asif Equbal

    Full Text Available Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in Leishmania donovani. In this study, TryS gene of L. donovani (LdTryS was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0-8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of L. major are conserved in L. donovani. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼ 2.0-folds than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H2O2 treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H2O2. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in L. donovani and involvement of TryS during oxidative stress to help the parasites survival.

  12. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    Science.gov (United States)

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

  13. Diversity among Leishmania isolates from the Sudan: isoenzyme homogeneity of L. donovani versus heterogeneity of L. major

    DEFF Research Database (Denmark)

    Ibrahim, M E; Evans, D A; Theander, T G

    1995-01-01

    Leishmania isolates from patients in the Sudan suffering from either visceral or cutaneous leishmaniasis were characterized using a battery of 12 enzymes. Aspartate aminotransferase separated the L. donovani isolates into 2 distinct zymodemes, but the overall results showed no significant...

  14. Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants.

    Directory of Open Access Journals (Sweden)

    Neha Biyani

    Full Text Available Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs annotated as AQP9 (230aa, AQP putative (294aa, AQP-like protein (279aa, AQP1 (314aa and AQP-like protein (596aa. We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis

  15. Identification and Characterization of miRNAs in Response to Leishmania donovani Infection: Delineation of Their Roles in Macrophage Dysfunction

    Science.gov (United States)

    Tiwari, Neeraj; Kumar, Vinod; Gedda, Mallikarjuna Rao; Singh, Ashish K.; Singh, Vijay K.; Gannavaram, Sreenivas; Singh, Surya P.; Singh, Rakesh K.

    2017-01-01

    The outcome of Leishmania infection depends on parasite abilities to evade host immune response and its survival in hostile environment of host macrophages. Despite a wealth of gained crucial information, parasite strategies by which it dampens host macrophage functions remain poorly understood. Micro RNAs (miRNAs) are evolutionarily conserved class of endogenous 22-nucleotide small non-coding RNA gene products, described to participate in the regulation of almost every cellular process investigated so far. In this study, we identified 940 miRNAs in Leishmania donovani infected macrophages by de novo sequencing out of which levels of 85 miRNAs were found to be consistently modified by parasite infection. Herein, we report the functional characteristics of 10 miRNAs i.e., mir-3620, mir-6385, mir-6973a, mir-6996, mir-328, mir-8113, mir-3473f, mir-763, mir-6540, and mir-1264 that were differentially but constantly regulated in infected macrophages for their role in regulation of macrophage effector functions. The target gene prediction and biological interaction analysis revealed involvement of these miRNAs in various biological processes such as apoptosis inhibition, phagocytosis, drug response, and T cell phenotypic transitions. These findings could contribute for the better understanding of macrophages dysfunction and leishmanial pathogenesis. Further, the identified miRNAs could also be used as biomarker/s in diagnosis, prognosis, and therapeutics of Leishmania infection. PMID:28303124

  16. Generating knock-in parasites: integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani.

    Science.gov (United States)

    Roberts, Sigrid C; Kline, Chelsey; Liu, Wei; Ullman, Buddy

    2011-06-01

    Leishmania null mutants created by targeted gene replacement are typically complemented with chimeric episomes harboring the replaced gene in order to validate that the observed phenotype is due to the specific gene deletion. However, the current inventory of available episomes for complementation of genetic lesions in Leishmania is unstable in the absence of drug selection, and levels of gene expression cannot be controlled, especially in vivo. To circumvent this impediment, a strategy to re-introduce the targeted gene into the original chromosomal locus to generate "knock-in" parasites within selectable null backgrounds has been developed. A genomic fragment encompassing the ornithine decarboxylase locus and lacking heterologous DNA sequences was transfected into ornithine decarboxylase-deficient Leishmania donovani. The construct randomly integrated into either chromosomal allele by homologous recombination restoring polyamine prototrophy and revealing that LdODC was functionally expressed in the knock-in clones. This strategy offers a mechanism for complementing a genetic lesion amenable to positive selection in a manner that facilitates stable gene expression from its original locus in the absence of continuous drug pressure.

  17. Leptin induces the phagocytosis and protective immune response in Leishmania donovani infected THP-1 cell line and human PBMCs.

    Science.gov (United States)

    Dayakar, Alti; Chandrasekaran, Sambamurthy; Veronica, Jalaja; Maurya, Radheshyam

    2016-01-01

    Visceral leishmaniasis (VL) is an infectious disease responsible for several deaths in malnourished children due to impaired cell-mediated immunity, which is accompanied by low circulating leptin levels. The cytokine function of leptin is implicated for several immune regulation activities such as hematopoiesis, angiogenesis, innate and adaptive immunity. Its deficiency associated with polarization of Th2 response, which coincides with VL pathogenesis. To determine the cytokine role of leptin in case of experimental VL, we tested the leptin associated Th1/Th2 type cytokine profile at mRNA level from Leishmania donovani infected human monocytic leukemia cell line (THP-1) and peripheral blood mononuclear cells (PBMCs). We also tested the effect of leptin on macrophages activation (viz. studying the phosphorylation of signaling moieties), phagocytic activity and intracellular reactive oxygen species (ROS) production during infection. We observed that leptin induced Th1 specific response by upregulation of IL-1α, IL-1β, IL-8 and TNF-α in THP-1 and IFN-γ, IL-12 and IL-2 in PBMCs. We also observed the downregulation of Th2 type cytokine i.e. IL-10 in THP-1 and unaltered expression of cytokines i.e. TGF-β, IL-10 and IL-4 in PBMCs. In addition, leptin stimulates the macrophages by inducing phosphorylation of Erk1/2 and Akt which are usually dephosphorylated in L. donovani infection. In concordance, leptin also induces the macrophage phagocytic activity by enhancing the intracellular ROS generation which helps in phagolysosome formation and oxidative killing of the parasite. In compilation, leptin is able to maintain the defensive environment against L. donovani infection through the classical macrophage activity.

  18. A new bisbenzylisoquinoline alkaloid isolated from Thalictrum foliolosum, as a potent inhibitor of DNA topoisomerase IB of Leishmania donovani.

    Science.gov (United States)

    Kumar, Ashish; Chowdhury, Somenath Roy; Sarkar, Tapas; Chakrabarti, Tulika; Majumder, Hemanta K; Jha, Tarun; Mukhopadhyay, Sibabrata

    2016-03-01

    Chemical investigation of the stem of Thalictrum foliolosum resulted in the isolation of two new bisbenzylisoquinoline alkaloids (1 and 2) along with known protoberberine group of isoquinoline alkaloids thalifendine (3) and berberine (4). The structures of the new compounds were established by detailed 2D NMR spectral analysis with their configurations determined from their optical rotation values and confirmed using circular dichroism. Inhibitory activities of these four compounds against DNA topoisomerase IB of Leishmania donovani were evaluated. Compound 2 exhibited almost complete inhibition of the enzyme activity at 50 μM concentration and it was found to be effective in killing both wild type as well as SAG resistant promastigotes of the parasite.

  19. l-Arginine Uptake by Cationic Amino Acid Transporter Promotes Intra-Macrophage Survival of Leishmania donovani by Enhancing Arginase-Mediated Polyamine Synthesis.

    Science.gov (United States)

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

    2017-01-01

    The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL), depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM) with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS) expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important therapeutic and

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

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    Wai-Lok Yau

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

  1. Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro

    Indian Academy of Sciences (India)

    Jayanta K Pal; Manisha Joshi-Purandare

    2001-06-01

    Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 M) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 M) reduced the level of protein synthesis in general and of -tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

  2. Identification of two natural compound inhibitors of Leishmania donovani Spermidine Synthase (SpdS) through molecular docking and dynamic studies.

    Science.gov (United States)

    V M, Vidhya; Dubey, Vikash Kumar; Ponnuraj, Karthe

    2017-09-05

    Visceral leishmaniasis caused by the protozoan Leishmania donovani is the most severe form of leishmaniasis and it is potentially lethal if untreated. Despite the availability of drugs for treating the disease, the current drug regime suffers from drawbacks like antibiotic resistance and toxicity. New drugs have to be discovered in order to overcome these limitations. Our aim is to identify natural compounds from plant sources as putative inhibitors considering the occurrence of structural diversity in plant sources. Spermidine Synthase (SpdS) was chosen as the target enzyme as it plays a vital role in growth, survival, and due to its contribution in virulence. Our initial investigation started with a literature survey in identifying natural compounds that showed antileishmanial activity. Subsequently, we identified two monoterpenoid compounds, namely Geraniol and Linalool, that were structurally analogous to one of the substrates (putrescine) of SpdS. In the present study, homology model of L. donovani SpdS was generated and the binding affinity of the identified compounds was analyzed and also compared with the putrescine through molecular docking and dynamic studies. The pharmacokinetic properties of the identified compounds were validated and the binding efficiency of these ligands over the original substrate has been demonstrated. Based on these studies, Geraniol and Linalool can be considered as lead molecules for future investigations targeting SpdS. This study further emphasizes the choice of natural compounds as a good source of therapeutic agents.

  3. Anticorpos antipeptídeos citrulinados e fator reumatoide em pacientes sudaneses com infecção por Leishmania donovani Anti-citrullinated peptide antibodies and rheumatoid factor in Sudanese patients with Leishmania donovani infection

    Directory of Open Access Journals (Sweden)

    Erik Ahlin

    2011-12-01

    Full Text Available OBJETIVO: Este estudo avaliou a presença de anticorpos antipeptídeos citrulinados cíclicos (anti-CCP, fator reumatoide (FR e imunocomplexos circulantes (ICC em pacientes sudaneses infectados por Leishmania donovani. PACIENTES E MÉTODOS: Os soros foram coletados de pacientes infectados por Leishmania (n = 116 e de sudaneses saudáveis (n = 93. Dezenove pacientes sudaneses com artrite reumatoide (AR e anti-CCP+ foram incluídos como controles positivos. Os níveis de ICC e anti-CCP foram medidos por ELISA. Para avaliar a reatividade citrulina-específica foi usada a placa-controle com peptídeos-controle cíclicos contendo arginina em vez de citrulina. RESULTADOS: Entre os pacientes infectados por Leishmania e os pacientes com AR e anti-CCP+, a maioria (86% era positiva para FR, enquanto a frequência de positividade para ICC foi maior entre pacientes com leishmaniose visceral (LV (LV 38%; AR e anti-CCP+ 24%. Quando foi analisada a reatividade anti-CCP, 12% dos pacientes com LV foram positivos. Os níveis de anti-CCP entre os pacientes com LV correlacionaram-se bem com os níveis de ICC encontrados (r = 0,65; P OBJECTIVE: The present study evaluated the presence of anti-cyclic citrullinated peptides antibodies (anti-CCP, rheumatoid factor (RF, and circulating immune complexes (CIC in Sudanese patients infected with the Leishmania donovani parasite. PATIENTS AND METHODS: Sera were collected from Leishmania infected patients (n = 116 and healthy Sudanese (n = 93. Nineteen Sudanese anti-CCP+ RA patients were included as positive controls. Levels of CIC and anti-CCP were measured by ELISA. Control plate with cyclic control peptides containing arginine instead of citrulline was used to evaluate citrulline specifi c reactivity. RESULTS: Among Leishmania-infected patients and anti-CCP+ RA patients, most were RF positive (86%, while the frequency of CIC positivity was higher among visceral leishmaniasis (VL patients (VL 38%; anti-CCP+ RA 24%. When

  4. Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

    Science.gov (United States)

    Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

    2017-01-01

    Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

  5. Developing imidazole analogues as potential inhibitor for Leishmania donovani trypanothione reductase: virtual screening, molecular docking, dynamics and ADMET approach.

    Science.gov (United States)

    Pandey, Rajan Kumar; Sharma, Drista; Bhatt, Tarun Kumar; Sundar, Shyam; Prajapati, Vijay Kumar

    2015-01-01

    Visceral leishmaniasis (VL) affects Indian subcontinent, African and South American continent, and it covers 70 countries worldwide. Visceral form of leishmaniasis is caused by Leishmania donovani in Indian subcontinent which is lethal if left untreated. Extensive resistance to antileishmanial drugs such as sodium stibogluconate, pentamidine and miltefosine and their decreased efficacy has been reported in the endemic region. Amphotericin B drug has shown good antileishmanial activity with significant toxicity, but its cost of treatment has limited the outreach of this treatment to affected people living in endemic zone. So, there is an urgent need to identify new antileishmanial drugs with excellent activity and minimal toxicity issues. Trypanothione reductase, a component of antioxidant system, is necessary for parasite growth and survival to raise infection. To develop potential inhibitor, we docked nine hundred and eighty-four 5-nitroimidazole analogues along with clomipramine which is a well-known inhibitor for TR. Total one hundred and forty-seven 5-nitroimidazole analogues with better docking score than clomipramine were chosen for ADMET and QikProp studies. Among these imidazole analogues, total twenty-four imidazole analogues and clomipramine were chosen on the basis of their ADMET, QikProp, and prime MM-GBSA study. Later on, two analogues with best MM-GBSA dG bind were undergone molecular dynamic simulation to ensure protein-ligand interactions. Using above approach, we confirm that ethyl 2-acetyl-5-[4-butyl-2-(3-hydroxypentyl)-5-nitro-1H-imidazol-1-yl]pent-2-enoate can be a drug candidate against L. donovani for the treatment of VL in the Indian subcontinent.

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

    Science.gov (United States)

    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.

  7. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  8. Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani.

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    Sanchita Das

    Full Text Available BACKGROUND: Sodium antimony gluconate (SAG unresponsiveness of Leishmania donovani (Ld had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a, identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. METHODOLOGY AND PRINCIPAL FINDINGS: The expression profile of 60s ribosomal L23a (60sRL23a was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III and was found to be altered towards the resistant mode. CONCLUSION/SIGNIFICANCE: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.

  9. Infecção experimental de Calomys callosus (Rodentia-Cricetidae com Leishmania donovani chagasi (Laison, 1982 Experimental infection of Calomys callosus (Rodentia-Cricetidae with Leishmania donovani

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    Dalva A. Mello

    1984-08-01

    Full Text Available Foi descrita a infecção experimental em Calomys callosus com uma cepa de Leishmania donovani chagasi de caso humano. Um grupo de 22 roedores foi inoculado por via intraperitoneal com 0,1 ml de um macerado de baço em salina, rico em amastigotas. Esses animais foram sacrificados três meses após as inoculações, tendo sido realizado: cultura "in vitro" em meio acelular (LIT e NNN e esfregaços, corados pelo Giemsa, de fígado, baço, medula óssea e sangue; cortes histológicos corados com hematoxilina-eosina de fígado e baço. Os resultados para fígado e baço foram: 67% de positividade nas culturas "in vitro"; esfregaços ricos em amastigotas intra e extra celular (inclui medula óssea; reações teciduais traduzidas por hepatomegalia com proliferação das células de Kupffer; reação granulomatosa das áreas portais, esplenomegalia com reações granulomatosas, abundância de formas amastigotas. Os resultados para o sangue foram negativos em todas as investigações.In the current paper experimental infection of Calomys callosus with Leishmania donovani is reported for the first time. A group of 22 C. callosus aged 20 months and weighing 25 g were inoculated with 0.1 of a homogeneous saline preparation of infected spleens of homologous animals. The L. donovani strain used in the experiments was isolated from a case of human visceral leishmaniasis from the state of Maranhão, Brazil. The animals infected were weighed and killed 3 months after the experimental infection. Spleens and livers were also weighed and pieces from them were fixed in 10% formaline and stained with hematoxilin-eosin for histological studies. Impression smears stained with Giemsa were made and cultivation "in vitro" (NNN and LIT was done, with material from blood, spleen, liver and bone marrow. At the end of the experiments the animals showed low of body weight. Splenomegaly was observed in all the inoculated animals. The "in vitro" cultures were positive from

  10. Leishmania donovani-derived lipophosphoglycan plus BCG induces a Th1 type immune response but does not protect Syrian golden hamsters (Mesocricetus auratus and BALB/c mice against Leishmania donovani

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    W.K. Tonui

    2003-11-01

    Full Text Available The efficacy of Leishmania donovani-derived lipophosphoglycan (LPG plus Mycobacterium bovis Bacille Calmette-Guérin (BCG as a vaccine candidate against visceral leishmaniosis in susceptible BALB/c mouse and Syrian golden hamster (Mesocricetus auratus models was investigated. Following a triple vaccination with a total dose of 150 µl BCG plus 60 µg or 30 µg of LPG for hamsters and BALB/c mice respectively, there were no noticeable side effects both locally and systemically; implying that the molecule was safe at this dosage level. Vaccinated animals demonstrated an activation of both the humoral as well as cell-mediated responses to LPG, which correlated with resistance against the disease. Protection by LPG plus BCG, was however, poor as the remaining immunized animals showed disease progression leading to severity of the disease as illustrated by emaciation, mass loss and heavy splenic parasitaemia in hamsters. These data nevertheless suggest that it may be rewarding to further evaluate the potential of LPG as a vaccine candidate in leishmaniosis using other adjuvants, which may enhance its immunogenicity.

  11. Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.

    Science.gov (United States)

    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.

  12. Febrifugine analogues as Leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking, ADMET and molecular dynamics simulation.

    Science.gov (United States)

    Pandey, Rajan Kumar; Kumbhar, Bajarang Vasant; Srivastava, Shubham; Malik, Ruchi; Sundar, Shyam; Kunwar, Ambarish; Prajapati, Vijay Kumar

    2017-01-01

    Visceral leishmaniasis affects people from 70 countries worldwide, mostly from Indian, African and south American continent. The increasing resistance to antimonial, miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an antileishmanial drug with excellent efficacy and safety profile. In this study we have docked series of febrifugine analogues (n = 8813) against trypanothione reductase in three sequential docking modes. Extra precision docking resulted into 108 ligands showing better docking score as compared to two reference ligand. Furthermore, 108 febrifugine analogues and reference inhibitor clomipramine were subjected to ADMET, QikProp and molecular mechanics, the generalized born model and solvent accessibility study to ensure the toxicity caused by compounds and binding-free energy, respectively. Two best ligands (FFG7 and FFG2) qualifying above screening parameters were further subjected to molecular dynamics simulation. Conducting these studies, here we confirmed that 6-chloro-3-[3-(3-hydroxy-2-piperidyl)-2-oxo-propyl]-7-(4-pyridyl) quinazolin-4-one can be potential drug candidate to fight against Leishmania donovani parasites.

  13. Interaccion de promastigotes de Leishmania donovani con celulas de exudado peritonial de ratones mediante la influencia de la quimotripsina

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    Lucila Arcay

    1986-06-01

    Full Text Available Se hace un estudio de la interacción de promastigotes de Leishmania donovani con células de exudado peritoneal de ratón (c e p mediante la influencia de la quimotripsina. La adhesión de los promastigotes a las c e p fue terminal y marginal, y en observaciones hechas a partir de los 10 minutos de enfrentamiento, esta adhesión fue nula hasta los 30 minutos en el grupo tratado, y sólo a las das horas hubo un pequeño incremento (2.4% con respecto al control. Se observa marcada disminución en todos los parámetro medidos, tales como enlace, penetración, multiplicación intracelular, división de formas flageladas, en el grupo tratado. La quimotripsina favorece la formación de formas intermedias flageladas, heciendose el parásito piriforme y esférico, apareciendo una forma aberrante de extremo anterior cilindrico que semeja a una forma coanoflagelada. Se sospecha que la enzima reduce efectivamente fragmentos proteicos o péptidos, los cuales pueden haber sido tan pequeños como para esconder a otros ligandos relacionados con la adhesión macrófago-parásito.

  14. Efficacy of protease inhibitor from marine Streptomyces sp. VITBVK2 against Leishmania donovani - An in vitro study.

    Science.gov (United States)

    Sreedharan, Veena; Bhaskara Rao, K V

    2017-03-01

    In the present study the leishmanicidal effect of potential protease inhibitor producing marine actinobacterial isolate has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Among 89 marine actinobacteria isolated from a salt pan in Kanyakumari, only one isolate (BVK2) showed 97% of protease inhibition activity against trypsin. Moderate to high protease inhibitor activity was shown by isolate BVK2 on proteinase (30%) and chymotrypsin (85%). In optimization study for protease inhibitor production glucose as carbon source and casein as nitrogen source showed the best activity. In the in-vitro Fluorescence-activated cell sorting (FACS) assay, 100 μg/ml of BVK2 extract was active against amastigotes in infected J774A.1 macrophages and showed 87% of parasitic inhibition. The isolate BVK2 showed significant anti-parasitic activity with an IC50 of 27.1 μg/ml after double doses were administered. The potential isolate was identified by molecular 16S rRNA gene sequencing as Streptomyces sp. VITBVK2. The results obtained suggest that the marine actinobacterial extract which have novel metabolites can be considered as a potential source for the development of drugs.

  15. Pharmacological activities of cilantro’s aliphatic Aldehydes against leishmania donovani

    Science.gov (United States)

    Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as ...

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

    Science.gov (United States)

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G.; Gannavaram, Sreenivas; Nakhasi, Hira L.

    2016-01-01

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

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

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    Jacqueline Araújo Fiuza

    2016-01-01

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

  18. IFN-γ-induced macrophage antileishmanial mechanisms in mice: A role for immunity-related GTPases, Irgm1 and Irgm3, in Leishmania donovani infection in the liver.

    Science.gov (United States)

    Murray, Henry W; Mitchell-Flack, Marisa; Taylor, Gregory A; Ma, Xiaojing

    2015-10-01

    In C57BL/6 mice, Leishmania donovani infection in the liver provoked IFN-γ-induced expression of the immunity-related GTPases (IRG), Irgm1 and Irgm3. To gauge the antileishmanial effects of these macrophage factors in the liver, intracellular infection was analyzed in IRG-deficient mice. In early- (but not late-) stage infection, Irgm3(-/-) mice failed to properly control parasite replication, generated little tissue inflammation and were hyporesponsive to pentavalent antimony (Sb) chemotherapy. Observations limited to early-stage infection in Irgm1(-/-) mice demonstrated increased susceptibility and virtually no inflammatory cell recruitment to heavily-parasitized parenchymal foci but an intact response to chemotherapy. In L. donovani infection in the liver, the absence of either Irgm1 or Irgm3 impairs early inflammation and initial resistance; the absence of Irgm3, but not Irgm1, also appears to impair the intracellular efficacy of Sb chemotherapy.

  19. MODELING AND MOLECULAR DOCKING STUDIES ON ASPERGILLUS RNASE NIGER AND LEISHMANIA DONOVANI ACTIN: ANTILEISHMANIAL ACTIVITY

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    Ravi Kumar Gundampati

    2013-01-01

    Full Text Available A.niger Rnase was designed from ACTBIND (PDB ID: 3D3Z. Yeast actin-human gelsolin segment 1 complex (PDB ID: 1YAG was used as template for L. donovani actin protein for 3D model in Modeller9v8. These models were testified by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. All evidences suggest that the geometric quality of the backbone conformation, energy profile, residue interaction and contact of the structures were well within the limits of reliable structures. The interaction energy of docking was calculated using the HEX server. Etotal and calculated RMSD values were -1.902, -9.323 kcal moL-1 and 0.402 Å, respectively. The study presented here has an advantage to design molecules that may have antileishmanial activity.

  20. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage

    OpenAIRE

    Morales, M. A.; R. WATANABE; Dacher, M.; Chafey, P.; Osorio y Fortea, J.; Scott, D A; Beverley, S. M.; van Ommen, G.; CLOS, J.; Hem, S.; Lenormand, P.; Rousselle, J.-C.; Namane, A.; Spath, G. F.

    2010-01-01

    Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions...

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

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    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.

  2. Characterization of glycolytic enzymes--rAldolase and rEnolase of Leishmania donovani, identified as Th1 stimulatory proteins, for their immunogenicity and immunoprophylactic efficacies against experimental visceral leishmaniasis.

    Science.gov (United States)

    Gupta, Reema; Kumar, Vikash; Kushawaha, Pramod Kumar; Tripathi, Chandradev Pati; Joshi, Sumit; Sahasrabuddhe, Amogh Anant; Mitra, Kalyan; Sundar, Shyam; Siddiqi, Mohammad Imran; Dube, Anuradha

    2014-01-01

    Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani--Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters' along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼ 90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼ 65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.

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

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    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

  4. 不同种株利什曼原虫抗原的多样性研究%Preliminary study of the antigenic diversity of different Leishmania spp.

    Institute of Scientific and Technical Information of China (English)

    王健; 张仁刚; 张洁; 敬保迁

    2013-01-01

    Objective To investigate the antigenic diversity of different Leishmania spp.Methods Leishmania donovani,Leishmania tropica,and Leishmania infantum promastigotes were cultured and transformed into amastigotes in vitro.Total protein from the promastigotes and amastigotes was separated with SDS-PAGE,and antigenic diversity was identified using polyclonal serum against L.donovani promastigotes and L.tropica promastigotes in Western blotting.Results Following SDS-PAGE electrophoresis and Coomassie brilliant blue staining,the total protein of different Leishmania spp.promastigotes and amastigotes resulted in similar visible bands,and proteins ranged in molecular weight from 12 ku to 150 ku.Western blotting showed that similar antigens were identified by polyclonal sera against L.donovani and L.tropica.Promastigotes and amastigotes of the 3 different Leishmania spp.had similar antigens of 48 ku,65 ku,and 35 ku.A 38-ku stage-specific antigen was present in L.donovani amastigotes and L.infantum amastigotes,and 45-ku and 24-ku species-specific antigens were present in L.tropica promastigotes and amastigotes.Conclusion Antigenic diversity is present in different Leishmania spp.This finding provides new insight into understanding immunity from leishmaniasis.%目的 观察不同种株利什曼原虫抗原的异质性.方法 培养杜氏利什曼原虫、热带利什曼原虫及婴儿利什曼原虫前鞭毛体及体外转化无鞭毛体,提取总蛋白,经SDS-PAGE电泳后,分别以抗杜氏利什曼原虫及热带利什曼原虫前鞭毛体血清进行Western blot,分析抗原的多样性.结果 经SDS-PAGE电泳和考马斯亮蓝染色,杜氏利什曼原虫、热带利什曼原虫及婴儿利什曼原虫前鞭毛体及体外转化无鞭毛体总蛋白呈现出分子质量单位为12~150 ku的相似蛋白带型;Western blot显示,抗杜氏利什曼原虫前鞭毛体多克隆血清识别的抗原组分与抗热带利什曼原虫前鞭毛体多克隆

  5. Heterogeneity of Leishmania donovani parasites complicates diagnosis of visceral leishmaniasis: comparison of different serological tests in three endemic regions.

    Directory of Open Access Journals (Sweden)

    Elfadil Abass

    Full Text Available Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT and two rapid tests of rK39 (IT LEISH and rKE16 (Signal-KA. Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96-100% and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001 and France (p<0.0037. Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT was high in Sudan (94% and India (92.3% but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2% but not Sudan (64-88% and France (73.1-88.5% and 63.6-81.8% in VL and VL/HIV patients, respectively. While the sensitivity varied, all tests showed high specificity in Sudan (96.7-100% and India (96.6%.Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests.

  6. A highly basic sequence at the N-terminal region is essential for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani.

    Science.gov (United States)

    Kumar, Devanand; Kumar, Diwakar; Saha, Swati

    2012-07-01

    The conserved eukaryotic DNA replication protein ORC1 is one of the constituents of pre-replication complexes that assemble at or very near origins prior to replication initiation. ORC1 has been shown to be constitutively nuclear in Leishmania major. This study investigates the sequences involved in nuclear localization of ORC1 in Leishmania donovani, the causative agent of visceral leishmaniasis. Nuclear localization signals (NLSs) have been reported in only a few Leishmania proteins. Functional analyses have delineated NLSs to regions of ~60 amino acids in length in the tyrosyl DNA phosphodiesterase I and type II DNA topoisomerase of L. donovani, and in the L. major kinesin KIN13-1. Using a panel of site-directed mutations we have identified a sequence essential for nuclear import of LdORC1. This sequence at the N terminus of the protein comprises residues 2-5 (KRSR), with K2, R3 and R5 being crucial. Independent mutation of the K2 residue causes exclusion of the protein from the nucleus, while mutating the R5 residue leads to diffusion of the protein throughout the cell. This sequence, however, is insufficient for targeting a heterologous protein (β-galactosidase) to the nucleus. Analysis of additional ORC1 mutations and reporter constructs reveals that while the highly basic tetra-amino acid sequence at the N terminus is essential for nuclear localization, the ORC1 NLS in its entirety is more complex, and of a distributive character. Our results suggest that nuclear localization signalling sequences in Leishmania nuclear proteins are more complex than what is typically seen in higher eukaryotes.

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

    Indian Academy of Sciences (India)

    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.

  8. Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India the LCNTDR Collection: Advances in scientific research for NTD control

    NARCIS (Netherlands)

    M.M. Cameron (Mary M.); A. Acosta-Serrano (Alvaro); C. Bern (Caryn); M. Boelaert (Marleen); M. Den Boer (Margriet); S. Burza (Sakib); L.A.C. Chapman (Lloyd A. C.); A. Chaskopoulou (Alexandra); M. Coleman (Michael); O. Courtenay (Orin); S. Croft (Simon); P.K. Das (P.); E. Dilger (Erin); G. Foster (Geraldine); R. Garlapati (Rajesh); L. Haines (Lee); A. Harris (Angela); J. Hemingway (Janet); T.D. Hollingsworth (T. Déirdre); S. Jervis (Sarah); G.F. Medley (Graham F.); M. Miles (Michael); M. Paine (Mark); A. Picado (Albert); R. Poché (Richard); P. Ready (Paul); M. Rogers (Matthew); M. Rowland (Mark); S. Sundar (Shyam); S.J. de Vlas (Sake); D. Weetman (David)

    2016-01-01

    textabstractVisceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination st

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

    Directory of Open Access Journals (Sweden)

    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 < 0.001, when compared to normal sera, and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7 amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001 and reduction of 85.5 % parasite liver burden (p<0.001 was detected in animals immunized with FML saponin, unrespectivety of the immunization route.

  10. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD).

    Science.gov (United States)

    Mauricio, Isabel L; Yeo, Matthew; Baghaei, Mehdi; Doto, Daniela; Pratlong, Francine; Zemanova, Eva; Dedet, Jean-Pierre; Lukes, Julius; Miles, Michael A

    2006-06-01

    Multilocus enzyme electrophoresis is the gold standard for identification of Leishmania species and strains. Drawbacks include: only amino acid polymorphisms affecting electrophoretic mobility are detected; distinct allozymes can have coincident mobilities; few characters are available; and parasites must be cultured in bulk. So far, thousands of Leishmania strains have been phenotyped by multilocus enzyme electrophoresis. Here, we sequence enzyme-coding genes to provide a PCR-based higher resolution equivalent of multilocus enzyme electrophoresis, particularly for Leishmania infantum. Of 15 enzymes used for multilocus enzyme electrophoresis (MON typing) we have sequenced aspartate aminotransferase, glucose-6-phosphate isomerase, nucleoside hydrolase 1, nucleoside hydrolase 2 and 6-phosphogluconate dehydrogenase. Heterozygous alleles were common, with multiple heterozygous sites within a single locus for several of the genes. Haplotypes were resolved by allele-specific PCR and allele-specific sequencing. Heterozygous haplotypes conformed to the haplotypes of putative parents. One strain appeared to be hybrid across two genetic groups of the Leishmania donovani complex. In most cases, a single amino acid polymorphism was responsible for change in enzyme mobility. Some indistinguishable phenotypes were produced by distinct genotypes. Silent genetic polymorphisms provided enhanced discrimination over multilocus enzyme electrophoresis, for example, by subdividing the zymodeme MON-1. The PCR-based genotyping that we describe could be applied directly to clinical samples or to small volume cultures and in a multilocus sequence typing format. Furthermore, it can be used to detect recombination indirectly and for population genetics studies.

  11. A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Bhavna Chawla

    Full Text Available Deoxyhypusine hydroxylase (DOHH catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N(€-(4-amino-2-hydroxybutyl lysine, which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A. We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ~42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp, in vitro. L. donovani DOHH (LdDOHH showed ~40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues and 174-183 (ten amino acid residues which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for

  12. A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani.

    Science.gov (United States)

    Chawla, Bhavna; Kumar, Ravi Ranjan; Tyagi, Nidhi; Subramanian, Gowri; Srinivasan, N; Park, Myung Hee; Madhubala, Rentala

    2012-01-01

    Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N(€)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ~42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ~40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure

  13. Deficiency of lymph node-resident dendritic cells (DCs) and dysregulation of DC chemoattractants in a malnourished mouse model of Leishmania donovani infection.

    Science.gov (United States)

    Ibrahim, Marwa K; Barnes, Jeffrey L; Osorio, E Yaneth; Anstead, Gregory M; Jimenez, Fabio; Osterholzer, John J; Travi, Bruno L; Ahuja, Seema S; White, A Clinton; Melby, Peter C

    2014-08-01

    Malnutrition is thought to contribute to more than one-third of all childhood deaths via increased susceptibility to infection. Malnutrition is a significant risk factor for the development of visceral leishmaniasis, which results from skin inoculation of the intracellular protozoan Leishmania donovani. We previously established a murine model of childhood malnutrition and found that malnutrition decreased the lymph node barrier function and increased the early dissemination of L. donovani. In the present study, we found reduced numbers of resident dendritic cells (conventional and monocyte derived) but not migratory dermal dendritic cells in the skin-draining lymph nodes of L. donovani-infected malnourished mice. Expression of chemokines and their receptors involved in trafficking of dendritic cells and their progenitors to the lymph nodes was dysregulated. C-C chemokine receptor type 2 (CCR2) and its ligands (CCL2 and CCL7) were reduced in the lymph nodes of infected malnourished mice, as were CCR2-bearing monocytes/macrophages and monocyte-derived dendritic cells. However, CCR7 and its ligands (CCL19 and CCL21) were increased in the lymph node and CCR7 was increased in lymph node macrophages and dendritic cells. CCR2-deficient mice recapitulated the profound reduction in the number of resident (but not migratory dermal) dendritic cells in the lymph node but showed no alteration in the expression of CCL19 and CCL21. Collectively, these results suggest that the malnutrition-related reduction in the lymph node barrier to dissemination of L. donovani is related to insufficient numbers of lymph node-resident but not migratory dermal dendritic cells. This is likely driven by the altered activity of the CCR2 and CCR7 chemoattractant pathways.

  14. Insilico Analysis unveils Putative Metabolic Pathways and Essential Genes within Leishmania donovani ‘Orfeome’

    Directory of Open Access Journals (Sweden)

    Nithin eRavooru

    2014-08-01

    Full Text Available Leishmaniasis is a parasitic disease caused by the protozoan Leishmania, which is active in two broad forms namely, Visceral Leishmaniasis (VL or Kala Azar and Cutaneous Leishmaniasis (CL. The disease is most prevalent in the tropical regions and poses a threat to more than 70 countries across the globe. In the Indian subcontinent, about 200 million people are estimated to be at risk of developing VL and this area harbours an estimated 67% of the global VL disease burden. The state of Bihar alone has captured almost 50% of the total cases in the Indian region. While no vaccination exists, several pentavalent antimonials and drugs like Paromomycin, Amphotericin, Miltefosine etc., are used in the treatment of Leishmaniasis. However, due to low efficacy of these drugs and the resistance developed by the bug to these medications, there is an urgent need to look into species specific targets. The proteome information available suggests that among the 7960 proteins, a staggering 65% of it remains to be annotated with clarity.Hence, in the present study, we have demonstrated a protocol to integrate the seqeunce and functional information from various databases, such as GO, PFAM, KEGG, String DB, COG and DEG, to assign putative functions to many of the hypothetical seqeucences present in this proteome. These crucial information related to pathways and essential genes show promise for exploring the design strategies towards developing drugs, to tackle this notorious parasitic disease.

  15. Molecular events leading to death of Leishmania donovani under spermidine starvation after hypericin treatment.

    Science.gov (United States)

    Singh, Shalini; Kumari, Ekta; Bhardwaj, Ruchika; Kumar, Ritesh; Dubey, Vikash Kumar

    2017-05-16

    We have previously reported that the hypericin treatment caused spermidine starvation and death of Leishmania parasite. Here, we report different molecular events under spermidine starvation and potential role of spermidine in processes other than redox homeostasis of the parasite. We have analyzed changes in expression of several genes by using quantitative gene expression analysis. Further, these changes at molecular level were also confirmed by using biochemical and cellular studies. Altered expression of several genes involved in redox metabolism, hypusine modification of eIF5A, DNA repair pathway and autophagy was observed. There was decrease in Sir2RP expression after hypericin treatment and this decrease has been found to be associated with induced ROS due to hypericin treatment as it has been rescued by either trypanothione or spermidine supplementation. Translation initiation in the parasite was decreased upon spermidine starvation. We also observed increased AMPK expression upon hypericin treatment. The increase in intracellular ATP and NAD(+) levels as well as decrease in Sir2RP expression of the parasite are cytoprotective mechanism towards generated ROS due to hypericin treatment possibly by inducing autophagy as indicated by increase in autophagy related gene expression and acridine orange staining. However, the autophagy needs to be established using more rigorous methodologies. © 2017 John Wiley & Sons A/S.

  16. Synthesis of Marine α-Methoxylated Fatty Acid Analogs that Effectively Inhibit the Topoisomerase IB from Leishmania donovani with a Mechanism Different from that of Camptothecin

    Directory of Open Access Journals (Sweden)

    Rosa M. Reguera

    2013-09-01

    Full Text Available Sponges biosynthesize α-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the α-methoxylated fatty acids have been less studied. In this work, we describe the total synthesis of (5Z,9Z-(±-2-methoxy-5, 9-eicosadienoic acid (1 and its acetylenic analog (±-2-methoxy-5,9-eicosadiynoic acid (2, and report that they inhibit (EC50 values between 31 and 22 µM the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB. The inhibition of LdTopIB (EC50 = 53 µM by the acid (±-2-methoxy-6-icosynoic acid (12 was studied as well. The potency of LdTopIB inhibition followed the trend 2 > 1 > 12, indicating that the effectiveness of inhibition depends on the degree of unsaturation. All of the studied α-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB at 100 µM. However, the α-methoxylated fatty acids were capable of inhibiting an active but truncated LdTopIB with which camptothecin (CPT cannot interact suggesting that the methoxylated fatty acids inhibit LdTopIB with a mechanism different from that of CPT. The diunsaturated fatty acids displayed low cytotoxicity towards Leishmania infantum promastigotes (EC50 values between 260 and 240 µM, but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC50 = 100 µM and a better therapeutic index.

  17. Comparative activities of the triterpene saponin maesabalide III and liposomal amphotericin B (AmBisome) against Leishmania donovani in hamsters.

    Science.gov (United States)

    Maes, Louis; Germonprez, Nils; Quirijnen, Ludo; Van Puyvelde, Luc; Cos, Paul; Vanden Berghe, Dirk

    2004-06-01

    Maesabalide III (MB-III), an oleane triterpene saponin isolated from the Vietnamese plant Maesa balansae, is a new antileishmanial lead compound whose activity against Leishmania donovani (MHOM/ET/67/L82) in groups of five golden hamsters was evaluated after administration of a single subcutaneous dose on either day 1 (prophylactic treatment) or day 28 (curative treatment) after infection. Liposomal amphotericin B (AmBisome), administered intravenously at 5 mg/kg of body weight, was used as the reference drug. Amastigote burdens in liver, spleen, and bone marrow were determined either 7 days (early effects) or 56 days (late effects) after treatment. Prophylactic administration of MB-III at 0.2 mg/kg reduced liver amastigote burdens by 99.8 and 83% within 7 and 56 days after treatment, respectively. In the latter group, however, all animals became ill and some died. Both MB-III at 0.8 mg/kg and liposomal amphotericin B were 100% effective against liver stages, but clearance from the spleen and bone marrow was not achieved. Curative administration of MB-III at 0.2 and 0.4 mg/kg was not protective, as no survivors were left at the termination of the experiment on day 84. Despite the high level of reduction of the liver amastigote burden after treatment with MB-III at 0.8 mg/kg (94.2%) or liposomal amphotericin B (99.4%), clinical protection could not be obtained in either group, with two deaths occurring and the residual liver burdens persisting. It is concluded that administration of a single dose of MB-III at 0.8 mg/kg has efficacy potential comparable to that of a single dose of liposomal amphotericin B at 5 mg/kg and is therefore considered a promising new antileishmanial lead compound. However, multiple-dose pharmacological, toxicological, and pharmacokinetic studies are still needed before it can become a valid drug candidate for development.

  18. Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Kurtzhals, J A; Bendtzen, K

    1993-01-01

    by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were...... analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced...

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Ensaio terapêutico com glucantime em sagüis (Callithrix jacchus infectados com uma cepa de Leishmania donovani aparentemente resistente ao tratamento

    Directory of Open Access Journals (Sweden)

    R. Dietze

    1985-03-01

    Full Text Available Os autores relatam o resultado de um ensaio terapêutico em sagüis com uma cepa de Leishmania donovani isolada de um caso humano fatal de calazar, clinicamente resistente aos antimoniais (Glucantime e Pentostam, Anfotericina B ePentamidina. Os testes cutâneos realizados no paciente para avaliação da imunidade celular foram negativos à exceção do DNCB a 2%. Quatro sangüis adultos (Callithrix jacchus foram inoculados por via intraperitoneal com uma suspensão deformas amastigotas de L. donovani. Duzentos e dez dias após, todos os animais mostravam formas amastigotas do protozoário, evidenciadas através de punção hepática por aspiração. Iniciou-se então um esquema terapêutico com glucantime (28 mgSb v/kg/dia em três séries de 10 dias com cinco dias de intervalo entre elas em três dosprimatas,ficando o quarto animal como controle. Nos três animais tratados houve curaparasitológica da doença, o mesmo não ocorrehdo com o controle. O fato de a amostra de L. donovani ter sido, no paciente, resistente aos vários tratamentos e ter sido sensível em modelo experimental à terapêutica com glucantime, sugere a possibilidade de que fatores imunológicos do paciente possam ter contribuído para a evolução fatal da doença.The authors describe a therapeutic study in marmosets (Callithrix jacchus of a strain of Leishmania donovani isolated from a fatal human case of Kala-azar. The patient failed to respond to antimoniais (Glucantime and Pentostam, Amphotericin B and pentamidine therapy. Skin tests for the evaluation of cellular immunity were negative with the exception of 2% dinitrochlobenzene. Four adult marmosets were inoculated intraperitoneally with a suspension of amastigotes of L. donovani isolated from the hamster liver and spleen. Two hundred and ten days later all the animals had amastigotes in aspirates obtained by liver puncture. Glucantime treatment 28 mg/kilogram body weightper day for 3 series of 10 days was begun in

  1. Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy.

    Science.gov (United States)

    Martin, M B; Grimley, J S; Lewis, J C; Heath, H T; Bailey, B N; Kendrick, H; Yardley, V; Caldera, A; Lira, R; Urbina, J A; Moreno, S N; Docampo, R; Croft, S L; Oldfield, E

    2001-03-15

    We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

  2. A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis.

    Science.gov (United States)

    Gupta, Reema; Kushawaha, Pramod K; Tripathi, Chandra Dev Pati; Sundar, Shyam; Dube, Anuradha

    2012-05-01

    The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (∼85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-β and IL-4, indicating its potential as a vaccine candidate against VL.

  3. Targeting the human parasite Leishmania donovani: discovery of a new promising anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series.

    Science.gov (United States)

    Castera-Ducros, Caroline; Paloque, Lucie; Verhaeghe, Pierre; Casanova, Magali; Cantelli, Christophe; Hutter, Sébastien; Tanguy, Floriane; Laget, Michèle; Remusat, Vincent; Cohen, Anita; Crozet, Maxime D; Rathelot, Pascal; Azas, Nadine; Vanelle, Patrice

    2013-11-15

    We report herein the discovery of antileishmanial molecules based on the imidazo[1,2-a]pyridine ring. In vitro screenings of imidazopyridines belonging to our chemical library, toward the promastigotes stage of Leishmania donovani, J774A.1 murine and HepG2 human cells, permitted to identify three selective hit-compounds (12, 20 and 28). New derivatives were then synthesized to allow structure-activity and -toxicity relationships analyses, enabling to characterize a lead-compound (44) displaying both a high potency (IC50=1.8 μM) and a good selectivity index, in comparison with three antileishmanial reference drug-compounds (amphotericin B, miltefosine and pentamidine). Moreover, lead-compound 44 also exhibits good in vitro activity against the intracellular amastigote stage of L. donovani. Thus, the 6-halo-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine scaffold appears as a new promising selective antileishmanial pharmacophore, especially when substituted at position 8 by a bromine atom.

  4. Pro-apoptotic effect of the landrace Bangla Mahoba of Piper betle on Leishmania donovani may be due to the high content of eugenol.

    Science.gov (United States)

    Misra, Pragya; Kumar, Awanish; Khare, Prashant; Gupta, Swati; Kumar, Nikhil; Dube, Anuradha

    2009-08-01

    In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar - a devastating parasitic disease caused by Leishmania donovani - the search for anti-leishmanial agents from natural resources in common use is imperative. Recently, the comparative in vitro anti-leishmanial activity of methanolic extracts from two landraces of Piper betle - P. betle landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) - has been reported. Here, the putative pathway responsible for death induced by the effective extract of PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L. donovani, was assessed using various biochemical approaches. It was found that PB-BM was capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic events by generation of reactive oxygen species targeting the mitochondria without any cytotoxicity towards macrophages. The study was extended to determine the presence or absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in essential oil composition present in the extract assessed by GC and MS. The essential oil from PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher content of eugenol in the active landrace. This observation emphasizes the need to extend studies related to traditional medicines from bioactive plants below the species level to the gender/landrace level for better efficacy and reproducibility.

  5. A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

    Science.gov (United States)

    Jain, Surendra K.; Sahu, Rajnish; Walker, Larry A.; Tekwani, Babu L.

    2012-01-01

    Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models. In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes. Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of

  6. Leishmania donovani: assessment of leishmanicidal effects of herbal extracts obtained from plants in the visceral leishmaniasis endemic area of Bihar, India.

    Science.gov (United States)

    Singh, Shubhankar K; Bimal, Sanjiva; Narayan, Shyam; Jee, Chandrawati; Bimal, Devla; Das, P; Bimal, Raageeva

    2011-02-01

    One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5mg/ml within 24h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    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....

  8. Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

    Science.gov (United States)

    Molecular tools enable the collection of accurate estimates of human blood index (HBI) in P. argentipes. The refinement of a metacyclic-specific qPCR assay to identify L. donovani in P. argentipes would enable quantification of the entomological inoculation rate (EIR) for the first time. Likewise, a...

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases....

  10. Molecular detection of infection homogeneity and impact of miltefosine treatment in a Syrian golden hamster model of Leishmania donovani and L. infantum visceral leishmaniasis.

    Science.gov (United States)

    Eberhardt, Eline; Mondelaers, Annelies; Hendrickx, Sarah; Van den Kerkhof, Magali; Maes, Louis; Caljon, Guy

    2016-10-01

    Control of visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani primarily relies on chemotherapy using an increasingly compromised repertoire of antileishmanial compounds. For evaluation of novel drugs, the Syrian golden hamster is considered as a clinically relevant laboratory model. In this study, two molecular parasite detection assays were developed targeting cathepsin-like cysteine protease B (CPB) DNA and 18S rRNA to achieve absolute amastigote quantification in the major target organs liver and spleen. Both quantitative PCR (qPCR) techniques showed excellent agreement with a strong correlation with the conventional microscopic reading of Giemsa-stained tissue smears. Using multiple single tissue pieces and all three detection methods, we confirmed homogeneity of infection in liver and spleen and the robustness of extrapolating whole organ burdens from a small single tissue piece. Comparison of pre- and post-treatment burdens in infected hamsters using the three detection methods consistently revealed a stronger parasite reduction in the spleen compared to the liver, indicating an organ-dependent clearance efficacy for miltefosine. In conclusion, this study in the hamster demonstrated high homogeneity of infection in liver and spleen and advocates the use of molecular detection methods for assessment of low (post-treatment) tissue burdens.

  11. Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

    Science.gov (United States)

    Campbell, S A; Alawa, J; Doro, B; Henriquez, F L; Roberts, C W; Nok, A; Alawa, C B I; Alsaadi, M; Mullen, A B; Carter, K C

    2012-02-08

    Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

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

    DEFF Research Database (Denmark)

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

    1992-01-01

    than 1:10,000 and varied considerably between individuals. Depletion of CD45R0-positive (memory) cells from the PBMC abolished proliferative responses induced by Leishmania antigen and by tetanus toxoid. In cell populations depleted of CD45RA-positive (naive) cells, only a small reduction in response...... likely memory cells recognizing antigens present both in the Leishmania preparations and in the environment....

  13. SOLiD™ sequencing of genomes of clinical isolates of Leishmania donovani from India confirm leptomonas co-infection and raise some key questions.

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    Neeloo Singh

    Full Text Available BACKGROUND: Known as 'neglected disease' because relatively little effort has been applied to finding cures, leishmaniasis kills more than 150,000 people every year and debilitates millions more. Visceral leishmaniasis (VL, also called Kala Azar (KA or black fever in India, claims around 20,000 lives every year. Whole genome analysis presents an excellent means to identify new targets for drugs, vaccine and diagnostics development, and also provide an avenue into the biological basis of parasite virulence in the L. donovani complex prevalent in India. METHODOLOGY/PRINCIPAL FINDINGS: In our presently described study, the next generation SOLiD™ platform was successfully utilized for the first time to carry out whole genome sequencing of L. donovani clinical isolates from India. We report the exceptional occurrence of insect trypanosomatids in clinical cases of visceral leishmaniasis (Kala Azar patients in India. We confirm with whole genome sequencing analysis data that isolates which were sequenced from Kala Azar (visceral leishmaniasis cases were genetically related to Leptomonas. The co-infection in splenic aspirate of these patients with a species of Leptomonas and how likely is it that the infection might be pathogenic, are key questions which need to be investigated. We discuss our results in the context of some important probable hypothesis in this article. CONCLUSIONS/SIGNIFICANCE: Our intriguing results of unusual cases of Kala Azar found to be most similar to Leptomonas species put forth important clinical implications for the treatment of Kala Azar in India. Leptomonas have been shown to be highly susceptible to several standard leishmaniacides in vitro. There is very little divergence among these two species viz. Leishmania sp. and L. seymouri, in terms of genomic sequence and organization. A more extensive perception of the phenomenon of co-infection needs to be addressed from molecular pathogenesis and eco

  14. Protective efficacy of antioxidants on cisplatin-induced tissue damage caused in Leishmania donovani infected BALB/c mice against murine visceral leishmaniasis

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    Meenakshi Sharma

    2013-06-01

    Full Text Available Objective: Therapeutic interventions against visceral leishmaniasis (VL are limited and facing serious concerns of toxicity, high cost and emerging resistance, there is a greater interest in new drug developments which are cost effective, efficient and easily available to people suffering from leishmaniasis. Cisplatin (cis-diamminedichloroplatinum II; CDDP has been found to have antileishmanial activity in vitro and in vivo which lead towards an apoptosis like cell death of both promastigotes and amastigotes and a significant reduction in parasite load and enhanced DTH responses which suggested the generation of protective cell-mediated immune responses. But, at higher doses it causes nephrotoxicity-a major side effect. The present study was designed to evaluate the protective efficacy of antioxidants on cisplatin induced tissue damage in Leishmania donovani infected BALB/c mice. Materials and methods: L. donovani infected and uninfected animals were treated with higher doses (5 and 2.5 mg/kg body weight of cisplatin alone and in combination with antioxidants (vitamin C, vitamin E and silibinin for 5 days. Mice were examined for the protective effects of antioxidants on cisplatin indiced tissue damage by DNA fragmentation and histological studies of kidneys, liver and spleen. Results: The damage caused by cisplatin was ameliorated after the supplementation of antioxidants showing a marked reduction in the extent of tubular damage, the focal reaction changes in liver were reversed and no signs of toxicity in the spleen were reported. Moreover, no DNA damage was observed in animals treated with cisplatin along with various antioxidants. Conclusion: The present results showed that antioxidants helped in the amelioration of drug induced toxic effects against murine visceral leishmaniasis, making the combination a potential anti-leishmanial therapy. [J Interdiscipl Histopathol 2013; 1(3.000: 121-136

  15. Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.

    Science.gov (United States)

    Barros-Álvarez, Ximena; Kerchner, Keshia M; Koh, Cho Yeow; Turley, Stewart; Pardon, Els; Steyaert, Jan; Ranade, Ranae M; Gillespie, J Robert; Zhang, Zhongsheng; Verlinde, Christophe L M J; Fan, Erkang; Buckner, Frederick S; Hol, Wim G J

    2017-07-01

    The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNA(Tyr). An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Th1-like response to lipophosphoglycan-associated protein in cells from cured visceral leishmaniasis patients

    DEFF Research Database (Denmark)

    Kurtzhals, J A; Hey, A S; Jardim, A

    1994-01-01

    The T cell response to different Leishmania donovani antigens was investigated using peripheral blood mononuclear cells (PBMC) from Kenyans cured of visceral leishmaniasis and non-exposed Danes. Crude promastigote and amastigote antigens both induced proliferation and interferon-gamma (IFN...... in five of 17 samples from cured patients. Four of the five responding cultures produced IL-4, i.e. the response to this antigen was of the Th2 type. Furthermore, sera from acutely ill visceral leishmaniasis patients contained high levels of IgG antibodies to gp63. The Th2-like response to gp63...... in patients cured of visceral leishmaniasis differs from the Th1-like response to the same antigen observed in patients cured of cutaneous leishmaniasis....

  17. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    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...

  18. Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage.

    Science.gov (United States)

    Gannavaram, Sreenivas; Connelly, Patricia S; Daniels, Mathew P; Duncan, Robert; Salotra, Poonam; Nakhasi, Hira L

    2012-10-01

    Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β-oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1-mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1(-/-)). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl-CoA, the end-product of the β-oxidation in the Ufm1(-/-) amastigote stage. The Ufm1(-/-) mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re-expression of wild-type Ufm1 with concomitant induction of acetyl-CoA but not by re-expressing the non-conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β-oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1(-/-) parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1(-/-) parasites as drug and vaccine targets.

  19. Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

    Science.gov (United States)

    Kashino, S S; Abeijon, C; Qin, L; Kanunfre, K A; Kubrusly, F S; Silva, F O; Costa, D L; Campos, D; Costa, C H N; Raw, I; Campos-Neto, A

    2012-07-01

    Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

  20. Toll-Like Receptor 2 Targeted Rectification of Impaired CD8⁺ T Cell Functions in Experimental Leishmania donovani Infection Reinstates Host Protection.

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    Syamdas Bandyopadhyay

    Full Text Available Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL, characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2 ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+, Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.

  1. An outbreak investigation of visceral leishmaniasis among residents of Dharan town, eastern Nepal, evidence for urban transmission of Leishmania donovani

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    Uranw Surendra

    2013-01-01

    Full Text Available Abstract Background Visceral leishmaniasis (VL is a predominantly rural disease, common in the low lands of eastern Nepal. Since 1997 VL cases have also been reported among residents of the city of Dharan. Our main research objective was to find out whether there had been local transmission of VL inside the city. Methods We conducted an outbreak investigation including a case–control study; cases were all urban residents treated for VL between 2000 and 2008 at BP Koirala Institute of Health Sciences, a university hospital in the city. For each case, we selected four random controls, with no history of previous VL; frequency-matched for age. Cases and controls were subjected to a structured interview on the main exposures of interest and potential confounders; a binominal multilevel model was used to analyze the data. We also collected entomological data from all neighborhoods of the city. Results We enrolled 115 VL patients and 448 controls. Cases were strongly clustered, 70% residing in 3 out of 19 neighborhoods. We found a strong association with socio-economic status, the poorest being most at risk. Housing was a risk factor independent from socio-economic status, most at risk were those living in thatched houses without windows. ‘Sleeping upstairs’ and ‘sleeping on a bed’ were strongly protective, OR of 0.08 and 0.25 respectively; proximity to a case was a strong risk factor (OR 3.79. Sand flies were captured in all neighborhoods; in collections from several neighborhoods presence of L. donovani could be demonstrated by PCR. Conclusion The evidence found in this study is consistent with transmission of anthroponotic VL within the city. The vector P. argentipes and the parasite L. donovani have both been identified inside the town. These findings are highly relevant for policy makers; in VL endemic areas appropriate surveillance and disease control measures must be adopted not only in rural areas but in urban areas as well.

  2. Differences in the antigenic profile and infectivity of murine macrophages of Leishmania (Viannia) parasites.

    Science.gov (United States)

    Matta, Nubia E; Cysne-Finkelstein, Lea; Machado, Gerzia Maria C; Da-Cruz, Alda Maria; Leon, Leonor

    2010-06-01

    The antigenic profile and infectivity were compared between 3 recent Leishmania (Viannia) isolates from the Amazonian region (Instituto Nacional de Pesquisas da Amazonia [INPA] strains) and 3 World Health Organization (WHO) reference species (Leishmania guyanensis, Leishmania braziliensis, and Leishmania naiffi). Differences were observed in the peak and extent of promastigote growth. The WHO reference strains exhibited significantly higher exponential growth as promastigotes than INPA strains. In the immunoblot analyses, the INPA strains revealed several specific peptide fragments, as well as the greatest recognition frequencies by sera from Leishmania sp.-infected patients; among the latter, antigens derived from L. naiffi were the most frequently recognized. In vitro infection was carried out using mice peritoneal macrophages; all strains were able to enter the macrophages, but only L. amazonensis was able to reproduce. A striking observation was that L. naiffi exhibited the longest survival time inside the macrophages. Our data strongly suggest the application of recently isolated parasites as sources of antigen for diagnosis procedures. Moreover, L. naiffi species possesses several characteristics relevant for its use as a source of novel antigens to be explored in the design of diagnostic tools and vaccines.

  3. An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

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    Márcia Cristina Aquino Teixeira

    2011-03-01

    Full Text Available Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

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

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    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

  5. Evaluation of the adjuvanticity of artemisinin with soluble Leishmania major antigens in BALB/c mice

    Institute of Scientific and Technical Information of China (English)

    Albert Kimutai; Milkah Mwangi; Lydia B. Nyamwamu; Willy K. Tonui; Michael M. Gicheru; Peter Kamau Ngure; Johnstone Ingonga; Stella Kepha; Laban Ireri Njeru; Dorcas Wachira; Robert Karanja Muhia

    2009-01-01

    Objective: To determine the adjuvant potential of artemisinin with a soluble leishmanial antigen in vaccinating BALB/c mice. Methods: Seventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble Leishmania antigens (SLA) alone, artemisinin co-administered with SLA, SLA and Bacille Calmette Gu rin (BCG) vaccine, and artemisinin and BCG alone. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4, IL-5 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad. Results: Mice receiving SLA plus artemisinin produced significantly high levels of IL-4 and IL-5 (P 0.05), resulting in exacerbated disease. Conclusion: These data suggest that artemisinin is not a suitable adjuvant for Leishmania vaccines. However, since artemisinin has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with Leishmania antigens.

  6. Advanced Developement of Leishmania Tropical Skin Test Antigen

    Science.gov (United States)

    2011-09-01

    ELIZA . The same inability to bind with antiserum was observed in lyophilized product. However, product stored at 2-8 C retained the ability to combine...Potency Experiments on Leishmania Drug Product Drug Product Storage Temp Results ELISA minimum acceptable S:N = 2.0 Results SDS-PAGE Results...these experiments were unsuccessful in that a correlation between specific SDS-PAGE bands and potency was not observed. We also compared the banding

  7. Restoration of IFNγR subunit assembly, IFNγ signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

    Directory of Open Access Journals (Sweden)

    Subha Sen

    2011-09-01

    Full Text Available Despite the presence of significant levels of systemic Interferon gamma (IFNγ, the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD infected macrophages (LD-MØs; the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3 phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1 and subunit 2 (IFNγR2 colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1 in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V-X(1-5-Y-X(1-5-(R/K] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of

  8. Target Oriented Drugs against Leishmania.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

    Directory of Open Access Journals (Sweden)

    Daniel P Depledge

    Full Text Available BACKGROUND: A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes. METHODS/PRINCIPAL FINDINGS: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. CONCLUSIONS/SIGNIFICANCE: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal

  11. A combination DNA vaccine encoding nucleoside hydrolase 36 and glycoproteine 63 protects female but not male hamsters against Leishmania mexicana

    Directory of Open Access Journals (Sweden)

    Chalé-balboa W.G.

    2009-09-01

    Full Text Available Leishmaniasis is a group of diseases caused by protozoan parasites of the Leishmania genus. Previous studies have shown that a DNA vaccine encoding Leishmania donovani antigen nucleoside hydrolase 36 and L. mexicana glycoprotein 63 is protective in mice. We investigated here the efficacy of this DNA vaccine to induce protection in golden hamsters. Male hamsters were more susceptible to infection by Leishmania mexicana than females. Following immunization with two doses of the DNA vaccine, only females resulted protected while males developed normal lesions.

  12. Leukodepletion Filters for Prevention of Transfusion Transmission Of Leishmania

    Science.gov (United States)

    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

  13. Field validation of a Leishmania (Leishmania) mexicana exo-antigens ELISA for diagnosing tegumentary leishmaniasis in regions of Leishmania (Viannia) predominance.

    Science.gov (United States)

    Soares, Killarney Ataide; Urdapilleta, Ada Amália Ayala; Santos, Gilcilene Maria dos; Carneiro, Andréa Lisboa; Gomes, Ciro Martins; Roselino, Ana Maria; Sampaio, Raimunda Nonata Ribeiro

    2015-01-01

    Several tests are performed to obtain better accuracy when diagnosing American tegumentary leishmaniasis (ATL). It is believed that antigens released via secretion, excretion and metabolism are more specific than are antigens released by the lysis of Leishmania parasites. Such antigens are known as exo-antigens (exo-Ag) and are formed from products released by cultured parasites in a way that is similar to that in which they cause infections in hosts. We attempted to validate a Leishmania mexicana ELISA exo-Ag for ATL diagnosis in Midwestern Brazil. A total of 281 patients were included in the study. We analysed pre-treatment blood from 98 ATL patients; out of those, 85.7% and 14.3% had cutaneous and mucosal forms, respectively. The exo-Ag accuracy was 83.99% (95% CI=79.24-87.81) with a sensitivity value of 90.82% (95% CI=83.46-95.09) and an overall specificity value of 80.33% (95% CI=73.97-85.44). The positive predictive value and negative predictive value were 71.20% (95% CI=62.72-78.41) and 94.23% (95% CI=89.40-96.94), respectively. Among healthy controls, exo-Ag had a specificity of 91.25% (95% CI=83.02-95.70); additionally, the test had specificity rates of 66.67% (95% CI=46.71-82.03) in Chagas disease patients, 60.61% (95% CI=43.68-75.32) in patients with rheumatic diseases, 76.92% (95% CI=49.74-91.82) in pemphigus foliaceus patients, 87.50% (95% CI=52.91-97.76) in leprosy patients, 87.50% (95% CI=63.98-96.50) in VRDL-positive patients, and 77.78 (95% CI=45.26-93.68) in deep mycosis patients. Based on the indicators of validity, we conclude that the results obtained in this study enable the recommendation of the exo-Ag ELISA for ATL diagnosis once it presented a reasonable accuracy compared to classical methods. Cost evaluations are necessary to completely define the role of this technique in large scale. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  14. Advanced Development of Leishmania Topical Skin Test Antigen

    Science.gov (United States)

    2012-09-28

    followed to assess the effectiveness of the cleaning and sanitization procedures. The filling, capping and assembly of final containers occurred in Class...disappeared after several days in both subjects. Topical steroid cream was administered to one subject to promote resolution. 2.5.2 Phase II Dose...al. (3) studied the effects of repeat skin tests in healthy volunteers residing in a leishmaniasis non-endemic area. The Leishmnaia antigen used in

  15. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    Science.gov (United States)

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

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

    Directory of Open Access Journals (Sweden)

    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. Leishmania Mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. Mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

    Directory of Open Access Journals (Sweden)

    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.

  18. Monocyte Chemotactic Protein 1 in Plasma from Soluble Leishmania Antigen-Stimulated Whole Blood as a Potential Biomarker of the Cellular Immune Response to Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Ana V. Ibarra-Meneses

    2017-09-01

    Full Text Available New biomarkers are needed to identify asymptomatic Leishmania infection as well as immunity following vaccination or treatment. With the aim of finding a robust biomarker to assess an effective cellular immune response, monocyte chemotactic protein 1 (MCP-1 was examined in plasma from soluble Leishmania antigen (SLA-stimulated whole blood collected from subjects living in a Leishmania infantum-endemic area. MCP-1, expressed 110 times more strongly than IL-2, identified 87.5% of asymptomatic subjects and verified some asymptomatic subjects close to the cutoff. MCP-1 was also significantly elevated in all patients cured of visceral leishmaniasis (VL, unlike IL-2, indicating the specific memory response generated against Leishmania. These results show MCP-1 to be a robust candidate biomarker of immunity that could be used as a marker of cure and to both select and follow the population in vaccine phase I–III human clinical trials with developed rapid, easy-to-use field tools.

  19. Fungus-Elicited Metabolites from Plants as an Enriched Source for New Leishmanicidal Agents: Antifungal Phenyl-Phenalenone Phytoalexins from the Banana Plant (Musa acuminata) Target Mitochondria of Leishmania donovani Promastigotes

    Science.gov (United States)

    Luque-Ortega, Juan Román; Martínez, Silvia; Saugar, José María; Izquierdo, Laura R.; Abad, Teresa; Luis, Javier G.; Piñero, José; Valladares, Basilio; Rivas, Luis

    2004-01-01

    Two antifungal phenyl-phenalenone phytoalexins isolated from the banana plant (Musa acuminata) elicited with the fungus Fusarium oxysporum, together with a methoxy derivative of one of them and two epoxide precursors of their chemical synthesis, were tested for leishmanicidal activity on Leishmania donovani promastigotes and L. infantum amastigotes. Drugs inhibited proliferation of both forms of the parasite with a 50% lethal concentration range between 10.3 and 68.7 μg/ml. Their lethal mechanism was found linked to the respiratory chain by a systematic approach, including electron microscopy, measurement of the oxygen consumption rate on digitonin-permeabilized promastigotes, and enzymatic assays on a mitochondrial enriched fraction. Whereas the whole set of compounds inhibited the activity of fumarate reductase in the mitochondrial fraction (50% effective concentration [EC50] between 33.3 and 78.8 μg/ml) and on purified enzyme (EC50 = 53.3 to 115 μg/ml), inhibition for succinate dehydrogenase was only observed for the two phytoalexins with the highest leishmanicidal activity: anigorufone and its natural analogue 2-methoxy-9-phenyl-phenalen-1-one (EC50 = 33.5 and 59.6 μg/ml, respectively). These results provided a new structural motif, phenyl-phenalenone, as a new lead for leishmanicidal activity, and support the use of plant extracts enriched in antifungal phytoalexins, synthesized under fungal challenge, as a more rational and effective strategy to screen for new plant leishmanicidal drugs. PMID:15105102

  20. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

  1. Cloning and Sequencing of Leishmania major Thiol-Specific-Antioxidant Antigen (TSA Gene

    Directory of Open Access Journals (Sweden)

    F Tabatabaie

    2007-08-01

    Full Text Available Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obli­gate intracellular parasite. TSA is the immuno-dominant antigen of Leishmania major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. Methods: Genomic DNA of TSA protein was extracted and amplificated as a template. Then the PCR product was cloned into pTZ57R/T vector. Finally, the recombinant plasmid was extracted from transformed Escherichia coli (TG1 strain and sequenced. Results: MRHO/IR/75/ER (an Iranian strain of L. major and TSA gene (Accession number LmjF15.1080 were used. Se­quence analysis of cloned TSA gene into pTZ57R/T vector showed high homology of 90% with LmjF15.1080 (TSA gene and strain "LV39" (Accession no. AF069386 and strain "Friedlin" (Accession no.AF044679. Conclusion: We cloned TSA gene of L. major successfully. Recombinant plasmid was confirmed. It is ready to express recombinant protein for further studies.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    Science.gov (United States)

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  4. Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

    Directory of Open Access Journals (Sweden)

    Bacellar O.

    1998-01-01

    Full Text Available It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC mAb (W6/32 suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

  5. Detection of IgG Anti-Leishmania Antigen by Flow Cytometry as a Diagnostic Test for Cutaneous Leishmaniasis

    Science.gov (United States)

    Schriefer, Albert; Magalhães, Andréa; Meyer, Roberto; Glesby, Marshall J.; Carvalho, Edgar M.; Carvalho, Lucas P.

    2016-01-01

    Diagnosis of cutaneous leishmaniasis (CL) relies on clinical presentation, parasite isolation, histopathologic evaluation and positive Montenegro skin test. However, the low amounts of parasites in the lesion of these individuals make parasite isolation and histopatologic diagnosis unreliable, often leading to false-negative results. Also, 15% of people living in endemic areas have sub-clinical infection characterized by positive Montenegro skin test, which may contribute to misdiagnosis. Although the main Leishmania killing mechanism is through cell-mediated immune response, antibodies against Leishmania antigens are found in infected individuals. Here our goal was to develop a new serological technique using polystyrene microspheres sensitized with soluble Leishmania antigens as a tool for the detection of IgG in serum from CL patients by flow cytometry. To validate the assay we carried out a comparative test (ELISA) commonly used as a diagnostic test for parasitic diseases. To determine cross-reactivity we used serum from patients with Chagas disease, caused by a trypanosome that has several proteins with high homology to those of the Leishmania genus. We observed that the flow cytometry technique was more sensitive than the ELISA, but, less specific. Our results show that the flow cytometry serologic test can be used to confirm CL cases in L. braziliensis transmission areas, however, presence of Chagas disease has to be ruled out in these individuals. PMID:27622535

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

    Science.gov (United States)

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

    2012-09-01

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

  7. In vitro susceptibilities of wild and drug resistant leishmania donovani amastigote stages to andrographolide nanoparticle: role of vitamin E derivative TPGS for nanoparticle efficacy.

    Directory of Open Access Journals (Sweden)

    Subhasish Mondal

    Full Text Available Visceral leishmaniasis (VL is a chronic protozoan infection in humans associated with significant global morbidity and mortality. There is an urgent need to develop drugs and strategy that will improve therapeutic response for effective clinical treatment of drug resistant VL. To address this need, andrographolide (AG nanoparticles were designed with P-gp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethyleneglycol 1000 succinate for sensitivity against drug resistant Leishmania strains. AG loaded PLGA (50∶50 nanoparticles (AGnps stabilized by vitamin E TPGS were prepared for delivery into macrophage cells infested with sensitive and drug resistant amastigotes of Leishmania parasites. Physico-chemical characterization of AGnps by photon correlation spectroscopy exhibited an average particle size of 179.6 nm, polydispersity index of 0.245 and zeta potential of -37.6 mV. Atomic force microscopy and transmission electron microscopy visualization revealed spherical nanoparticles with smooth surfaces. AGnps displayed sustained AG release up to 288 hours as well as minimal particle aggregation and drug loss even after three months study period. Antileishmanial activity as revealed from selectivity index in wild-type strain was found to be significant for AGnp with TPGS in about one-tenth of the dosage of the free AG and one-third of the dosage of the AGnp without TPGS. Similar observations were also found in case of in vitro generated drug resistant and field isolated resistant strains of Leishmania. Cytotoxicity of AGnp with and without TPGS was significantly less than standard antileishmanial chemotherapeutics like amphotericin B, paromomycin or sodium stibogluconate. Macrophage uptake of AGnps was almost complete within one hour as evident from fluorescent microscopy studies. Thus, based on these observations, it can be concluded that the low-selectivity of AG in in vitro generated drug resistant and field isolated resistant strains was

  8. Seroprevalence of dogs to Leishmania infantum antigens in the territory of Crna Gora

    Directory of Open Access Journals (Sweden)

    Pavićević Zorica

    2014-01-01

    Full Text Available Due to expansion of vector borne diseases and their importance for human health we have investigated the prevalence of antibodies to Leishmania infantum antigens in serum samples of dogs. Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. According to the World Health Organization the disease is a constant threat to 350 million people in 88 countries, provided that most Mediterranean countries belong to the area designated as endemic. Since Crna Gora is a country of Mediterranean basin, it is important to present the results of the investigation on the presence of antibodies on L. infantum antigens in blood serum of dogs, considering that dogs can be reservoirs and source of infection for humans. In the period from 2006. to 2010., there were tested 433 serum samples of dogs with clinical symptoms and 68 serum samples of dogs without symptoms of the disease. Those were dogs with visible symptoms of the disease such as alopecia, weight loss and lymphadenomegalia. In 66% of cases, there was known hematological profile with predominant hypochromic anemia and neutrophilic leukocytosis. As mentioned above, the investigation included also 68 asymptomatic dogs in good condition, out of which 45 were service dogs in use in military and police as well as 23 dogs from the asylum. By Indirect immunofluorescence test or Indirect Fluorescent Antibody Technique (IF-AT and Direct Agglutination Test (DAT, there was determined the presence of specific antibodies to L. infantum antigens. Positive findings in the serum dilution 1:160 was deter­mined in 258 samples (59.58% in the group of dogs with clinical symptoms, and in 5.9% in the tested asymptomatic dogs. A part of the territory of Crna Gora with its average annual air temperature of 14.6 - 17.8°C and mediterranean climate (long, warm and dry summer and mild, rainy winter has all the prerequisites for the development and maintenance of the infection. The confirmed presence of

  9. Lymphoproliferative response after stimulation with soluble leishmania antigen (SLA) as a predictor of visceral leishmaniasis (VL) relapse in HIV+ patients.

    Science.gov (United States)

    Castro, Alicia; Carrillo, Eugenia; San Martín, Juan V; Botana, Laura; Molina, Laura; Matía, Belén; Fernandez, Laura; Horrillo, Luis; Ibarra-Meneses, Ana; Sanchez, Carmen; Ruiz-Giardin, Jose M; Moreno, Javier

    2016-12-01

    The introduction of HAART resulted in the decrease of Leishmania/HIV co-infection cases; nevertheless, the number of relapses remains high and secondary prophylaxis is recommended. However, secondary prophylaxis is not necessary in all patients, and presents a high risk of toxicity and an elevated cost. Our aim was to study whether specific cellular response to Leishmania infantum (measured by cell proliferation response after stimulation with soluble Leishmania antigen (SLA)), could be a useful tool to attempt a secondary prophylaxis withdrawal. In June 2009 an outbreak of leishmaniasis by Leishmania infantum was declared in the southeast of Madrid, and since January 2013, we recruited 10 HIV+ patients that had been treated for visceral leishmaniasis. 6 patients had positive SLA-cell proliferation test. The mean CD4 cell counts of those patients with positive SLA were 140 cel/mm3 and 40 cel/mm3 in those with negative SLA test. 3 patients with positive SLA-cell proliferation test (CD4 count: 336, 307, 625) were not on prophylaxis, and the other 3 patients (CD4 count: 152, 189, 359) were on secondary prophylaxis that was withdrawn after the positive SLA-cell proliferation test with no posterior relapses (mean follow up 60 weeks). From the 4 patients, which had negative SLA-cell proliferation test and continued on prophylaxis, 3 had positive PCR for Leishmania at the end of the follow-up and 2 presented clinical relapses. The performance of SLA-cell proliferation test can be a useful tool that can permit us to try withdrawal of the prophylaxis in Leishmania/HIV co-infected patients with low CD4(+) counts under clinical supervision, diminishing risk of toxicity and cost. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte.

    Science.gov (United States)

    Rodrigues, A; Claro, M; Alexandre-Pires, G; Santos-Mateus, D; Martins, C; Valério-Bolas, A; Rafael-Fernandes, M; Pereira, M A; Pereira da Fonseca, I; Tomás, A M; Santos-Gomes, G

    2017-02-01

    In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8(+) T cell subset, but inhibit the proliferation of CD8(+) T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins

  11. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity.

    Science.gov (United States)

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A; Schreiber, Stuart L; Ioset, Jean-Robert; Gray, David W

    2015-09-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Structure-based virtual screening, molecular docking, ADMET and molecular simulations to develop benzoxaborole analogs as potential inhibitor against Leishmania donovani trypanothione reductase.

    Science.gov (United States)

    Pandey, Rajan Kumar; Kumbhar, Bajarang Vasant; Sundar, Shyam; Kunwar, Ambarish; Prajapati, Vijay Kumar

    2017-02-01

    Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis and it affects 70 countries worldwide. Increasing drug resistant for antileishmanial drugs such as miltefosine, sodium stibogluconate and pentamidine has been reported in the VL endemic region. Amphotericin B has shown potential antileishmanial activity in different formulations but its cost of treatment and associated nephrotoxicity have limited its use by affected people living in the endemic zone. To control the VL infection in the affected countries, it is necessary to develop new antileishmanial compounds with high efficacy and negligible toxicity. Computer aided programs such as binding free energy estimation; ADMET prediction and molecular dynamics simulation can be used to investigate novel antileishmanial molecules in shorter duration. To develop antileishmanial lead molecule, we performed standard precision (SP) docking for 1160 benzoxaborole analogs along with reference inhibitors against trypanothione reductase of Leishmania parasite. Furthermore, extra precision (XP) docking, ADMET prediction, prime MM-GBSA was conducted over 115 ligands, showing better docking score than reference inhibitors to get potential antileishmanial compounds. Simultaneously, area under the curve (AUC) was estimated using ROC plot to validate the SP and XP docking protocol. Later on, two benzoxaborole analogs with best MM-GBSA ΔG-bind were subjected to molecular simulation and docking confirmation to ensure the ligand interaction with TR. The presented drug discovery based on computational study confirms that BOB27 can be used as a potential drug candidate and warrants further experimental investigation to fight against VL in endemic areas.

  15. Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.

    Science.gov (United States)

    Mukherjee, Sandip; Mukherjee, Budhaditya; Mukhopadhyay, Rupkatha; Naskar, Kshudiram; Sundar, Shyam; Dujardin, Jean-Claude; Roy, Syamal

    2014-10-15

    The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.

  16. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    Science.gov (United States)

    Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. PMID:24675711

  17. Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

    Directory of Open Access Journals (Sweden)

    Farnaz Zahedifard

    2014-03-01

    Full Text Available BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15 and represents a novel promising vaccination approach against leishmaniasis.

  18. Mimotope-based vaccines of Leishmania infantum antigens and their protective efficacy against visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Lourena Emanuele Costa

    Full Text Available BACKGROUND: The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL. Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. METHODOLOGY/MAIN FINDINGS: Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin, showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. CONCLUSIONS/SIGNIFICANCE: This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the

  19. Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis.

    Science.gov (United States)

    Heravi Shargh, Vahid; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jalali, Seyed Amir; Firouzmand, Hengameh; Abbasi, Azam; Badiee, Ali

    2012-07-01

    Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.

  20. Interferon-¿ production by human T cells and natural killer cells in vitro in response to antigens from the two intracellular pathogens Mycobacterium tuberculosis and Leishmania major

    DEFF Research Database (Denmark)

    Kemp, K; Hviid, L; Kharazmi, A

    1997-01-01

    protein derivative of tuberculin (PPD) and Leishmania antigens. It was found that IFN-gamma was produced in response to both PPD and Leishmania stimulant by T cells in the cultures. Activation of IFN-gamma producing natural killer (NK) cells was demonstrated only in some cultures, and only......Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-gamma (IFN-gamma), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-gamma production was investigated in human peripheral blood...... mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-gamma and surface markers allowed flow cytometric identification of the cellular sources of IFN-gamma in cell cultures incubated with purified...

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

    Science.gov (United States)

    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

  2. Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis.

    Science.gov (United States)

    Lakshmi, Bhavana Sethu; Wang, Ruobing; Madhubala, Rentala

    2014-06-24

    Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform.

  3. Antigen-Experienced T cells Limit the Priming of Naïve T cells During Infection with Leishmania major1

    Science.gov (United States)

    Gray, Peter M.; Reiner, Steven L.; Smith, Deborah F.; Kaye, Paul M.; Scott, Phillip

    2009-01-01

    One mechanism to control immune responses following infection is to rapidly down regulate antigen presentation, which has been observed in acute viral and bacterial infections. Here we describe experiments designed to address whether antigen presentation is decreased after an initial response to Leishmania major. Naïve α-β-Leishmania-specific (ABLE) T cell receptor transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived antigens. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells or antigen load. However, antigen-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naïve ABLE cell responses was not due to lack of antigen presentation. Since naïve T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of antigen pulsed dendritic cells, it appears that T cells that have previously encountered antigen during infection compete with naïve antigen-specific T cells. These results suggest that during L. major infection antigen-experienced T cells, rather than naïve T cells, may be primarily responsible for sustaining the immune response. PMID:16818747

  4. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

    DEFF Research Database (Denmark)

    Kemp, M; Handman, E; Kemp, K

    1998-01-01

    . Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor...... and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen...

  5. Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

    Science.gov (United States)

    Duthie, Malcolm S; Favila, Michelle; Hofmeyer, Kimberley A; Tutterrow, Yeung L; Reed, Steven J; Laurance, John D; Picone, Alessandro; Guderian, Jeffrey; Bailor, H Remy; Vallur, Aarthy C; Liang, Hong; Mohamath, Raodoh; Vergara, Julie; Howard, Randall F; Coler, Rhea N; Reed, Steven G

    2016-05-27

    Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  6. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania...

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

    Science.gov (United States)

    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

  8. KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi.

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    Regis Gomes

    Full Text Available BACKGROUND: Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. METHODS: Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge. RESULTS: Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4(+CD62L(lowCCR7(low effector memory T cells pre- and post-sand fly challenge. CONCLUSIONS: This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection.

  9. Immunoliposomes containing Soluble Leishmania Antigens (SLA) as a novel antigen delivery system in murine model of leishmaniasis.

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    Eskandari, Faeze; Talesh, Ghazal Alipour; Parooie, Maryam; Jaafari, Mahmoud Reza; Khamesipour, Ali; Saberi, Zahra; Abbasi, Azam; Badiee, Ali

    2014-11-01

    Development of new generation of vaccines against leishmaniasis requires adjuvants to elicit the type and intensity of immune response needed for protection. The coupling of target-specific antibodies to the liposomal surface to create immunoliposomes has appeared as a promising way in achieving a liposome active targeting. In this study, immunoliposomes were prepared by grafting non-immune mouse IgG onto the liposomal surface. The influence of active targeted immunoliposomes on the type and intensity of generated immune response against Leishmania was then investigated and compared with that of liposomes and control groups which received either SLA or HEPES buffer alone. All formulations contained SLA and were used to immunize the mice in the left hind footpad three times in 3-week intervals. Evaluation of lesion development and parasite burden in the foot and spleen after challenge with Leishmania major, evaluation of Th1 cytokine (IFN-γ), and titration of IgG isotypes were carried out to assess the type of generated immune response and the extent of protection. The results indicated that liposomes might be effective adjuvant systems to induce protection against L. major challenge in BALB/c mice, but stronger cell mediated immune responses were induced when immunoliposomes were utilized. Thus, immune modulation using immunoliposomes might be a practical approach to improve the immunization against L. major. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Atypical Mucocutaneous Leishmaniasis Caused by Leishmania braziliensis in an Acquired Immunodeficiency Syndrome Patient: T-cell Responses and Remission of Lesions Associated with Antigen Immunotherapy

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    Da-Cruz Alda M

    1999-01-01

    Full Text Available An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B. Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens. Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR. Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4. After the first course of combined therapy it became positive (SI=4.17. The antigen responding cells were predominantly T-cells (47.5% most of them with CD8+ phenotype (33%. Very low CD4+ cells (2.2% percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

  11. Leishmania Skin Test

    Science.gov (United States)

    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

  12. IFN-γ, IL-2, IP-10, and MIG as Biomarkers of Exposure to Leishmania spp., and of Cure in Human Visceral Leishmaniasis

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    Ana V. Ibarra-Meneses

    2017-05-01

    Full Text Available New biomarkers are needed for monitoring the effectiveness of treatment for visceral leishmaniasis (VL. They might also improve the detection of the asymptomatic population in Leishmania-endemic areas. This paper examines the IL-2, IFN-γ, IFN-γ-induced protein 10 (IP-10, and monokine-induced-by-IFN-γ (MIG levels in whole blood—stimulated in vitro with soluble Leishmania antigen (SLA—taken from asymptomatic individuals and patients treated for VL living in a post-outbreak (Leishmania infantum area in Spain, and in an endemic (Leishmania donovani area of Bangladesh. IP-10 was found to be an accurate global marker of asymptomatic subjects with positive cellular/humoral tests, while MIG was found to be a better marker of contact with L. donovani than IL-2 but no for those with L. infantum. Determining IP-10, MIG, and IFN-γ levels proved useful in monitoring the cellular immune response following treatment for active disease caused by L. infantum.

  13. Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus.

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    da Silveira Neto, Luiz; Sobrinho, Ludmila Silva Vicente; Martins, Camille Oliveira; Machado, Rosangela Zacarias; Marcondes, Mary; de Lima, Valéria Marçal Felix

    2011-05-11

    Visceral leishmaniasis is a disease caused by Leishmania (Leishmania) chagasi and represents a serious public health problem. The dog is the main urban reservoir of the disease; however, investigations regarding the occurrence and epidemiological importance of leishmaniasis in cats have recently been initiated. This study aimed to detect cats seropositive for Leishmania spp. using different antigens. Additional studies were performed using sera from cats with Toxoplasma gondii (n=15) to evaluate cross-reactivity. Serum samples (n=113) from cats living in the town of Araçatuba, State of São Paulo, Brazil, an endemic area for human and canine visceral leishmaniasis, were tested by indirect ELISA using different antigens: crude (CAG-ELISA), fucose-mannose ligand (FML-ELISA) and K39 (rK39-ELISA). Anti-Leishmania spp. antibodies were detected in 23.0% of samples evaluated by CAG-ELISA, 13.3% by FML-ELISA and 15.9% by RK39-ELISA. Only reactive sera in all three tests were considered truly positive. No disagreement occurred among the tests (pfeline leishmaniasis in Araçatuba. Further studies are required to clarify the role of cats in the epidemiological cycle of leishmaniasis. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Lesion Size Correlates with Leishmania Antigen-Stimulated TNF-Levels in Human Cutaneous Leishmaniasis

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    Oliveira, Fabiano; Bafica, Andre; Rosato, Andrea B.; Favali, Cecilia B. F.; Costa, Jackson M.; Cafe, Virginia; Barral-Netto, Manoel; Barral, Aldina

    2011-01-01

    Cutaneous leishmaniasis (CL) is a worldwide disease endemic in several regions of the globe. The hallmark of CL is skin ulcers likely driven by efforts of the immune system to control Leishmania growth. Cytokines, such as tumor necrosis factor (TNF) and interferon-gamma can control disease progression in animal models. Nevertheless, the impact of these cytokines in CL ulcer outcome is not well established in humans. In this study, 96 CL patients from an endemic area of Leishmania braziliensis were enrolled for a follow-up study that consisted of clinical and immunological evaluations in a 2-year period. Statistical analysis revealed that healing time (P = 0.029), age (P = 0.002), and TNF levels (P = 0.0002) positively correlate with ulcer size at the time of the first clinical evaluation. Our findings suggest that ulcer size correlates with healing time and TNF levels support the use of TNF inhibitors combined with standard therapy to improve healing in CL patients with severe lesions. PMID:21734128

  15. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination

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    Bañuls Anne-Laure

    2008-10-01

    Full Text Available Abstract Background PSA (promastigote surface antigen is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results From the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes. Conclusion PSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs

  16. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

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    Iris J Gonzalez-Leal

    2014-09-01

    Full Text Available Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb and L (Ctsl play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC and macrophages (BMM from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12 expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  17. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

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    Gonzalez-Leal, Iris J; Röger, Bianca; Schwarz, Angela; Schirmeister, Tanja; Reinheckel, Thomas; Lutz, Manfred B; Moll, Heidrun

    2014-09-01

    Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  18. Pegylation of Cationic Liposomes Encapsulating Soluble Leishmania Antigens (SLA) Reduces the Adjuvant Efficacy of Liposomes in Murine Model.

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    Naseri, Hadi; Eskandari, Faezeh; Jaafari, Mahmoud Reza; Khamesipour, Ali; Abbasi, Azam; Badiee, Ali

    2017-09-16

    Although there have been several attempts to develop a vaccine against leishmaniasis; no vaccine in human has been developed yet. Liposomes consisting of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) encapsulating soluble Leishmania antigens (SLA) enhance protective immunity of SLA against L. major infection in mice. However, they immobilized at the injection site because of their positive charge. To overcome the problem, shielding the surface charge with polyethylene glycol (PEGylation) was chosen in this study. Liposomal SLA consisting different concentrations of PEG (1.9-15% mol) were prepared. BALB/c mice were immunized three times in three weeks intervals with different formulations. Lesion development and parasite burden in footpad and spleen were evaluated to specify the type of generated immune response and extent of protection. Th1/Th2 cytokine profiles and IgG isotypes were also analyzed. The maximum protection was observed in mice immunized with Lip-SLA or pLip-SLA (1.9%) due to smaller footpad swelling, reduction in parasite load, an increase in IgG2a and IFN-γ production. Our results showed that immunization of mice with high level of PEG (>7.5%) did not improve protective immunity of liposomal SLA. The presence of PEG, particularly more than 3.75%, is not recommended for protection against leishmaniasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis

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    Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  20. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

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    Vivian T Martins

    Full Text Available The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1, previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1 was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL, but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin, showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.

  1. Humoral and In Vivo Cellular Immunity against the Raw Insect-Derived Recombinant Leishmania infantum Antigens KMPII, TRYP, LACK, and papLe22 in Dogs from an Endemic Area

    Science.gov (United States)

    Todolí, Felicitat; Solano-Gallego, Laia; de Juan, Rafael; Morell, Pere; del Carmen Núñez, Maria; Lasa, Rodrigo; Gómez-Sebastián, Silvia; Escribano, José M.; Alberola, Jordi; Rodríguez-Cortés, Alhelí

    2010-01-01

    Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs. PMID:21118936

  2. Induction of protection against leishmaniasis in susceptible BALB/c mice using simple DOTAP cationic nanoliposomes containing soluble Leishmania antigen (SLA).

    Science.gov (United States)

    Firouzmand, Hengameh; Badiee, Ali; Khamesipour, Ali; Heravi Shargh, Vahid; Alavizadeh, Seyedeh Hoda; Abbasi, Azam; Jaafari, Mahmoud Reza

    2013-12-01

    A suitable adjuvant and delivery system are needed to develop an effective vaccine against leishmaniasis. To induce a Th1 type of response and protection in BALB/c mice against Leishmania major infection, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) nanoliposomes bearing an intrinsic adjuvanticity, were used as an antigen delivery system and immunoadjuvant for soluble Leishmania antigens (SLA). DOTAP liposomes containing different concentrations of SLA were prepared by using lipid film method followed by sonication. The prepared vesicles showed a diameter of about 100nm, a positive zeta potential and approximately 70% encapsulation efficiency of SLA. BALB/c mice were immunized subcutaneously (SC), three times in a 3-week interval with different concentrations of liposomal SLA (12.5, 25, and 50μg of SLA/50μl/mice), free SLA and as well as free liposome. The group of mice received 50μg of SLA in DOTAP-nanoliposomes showed a significantly (pSLA are appropriate delivery systems to induce a Th1 type of immune response and protection against L. major infection in BALB/c mice. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Mechanisms of the natural reactivity of lymphocytes from noninfected individuals to membrane-associated Leishmania infantum antigens.

    Science.gov (United States)

    Sassi, Atfa; Larguèche-Darwaz, Beya; Collette, Alexis; Six, Adrien; Laouini, Dhafer; Cazenave, Pierre André; Dellagi, Koussay

    2005-03-15

    Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRalphabeta+, CD45RO+, and CD45RA+ and secrete IFN-gamma and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-alpha, but have no effect on IL-1alpha or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable beta segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host's natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.

  4. Qualitative and semi-quantitative comparison of an rK39 strip test and direct agglutination test for detection of anti-Leishmania donovani antibodies in the Sudan.

    Science.gov (United States)

    Mansour, Durria; Abass, Elfadil M; Mahamoud, Abdelhafeiz; el Harith, Abdallah

    2009-12-01

    Until now, the comparison of the rK39 strip test (RKT) and direct agglutination test (DAT) for detection of visceral leishmaniasis (VL) is exclusively based on either positive or negative qualification of the reaction outcome. In this study, we compared the diagnostic performance of RKT and DAT for VL both qualitatively and semi-quantitatively. For comparison based on semi-quantitative grounds, the execution of RKT and DAT was according to the standard procedures. For comparison on semi-qualitative grounds with DAT, the RKT was applied to aliquots from positive samples that were two-fold serially diluted in saline to determine, as for the DAT, the end-point reaction in RKT. While qualitatively both RKT and DAT demonstrated comparable reliability for VL detection (sensitivity = 96% and specificity = 98.7% or 99.3%), no significant correlation (r = 0.13) could be established between intensities of their positive reactions in 25 cases studied. A negative correlation was further determined in those 25 VL cases between the positive intensities of the RKT and antibody levels measured semi-quantitatively with the same procedure (r = -0.36) or the DAT (r = -0.30). Irrespective of the low, moderate or high antibody levels measured with RKT ( or = 1:256) or DAT ( or = 1:3,276,800) in patients with confirmed or unconfirmed VL infection, exclusively strong positive intensities were obtained with RKT. For further optimizing diagnosis and simultaneously assessing magnitude of immune response to L. donovani infection in Sudanese patients, the combined application of RKT and DAT is recommended.

  5. Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis.

    Science.gov (United States)

    Mizbani, Amir; Taheri, Tahereh; Zahedifard, Farnaz; Taslimi, Yasaman; Azizi, Hiva; Azadmanesh, Kayhan; Papadopoulou, Barbara; Rafati, Sima

    2009-12-10

    Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccine against this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, but none of them conferred complete long-term protection. The use of live attenuated vaccines has recently emerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovani A2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated its protective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperitoneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-gamma production prior and after challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to a potent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, characterized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection. All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against visceral leishmaniasis.

  6. DDA/TDB liposomes containing soluble Leishmania major antigens induced a mixed Th1/Th2 immune response in BALB/c mice

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    Mansure Hojatizade

    2017-04-01

    Full Text Available Objective(s: Leishmaniasis is a complex parasitic disease that represents a major public health problem. Despite numerous attempts over the past decades, yet there is no effective vaccine against human leishmaniasis probably due to the lack of suitable adjuvants. In this study, a first generation liposomal-based Leishmania vaccine was developed using soluble Leishmania major antigens (SLA and á, Ü-trehalose6, 6'-dibehenat (TDB as an immunostimulatory adjuvant. In this liposome structure, the cationic lipid Dimethyldioctadecylammonium (DDA provides intrinsic adjuvant activity and cholesterol was added as a membrane stabilizer. Liposomes containing SLA were prepared.Materials and Methods: BALB/c mice were subcutaneously (sc immunized with Lip (DDA/TDB/CHOL-SLA+, Lip (DDA/TDB-SLA+, Lip (DDA-SLA+, Lip (DDA/CHOL-SLA+, SLA or Tris-HCl buffer. Immunization was done every two weeks for three weeks. The immunized mice were then challenged sc in the left footpad with 1×106 stationary phase L. major promastigotes (50 ìl, at 2 weeks after last booster injection.Results: mice immunized with any of the liposomal formulations containing SLA (Lip-SLA+, substantially increased footpad swelling and parasite loads of foot and spleen with no significant difference compared to Tris-HCl buffer or SLA alone. Lip-SLA+ formulations induced a mixed Th1/Th2 immune response characterized by IFN-ã and IL-4 production as well as high levels of IgG1 anti-Leishmania antibody. Conclusion: immunization with liposomes containing DDA and/or TDB in combination with SLA induces a mixed Th1/Th2 immune response and is not an appropriate strategy for preferential induction of a Th1 response and protection against leishmaniasis.

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil.

    Directory of Open Access Journals (Sweden)

    Mauro Maciel de Arruda

    Full Text Available BACKGROUND: American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. METHODS: A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. RESULTS: The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. INTERPRETATION: ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

  9. Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis.

    Science.gov (United States)

    Datta, Sanchita; Manna, Madhumita; Khanra, Supriya; Ghosh, Moumita; Bhar, Radhaballav; Chakraborty, Anindita; Roy, Syamal

    2012-07-01

    After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman-Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further.

  10. Phylogenetic position of Leishmania isolates from Khyber Pakhtunkhwa province of Pakistan.

    Science.gov (United States)

    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.

  11. Development and Production of a Leishmania Skin Test

    Science.gov (United States)

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies...... 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....

  13. A soro-aglutinação das Leishmanias Agglutination of Leishmanias

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    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

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

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    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.

  15. 利什曼原虫胞内寄生相关基因的研究%The research on genes related to the endoparasitic Leishmania

    Institute of Scientific and Technical Information of China (English)

    曹得萍; 陈建平

    2010-01-01

    利什曼病是由利什曼原虫无鞭毛体寄生在包括人在内的哺乳动物巨噬细胞而引起的疾病,由杜氏利什曼原虫引起的内脏利什曼病若不治疗则会致命.研究者对寄生在白蛉属消化道内的前鞭毛体和寄生在巨噬细胞内的无鞭毛体胞内高表达基因或蛋白进行研究,筛选出一些特异基因,为利什曼原虫疫苗候选抗原的确定和靶作用药物的确定提供了科学依据.%Leishmaniasis is a disease caused by Leishmania spp. amastigotes which parasitize in the macrophage cells of mammals including human being. Visceral leishmaniasis caused by Leishmania donovani is usually fatal if not properly treated. Parasitological studies have found some special genes by screening high expressed genes or proteins in motile promastigotes existing in the midgut of sandflies and non-motile amastigotes residing in macrophages. These findings may provide a scientific foundation to determine target drugs and candidate antigens of Leishmania vaccine.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections

    DEFF Research Database (Denmark)

    Gaafar, A; Kharazmi, A; Ismail, A

    1995-01-01

    The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n = 10) and mild (n = 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP...

  19. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases.

    Science.gov (United States)

    Seyyedtabaei, Seyyed Javad; Rostami, Ali; Haghighi, Ali; Mohebali, Mehdi; Kazemi, Bahram; Fallahi, Shirzad; Spotin, Adel

    2017-01-01

    Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB) compared with indirect immunofluorescence test (IFAT) to serodiagnosis of leishmaniasis. This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups. WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis.

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    Vivian Tamietti Martins

    Full Text Available In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF; to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine, were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells, correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of

  2. Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

    Science.gov (United States)

    Martins, Vivian Tamietti; Chávez-Fumagalli, Miguel Angel; Lage, Daniela Pagliara; Duarte, Mariana Costa; Garde, Esther; Costa, Lourena Emanuele; da Silva, Viviane Gomes; Oliveira, Jamil Silvano; de Magalhães-Soares, Danielle Ferreira; Teixeira, Santuza Maria Ribeiro; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2015-01-01

    In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite

  3. Multilocus microsatellite typing (MLMT of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group.

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    Evi Gouzelou

    Full Text Available BACKGROUND: New foci of human CL caused by strains of the Leishmania donovani (L. donovani complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE using the Montpellier (MON system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. METHODOLOGY/PRINCIPAL FINDINGS: The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains and from a VL patient in the south-west (Kuşadasi; EP59 strain. These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains and MON-308 (EP59. A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. CONCLUSION: The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding

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

    Science.gov (United States)

    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.

  5. Enhanced efficacy and immunogenicity of 78kDa antigen formulated in various adjuvants against murine visceral leishmaniasis.

    Science.gov (United States)

    Nagill, Rajeev; Kaur, Sukhbir

    2010-05-21

    Leishmania infection causes localized cutaneous to severe visceral disease in humans and animals. Current control measures, based on antimonial compounds, are not effective because of resistance in Leishmania. Vaccination would be a feasible alternative, but as yet no vaccine to protect humans against infection has been commercialized. Parasite antigens that preferentially stimulate the induction of significant protection through Th1 response presents a rational approach for a vaccine against leishmaniasis. With this view in mind, we investigated the potential of 78kDa antigen of Leishmania donovani alone and along with different adjuvants against murine visceral leishmaniasis. Various adjuvants used along with 78kDa antigen include monophosphoryl lipid A (MPL-A), liposomal encapsulation, recombinant IL-12, autoclaved Leishmania antigen (ALD) and Freund's adjuvant (FCA). BALB/c mice were immunized subcutaneously thrice with respective vaccine formulation. Challenge infection was given intracardially after 2 weeks of second booster. A significant decrease in parasite burden was seen in vaccinees over the infected controls on all post challenge days and was found that maximum protection was provided by 78kDa+rIL-12 vaccine and it was highly immunogenic as depicted by the reduction in parasite load (71-94.8%), reduction in infection rate of peritoneal macrophages (92.9-98%), enhanced DTH response (6.5-10.5 fold), increase in IgG2a anti-leishmanial antibody production (3-3.7 fold) and up-regulation of IFN-gamma (3.7-6.5 fold) and IL-2 levels (7.7-12.3 fold), which demonstrate the generation of protective Th1 type of immune response. Comparable results were also observed in 78kDa+MPL-A and liposome-encapsulated 78kDa vaccines with 56.5-92% and 62.9-93.4% reduction in parasite load respectively. Significant results have also been obtained with 78kDa antigen+ALD, 78kDa antigen+FCA and 78kDa antigen alone group but the protective efficacy was reduced as compared to the

  6. In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response.

    Directory of Open Access Journals (Sweden)

    Negar Seyed

    2011-09-01

    Full Text Available BACKGROUND: As a potent CD8(+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. METHODS AND FINDINGS: Six Leishmania (L. major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3 were screened for potential CD8(+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele. Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2(+ individuals recovered from L. major. HLA-A2(- individuals recovered from L. major and HLA-A2(+ healthy donors were included as control groups. Individual response of HLA-A2(+ recovered volunteers as percent of CD8(+/IFN-γ(+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2(- recovered individuals. Based on cutoff scores calculated from the response of HLA-A2(- recovered individuals, 31.6% and 13.3% of HLA-A2(+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2(- recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2(+ recovered individuals. CONCLUSION: Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II and LPG-3- (pool IV related peptides specifically presented in HLA-A*0201 context. This is among the very few reports

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

    Directory of Open Access Journals (Sweden)

    B Bastrenta

    2003-03-01

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

  8. Optimizing Immunization Strategies for the Induction of Antigen-Specific CD4 and CD8 T Cell Responses for Protection against Intracellular Parasites.

    Science.gov (United States)

    Hofmeyer, Kimberly A; Duthie, Malcolm S; Laurance, John D; Favila, Michelle A; Van Hoeven, Neal; Coler, Rhea N; Reed, Steven G

    2016-09-01

    Immunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such as Leishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against the Leishmania vaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimental L. donovani infection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic. Copyright © 2016 Hofmeyer et al.

  9. Characterization of microRNA expression profiles in Leishmania-infected human phagocytes.

    Science.gov (United States)

    Geraci, N S; Tan, J C; McDowell, M A

    2015-01-01

    Leishmania are intracellular protozoa that influence host immune responses eliciting parasite species-specific pathologies. MicroRNAs (miRNAs) are short single-stranded ribonucleic acids that complement gene transcripts to block protein translation and have been shown to regulate immune system molecular mechanisms. Human monocyte-derived dendritic cells (DC) and macrophages (MP) were infected in vitro with Leishmania major or Leishmania donovani parasites. Small RNAs were isolated from total RNA and sequenced to identify mature miRNAs associated with leishmanial infections. Normalized sequence read count profiles revealed a global downregulation in miRNA expression among host cells following infection. Most identified miRNAs were expressed at higher levels in L. donovani-infected cells relative to L. major-infected cells. Pathway enrichments using in silico-predicted gene targets of differentially expressed miRNAs showed evidence of potentially universal MAP kinase signalling pathway effects. Whereas JAK-STAT and TGF-β signalling pathways were more highly enriched using targets of miRNAs upregulated in L. donovani-infected cells, these data provide evidence in support of a selective influence on host cell miRNA expression and regulation in response to differential Leishmania infections.

  10. Cutaneous leishmaniasis caused by Leishmania infantum in Southern Israel.

    Science.gov (United States)

    Ben-Shimol, Shalom; Sagi, Orli; Horev, Amir; Avni, Yonat Shemer; Ziv, Mati; Riesenberg, Klaris

    2016-12-01

    Cutaneous leishmaniasis (CL) caused by Leishmania major is common in southern Israel, while Leishmania infantum (sub-strain of L. donovani, causing zoonotic visceral leishmaniasis) infections were rarely reported in Israel and only in other regions. We report the first case of L. infantum infection in southern Israel, presented atypically as CL in an immunosuppressed 47-year old male. The patient was treated with liposomal amphotericin-B and recovered, without extra-cutaneous complications. Diagnosis of L. infantum CL was confirmed by microscopic identification of amastigotes in Gimsa-stained smear of skin lesion, positive blood serology and a positive polymerase chain reaction (PCR) amplification of the internal transcribed spacer 1 genes (ITS1) and restriction fragment length polymorphism (ITS1 PCR-RFLP). We also review the medical literature on old-world CL caused by L. infantum. Multiple L. donovani/infantum CL cases were identified in the literature search. These can be divided schematically to two: 1) In several endemic countries, L. infantum strains are the main causative agents of CL; 2) In other regions, CL is almost exclusively caused by L. major or L. tropica, while L. donovani strains CL cases were reported sporadically or as imported disease.

  11. A Mr 90 000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen.

    Science.gov (United States)

    Dragon, E A; Brothers, V M; Wrightsman, R A; Manning, J

    1985-09-01

    Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.

  12. 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

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    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

  13. Detection and characterization of Leishmania in tissues of patients with post kala-azar dermal leishmaniasis using a specific monoclonal antibody

    DEFF Research Database (Denmark)

    Ismail, A; Kharazmi, A; Permin, H

    1997-01-01

    Sections from skin lesions and draining lymph nodes of patients with post kala-azar dermal leishmaniasis were examined using an immunoperoxidase method and a monoclonal antibody directed against Leishmania donovani. Parasites were detected in 22 of 25 biopsies (88%). In parallel sections stained...

  14. Evaluación del efecto del ácido nalidíxico, ampicilina, kanamicina, penicilina G y polimixina B en los cultivos de promastigotes de leishmania

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    Sofía Duque Beltrán

    1992-06-01

    Full Text Available Se evaluó el efecto de diferentes concentraciones de ácido nalidíxico, ampicilina, kanarnicina, penicilina G y polimixina B, sobre la población de promastigotes de Leishmania braziliensis braziliensis , Leishmania donovani chagasi y Leishmania mexicana amazonensis in vitro. La penicilina G y la ampicilina se pueden utilizar hasta concentraciones de 1000 ug/ml y 500 ug/ml respectivamente en cultivo de promastigotes de cualquier cepa de Leishmania sin que éstos se afecten. La polimixina B disminuye la población de prosmastigotes por lo cual es preferible no usarse en cultivos de Leishmania. El ácido nalidíxico y kanamicina pueden ser utilizados in vitro pero teniéndose en cuenta la especie de Leishmania y la concentración de anfimicrobiano recomendado para la misma.

  15. Cysteine proteinase type III is protective against Leishmania infantum infection in BALB/c mice and highly antigenic in visceral leishmaniasis individuals.

    Science.gov (United States)

    Khoshgoo, Naghmeh; Zahedifard, Farnaz; Azizi, Hiva; Taslimi, Yasaman; Alonso, Maribel Jiménez; Rafati, Sima

    2008-10-29

    Visceral leishmaniasis is the most acute form of leishmaniasis and vaccination is the best approach to control it. One of the major groups of virulence factors in Leishmania belongs to cysteine proteinase family. In this study, for the first time, the protective potential of Leishmania infantum cysteine proteinase type III (CPC) by using a prime-boost strategy is evaluated in BALB/c mice. The experiment was carried out in three groups of mice. Vaccinated group was primed with pcDNA-cpc and boosted with rCPC-DHFR in combination with CpG motif and Montanide 720 as adjuvant. Control groups received pcDNA and rDHFR or PBS. The ratio of IgG2a/IgG1, nitric oxide concentration and IFN-gamma induction in vaccinated group is significantly higher than controls. Furthermore, the parasite load of vaccinated group is significantly lower than controls. In addition, sera reactivity of visceral leishmaniasis individuals was examined and showed considerable reactivities toward rCPC in comparison with cutaneous leishmaniasis. The achieved result is highly encouraging the use of cysteine proteinases types I, II and III as vaccine candidate against visceral leishmaniasis.

  16. Vaccination with liposomal leishmanial antigens adjuvanted with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) confers long-term protection against visceral leishmaniasis through a human administrable route.

    Science.gov (United States)

    Ravindran, Rajesh; Maji, Mithun; Ali, Nahid

    2012-01-01

    The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8⁺ T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route.

  17. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. PMID:26197085

  18. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Mehdi Shahbazi

    Full Text Available Canine Visceral Leishmaniasis (CVL is a major veterinary and public health problem caused by Leishmania infantum (L. infantum in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

  19. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

  20. 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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Screening and characterization of RAPD markers in viscerotropic Leishmania parasites.

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    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

  3. Generation of growth arrested Leishmania amastigotes: a tool to develop live attenuated vaccine candidates against visceral leishmaniasis.

    Science.gov (United States)

    Selvapandiyan, Angamuthu; Dey, Ranadhir; Gannavaram, Sreenivas; Solanki, Sumit; Salotra, Poonam; Nakhasi, Hira L

    2014-06-30

    Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis.

  4. Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone.

    Science.gov (United States)

    Vila-Nova, Nadja Soares; de Morais, Selene Maia; Falcão, Maria José Cajazeiras; Alcantara, Terezinha Thaize Negreiros; Ferreira, Pablito Augusto Travassos; Cavalcanti, Eveline Solon Barreira; Vieira, Icaro Gusmão Pinto; Campello, Cláudio Cabral; Wilson, Mary

    2013-03-01

    Leishmaniasis is a zoonotic disease that can manifest itself in visceral and cutaneous form. The aim of this study was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied to compounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmania mexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form were used to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundum and two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities in relation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. major and L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetogenin annonacinone (EC(50)=6.72-8.00 μg/mL) indicated high leishmanicidal activity; corossolone (EC(50)=16.14-18.73 μg/mL) and scoparone (EC(50)=9.11-27.51 μg/mL) moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate these compounds for further studies aiming the development of new leishmanicidal agents.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    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...

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

    Science.gov (United States)

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

    2016-10-01

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

  9. Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

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    Daniel Paape

    2014-12-01

    Full Text Available We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT. These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646 against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.

  10. Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain.

    Science.gov (United States)

    Rodríguez-González, Isabel; Marín, Clotilde; Vargas, Franklin; Córdova, Ofelia; Barrera, Mario; Gutiérrez-Sánchez, Ramón; Alunda, Jose María; Sánchez-Moreno, Manuel

    2006-01-01

    Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.

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

    Directory of Open Access Journals (Sweden)

    Ameer Khusro

    2017-07-01

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

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

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    Sara María Robledo Restrepo

    2000-03-01

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

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

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    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

  14. Naloxonazine, an Amastigote-Specific Compound, Affects Leishmania Parasites through Modulation of Host-Encoded Functions

    Science.gov (United States)

    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

  15. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

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    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  16. Molecular detection, identification and phylogenetic inference of Leishmania spp. in some desert lizards from Northwest China by using internal transcribed spacer 1 (ITS1) sequences.

    Science.gov (United States)

    Zhang, Jun-Rong; Guo, Xian-Guang; Liu, Jin-Long; Zhou, Tian-He; Gong, Xiong; Chen, Da-Li; Chen, Jian-Ping

    2016-10-01

    Leishmaniasis caused by Leishmania is still endemic in Northwest China. It has been thought that reptiles could be a reservoir for mammalian leishmaniasis. However, data are still scarce on natural infection of lizards with Leishmania spp. in China. The present study deals with detection, identification and phylogenetic inference of Leishmania parasites at species and intraspecies levels isolated from six desert lizard species from 10 geographical locations in Northwest China using amplification and sequencing of ITS-rDNA. In total, 83 haplotypes were found among 137 ITS1 sequences obtained from up to 64.6% of all captured lizards. Representative sequences of Leishmania available in GenBank were compiled for comparison with the obtained haplotypes. Tree-based species delimitation was achieved by using Bayesian phylogenitc analyses and maximum parsimony approach. Phylogenetic trees congruently supported that the haplotypes were found to belong to three Leishmania species including L. (sauroleishmania) sp., Leishmania tropica and Leishmania donovani complex. A network approach revealed paraphyletic populations of L. (sauroleishmania) sp. and L. tropica at intraspecies level regarding geographical origin and low host specificity. Chinese L. tropica from lizards showed significant heterogeneity as the obtained haplotypes were distributed in different clusters from other countries. Common ancestry was observed between some sequences of L. tropica from lizards and other sequence types from clinical samples from other countries. This may lend support to the potential reservoir role of lizards for human leishmaniasis. Our results appear to be the first molecular evidence for natural infection of lizards in Northwest China with reptilian Leishmania and mammalian Leishmania species. Desert lizards may be considered as putative reservoir hosts for Leishmania in China. Further studies on persistence of the Leishmania parasites in lizards and sandflies are recommended for the

  17. CD271+ Mesenchymal Stem Cells as a Possible Infectious Niche for Leishmania infantum

    Science.gov (United States)

    Lopes, Carolina S.; Daifalla, Nada; Das, Bikul; Dias da Silva, Valdo; Campos-Neto, Antonio

    2016-01-01

    Visceral leishmaniasis (VL) is a serious and fatal disease. Therapeutic drugs are toxic and non-sterilizing. The etiological agents Leishmania infantum and Leishmania donovani cause active and asymptomatic diseases. Effective drugs to treat VL exist but unfortunately, post-treatment relapses are common. Little is known why drugs are non-sterilizing or how these intracellular pathogens can escape treatment. Here, using a murine model of VL we found that CD271+/Sca1+ bone marrow mesenchymal stem cells (BM-MSCs) are readily infected in vitro and in vivo by L. infantum. Because BM-MSCs express potent drug efflux pumps, e.g., ABCG2 it is possible that this unique intracellular infectious niche could allow L. infantum to escape anti-parasite drugs. PMID:27622907

  18. Identification and Characterization of Genes Involved in Leishmania Pathogenesis: The Potential for Drug Target Selection

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    Robert Duncan

    2011-01-01

    Full Text Available Identifying and characterizing Leishmania donovani genes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers in Leishmania show how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented.

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

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    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.

  20. Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population

    Science.gov (United States)

    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

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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    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.

  4. Molecular probes and the polymerase chain reaction for detection and typing of Leishmania species in Mexico.

    Science.gov (United States)

    Monroy-Ostria, Amalia; Sanchez-Tejeda, Gustavo

    2002-04-01

    Leishmaniasis in Mexico is a public health problem because all the clinical forms have been recorded in most Mexican states. We studied patients showing clinical symptoms of any form of leishmaniasis, from several endemic areas. Bone marrow samples, aspirates or skin biopsies were taken and deoxyribonucleic acid (DNA) was extracted and amplified by the polymerase chain reaction (PCR) with universal primers AJS1 and DeB8, specific for the Leishmania subgenus Leishmania. The PCR products were then hybridized by dot- or Southern blotting and probed with probe 9.2, specific for the L. mexicana complex. If hybridization did not occur, the DNA was amplified with primers D1 and D2, specific for members of the L. donovani complex, and PCR products were hybridized with probe B4Rsa, also specific for the L. donovani complex. DNA was also amplified with primers B1 and B2, specific for the subgenus Viannia, and the PCR products were hybridized with probe B18, specific for the L. braziliensis complex. It was found that in Tabasco and Veracruz, Mexico, localized cutaneous leishmaniasis (LCL) is caused by infection with members of the L. mexicana complex, whereas in the states of Nayarit and Campeche it was due to infection with the L. mexicana and/or L. braziliensis complexes. Visceral leishmaniasis was caused by L. (L.) chagasi, mainly in the states of Chiapas and Guerrero, and by L. (L.) mexicana in one immunocompromised patient from Tabasco.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Role of the domestic dog as a reservoir host of Leishmania donovani in eastern Sudan

    NARCIS (Netherlands)

    M.M. Hassan; O.F. Osman; F.M.A. El-Raba'a; H.D.F.H. Schallig; D.E.A. Elnaiem

    2009-01-01

    Background: The study aims to determine the role of domestic dogs in transmission of visceral leishmaniasis in eastern Sudan. A cross-sectional survey was conducted in 10 villages along the River Rahad in eastern Sudan to elucidate the role of domestic dogs (Canis familiaris, Linnaeus, 1758) as a re

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

    Science.gov (United States)

    de Almeida, Marcos E; Koru, Ozgur; Steurer, Francis; Herwaldt, Barbara L; da Silva, Alexandre J

    2017-01-01

    Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (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. Copyright © 2016 American Society for Microbiology.

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

    Directory of Open Access Journals (Sweden)

    Elsy Nalleli Loría-Cervera

    2013-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Zampieri

    2016-02-01

    Full Text Available Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.Exploring the High Resolution Melting (HRM dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR targeting heat-shock protein 70 coding gene (hsp70 revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania infantum chagasi, L. (L. amazonensis, L. (L. mexicana, L. (Viannia lainsoni, L. (V. braziliensis, L. (V. guyanensis, L. (V. naiffi and L. (V. shawi, and three species found in Eurasia and Africa, including L. (L. tropica, L. (L. donovani and L. (L. major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  10. Complete conservation of an immunogenic gene (lcr1 in Leishmania infantum and Leishmania chagasi isolated from Iran, Spain and Brazi

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    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.

  11. Leishmania Interferes with Host Cell Signaling to Devise a Survival Strategy

    OpenAIRE

    Suvercha Bhardwaj; Neetu Srivastava; Raki Sudan; Bhaskar Saha

    2010-01-01

    The protozoan parasite Leishmania spp. exists as extracellular promastigotes in its vector whereas it resides and replicates as amastigotes within the macrophages of its mammalian host. As a survival strategy, Leishmania modulates macrophage functions directly or indirectly. The direct interference includes prevention of oxidative burst and the effector functions that lead to its elimination. The indirect effects include the antigen presentation and modulation of T cell functions in such a wa...

  12. Ability of immunodiagnostic tests to differentiate between dogs naturally infected with Leishmania infantum and Leishmune(®)-vaccinated dogs.

    Science.gov (United States)

    Ribeiro, R A N; Teixeira-Neto, R G; Belo, V S; Ferreira, E C; Schallig, H D F H; Silva, E S

    2015-06-01

    Visceral leishmaniasis (VL) is a serious chronic disease with a lethality rate of up to 10% in humans. In urban areas of Brazil, dogs are the main reservoirs of the etiological agent (Leishmania infantum) of VL, and the Brazilian Ministry of Health recommends the euthanasia of animals that are seropositive in both the immunochromatographic dual path platform rapid test (DPP(®); Bio-Manguinhos) and the enzyme-linked immunosorbent assay (ELISA) with an L. major-like antigen (Bio-Manguinhos). Vaccination is an additional tool in the control of canine VL, but the use of Leishmune(®) (Zoetis Indústria de Produtos Veterinários, São Paulo, SP, Brazil), which contains the fucose mannose ligand (FML) isolated from L. donovani, is not currently recommended by the Brazilian Ministry of Health because vaccinated animals may exhibit positive serology and there are reservations regarding the efficacy of the vaccine. The aims of the present study were: (i) to verify the abilities of the fast agglutination screening test (FAST), the direct agglutination test (DAT), the indirect fluorescent-antibody test (IFAT), the DPP rapid test, and ELISA tests with L. major-like and FML antigens to differentiate between L. infantum-infected and Leishmune(®)-vaccinated dogs, and (ii) to analyze the sensitivities and specificities of the different methods. The reactivities to these tests of Leishmune(®)-vaccinated dogs (n = 71), asymptomatic (n = 20) and symptomatic (n = 20) naturally infected dogs, and unvaccinated healthy control dogs (n = 5) were compared. None of the Leishmune(®)-vaccinated dogs tested seropositive in FAST and DAT, although one dog was reactive to DPP and four dogs to ELISA/L. major-like and IFAT tests. While 69 (97%) of vaccinated dogs reacted to ELISA/FML, only one was seropositive in both ELISA/L. major-like and IFAT tests. Individually, all immunodiagnostic tests presented high specificities and positive likelihood ratios (LR+), and high specificity values were

  13. Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

    Science.gov (United States)

    Zhang, Wen-Wei; Lypaczewski, Patrick

    2017-01-01

    ABSTRACT CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all tested Leishmania species, including L. donovani, L. major, and L. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles of RAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential for L. donovani survival based on direct observation of the death of mutants with both RAD51 alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair in L. donovani. IMPORTANCE Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting

  14. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests

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    Chen Hai-Tang

    2011-05-01

    Full Text Available Abstract Background Canine leishmaniasis (CanL is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China, which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp fragment of the conserved region in the minicircle kinetoplast DNA (kDNA and the results of phylogenetic analyses based on the sequence of the amplified fragment. Results The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease. Conclusions More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of

  15. The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

    DEFF Research Database (Denmark)

    Gasim, S; Elhassan, A M; Kharazmi, A

    2000-01-01

    assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack...

  16. Identification of Leishmania by MALDI-TOF mass spectrometry using a free web-based application and a dedicated mass spectral library.

    Science.gov (United States)

    Lachaud, Laurence; Fernández-Arévalo, Anna; Normand, Anne Cécile; Lami, Patrick; Nabet, Cécile; Donnadieu, Jean Luc; Piarroux, Martine; Djenad, Farid; Cassagne, Carole; Ravel, Christophe; Tebar, Silvia; Llovet, Teresa; Blanchet, Denis; Demar, Magalie; Harrat, Zoubir; Aoun, Karim; Bastien, Patrick; Muñoz, Carmen; Gállego, Montserrat; Piarroux, Renaud

    2017-07-19

    Human leishmaniasis are widely spread diseases with different clinical forms caused by about 20 species within the Leishmania genus. Leishmania species identification is relevant for therapeutic management and prognosis, especially for cutaneous and muco-cutaneous forms. Several methods are available to identify Leishmania species from culture, but they have not been standardized for the majority of the currently described species, with the exception of MultiLocus Enzyme Electrophoresis. Moreover, these techniques are expensive, time-consuming and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for microorganism identification, including Leishmania However, no commercial reference mass spectral database is available. In this study, a reference mass spectral library (MSL) for Leishmania isolates, accessible through a free web-based application (mass spectral identification, MSI), was constructed and tested. It includes mass spectral data for 33 different Leishmania species, including species that infect humans, animals and phlebotomine vectors. Four laboratories on two continents evaluated MSI performance using 268 samples of which 231 were Leishmania strains. All Leishmania strains, but one, were correctly identified at least at the complex level. A risk of species misidentification within the L. donovani, L. guyanensis and L. braziliensis complexes was observed, as already reported with other techniques. The tested application was reliable, with identification results comparable to those obtained with reference methods, but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in the user's computers. Copyright © 2017 American Society for Microbiology.

  17. EFFECT OF IMMUNOMODULATOR DARAPRIM ON POTENTIATION OF VACCINE PROTECTION OF LEISHMANIA MAJOR IN BALB/c MICE

    OpenAIRE

    M. Keshavarzi; Z.M. Hassan

    2003-01-01

    In this study, a crude leishmanial antigen, was prepared by sonicating Leishmania major promastigotes used to induce immunity in BALB/c mice against cutaneous leishmaniasis. Correlation between the route of antigen injection and the efficacy of induced protection was examined. To enhance the effectiveness of the antigen, an immunostimulant drug, daraprim (pyrimethamine), was administered simultaneously with the antigen. The experiment demonstrated that simultaneous intraperitoneal injection o...

  18. Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern.

    Directory of Open Access Journals (Sweden)

    Deborah E Dobson

    Full Text Available Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG, which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite

  19. Clinical manifestations and genetic variation of Leishmania infantum and Leishmania tropica in Southern Turkey.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Three-dimensional reconstructions of the mitotic spindle and dense plaques in three species of Leishmania.

    Science.gov (United States)

    Ureña, F

    1986-01-01

    The ultrastructure of the mitotic nucleus in Leishmania braziliensis braziliensis, L. mexicana and L. donovani was studied by serial thin sections and three-dimensional reconstructions of each divisional stage. The structures of the interphase and four stages of dividing nuclei were described. Attention was paid to dense plaques and spindle microtubules. At the beginning of the nuclear division, a set of six dense plaques was found in association with spindle microtubules in the vicinity of the equatorial region of the nucleus. The number of the plaques was the same in the three species examined. Each plaque was divided into two, forming hemiplaques at the elongational stage of the division; these two sets then migrate to the poles. The plaques appeared to correspond with centromeres of metazoan cells and play an important role in the process of nuclear division.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

    Directory of Open Access Journals (Sweden)

    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

  6. KDNA genetic signatures obtained by LSSP-PCR analysis of Leishmania (Leishmania infantum isolated from the new and the old world.

    Directory of Open Access Journals (Sweden)

    Janaína Sousa Campos Alvarenga

    Full Text Available BACKGROUND: Visceral Leishmaniasis (VL caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR technique for analyzing the kinetoplastid DNA (kDNA minicircles hypervariable region. PRINCIPAL FINDINGS: KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine. To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. CONCLUSIONS: LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.

  7. Homologous recombination in Leishmania enriettii.

    OpenAIRE

    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...

  8. Leishmania vaccine development: exploiting the host-vector-parasite interface.

    Science.gov (United States)

    Reed, S G; Coler, R N; Mondal, D; Kamhawi, S; Valenzuela, J G

    2016-01-01

    Visceral leishmaniasis (VL) is a disease transmitted by phlebotomine sand flies, fatal if untreated, and with no available human vaccine. In rodents, cellular immunity to Leishmania parasite proteins as well as salivary proteins of the sand fly is associated with protection, making them worthy targets for further exploration as vaccines. This review discusses the notion that a combination vaccine including Leishmania and vector salivary antigens may improve vaccine efficacy by targeting the parasite at its most vulnerable stage just after transmission. Furthermore, we put forward the notion that better modeling of natural transmission is needed to test efficacy of vaccines. For example, the fact that individuals living in endemic areas are exposed to sand fly bites and will mount an immune response to salivary proteins should be considered in pre-clinical and clinical evaluation of leishmaniasis vaccines. Nevertheless, despite remaining obstacles there is good reason to be optimistic that safe and effective vaccines against leishmaniasis can be developed.

  9. A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene

    Directory of Open Access Journals (Sweden)

    Derouin Francis

    2005-03-01

    Full Text Available Abstract Background Leishmaniases are among the most proteiform parasitic infections in humans ranging from unapparent to cutaneous, mucocutaneous or visceral diseases. The various clinical issues depend on complex and still poorly understood mechanisms where both host and parasite factors are interacting. Among the candidate factors of parasite virulence are the A2 genes, a family of multiple genes that are developmentally expressed in species of the Leishmania donovani group responsible for visceral diseases (VL. By contrast, in L. major determining cutaneous infections (CL we showed that A2 genes are present in a truncated form only. Furthermore, the A2 genomic sequences of L. major were considered subsequently to represent non-expressed pseudogenes 1. Consequently, it was suggested that the structural and functional properties of A2 genes could play a role in the differential tropism of CL and VL leishmanias. On this basis, it was of importance to determine whether the observed structural/functional particularities of the L. major A2 genes were shared by other CL Leishmania, therefore representing a proper characteristic of CL A2 genes as opposed to those of VL isolates. Methods In the present study we amplified by PCR and sequenced the A2 genes from genomic DNA and from clonal libraries of the four Old World CL species comparatively to a clonal population of L. infantum VL parasites. Using RT-PCR we also amplified and sequenced A2 mRNA transcripts from L. major. Results A unique A2 sequence was identified in Old World cutaneous Leishmania by sequencing. The shared sequence was highly conserved among the various CL strains and species analysed, showing a single polymorphism C/G at position 58. The CL A2 gene was found to be functionally transcribed at both parasite stages. Conclusion The present study shows that cutaneous strains of leishmania share a conserved functional A2 gene. As opposed to the multiple A2 genes described in VL isolates

  10. Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantumin Mato Grosso do Sul, Brazil

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    Keyla Carstens Marques de Sousa

    Full Text Available Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi, a serological and molecular study was conducted to detectEhrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis ofL. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies toEhrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%, 49 (81.6% and 60 (100% of the sampled dogs. Twenty-seven (45%, fifty-four (90%, fifty-three (88.3%, two (3.3% and one (1.6% dog were positive, respectively, forE. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys andLeishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

  11. Leishmaniasis in Turkey: Determination of Leishmania Species by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS.

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    Gülnaz Culha

    2014-06-01

    Full Text Available Cutaneous leishmaniasis (CL is endemic in Southeastern Anatolia, mainly in Sanliurfa and Hatay provinces, and the causative agents are mostly Leishmania tropica and less frequently L. infantum. Here, we report the first MALDI-TOF analyses of Leishmania promastigotes obtained from the cultures of two CL cases from Osmaniye and Hatay provinces who were initially diagnosed by microscopy, culture and identified as L. infantum with Real-Time PCR (RT-PCR.Samples obtained from the skin lesions of patients were initially stained with Giemsa and cultivated in NNN medium. Examination of the smears and cultures revealed Leishmania amastigotes and promastigotes, respectively. The promastigotes (MHOM/TR/2012/CBU15 and MHOM/TR/2012/MK05 obtained from the cultures of both patients were used for RT-PCR targeting the ITS-1 region in the SSU of rRNA. The reference strains of four Leishmania species (L. infantum, L. donovani, L. tropica and L. major were initially assessed with MALDI-TOF and their data were added to MALDI-TOF Biotyper Library.Both RT-PCR and MALDI-TOF analyses indicated that the causative agent in both patient samples was L. infantum.Despite disadvantages such as requirement of culture fluid with nothing but promastigotes and high cost, MALDI-TOF analysis may be a fast, sensitive and specific diagnostic tool in especially large-scale research studies, where the cost declines, relatively.

  12. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  13. Activation of human T lymphocytes by Leishmania lipophosphoglycan

    DEFF Research Database (Denmark)

    Kemp, M; Theander, T G; Handman, E

    1991-01-01

    This study describes Leishmania antigen-induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis, and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala-Azar patients proliferated and produced Interferon...... 63 failed to activate PBMC from any of the donors tested. These results show that the individuals cured from visceral leishmaniasis had expanded T-cell clones recognizing LPG, conceivably as a result of Leishmania infection. The LPG preparation was without detectable protein contamination. Thus...

  14. Intradermorreação de Montenegro em cães (Mammalia: Canidae experimentalmente inoculados por Leishmaniaguyanensis e Leishmania braziliensis (Kinetoplastida: Trypanosomatidae, principais agentes causadores de Leishmaniose Tegumentar na Amazônia Montenegro's skin test in dogs (Mammalia: Canidae experimentally inoculated with Leishmaniaguyanensis and Leishmania braziliensis (Kinetoplastida: Trypanosomatidae the main agents of Amazônia Tegumentary Leishmaniasis

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    Sônia Rolim Reis

    2008-01-01

    Full Text Available O teste de intradermorreação de Montenegro é utilizado para detectar infecção por Leishmania em humanos. A técnica se baseia numa reação de hipersensibilidade tardia. Os antígenos de Montenegro utilizados no experimento são soluções de antígenos homólogos brutos de Leishmania (Viannia braziliensis e L.(Vguyanensis. Este experimento demonstrou que os animais inoculados com as espécies de Leishmania inoculadas desenvolveram enduração no local do teste mais acentuada que os animais controle. Os resultados sugerem que o teste cutâneo pode vir a ser indicado como método auxiliar de diagnóstico em cães infectados por Leishmania sp.Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of infection with Leishmania. Montenegro's antigen consisted of a crude Leishmania homologous antigen solution that was used as a skin test in five dogs experimentally inoculated with Leishmania (Viannia spp. In this work it is shown that all animals infected presented an induration at the site of injection in contrast of the dogs non infected used as a control group. This demonstrated that the skin tests in dogs could be used to make an diagnosis of the Leishmania infection.

  15. Leishmania-specific T cells expressing interferon-¿(IFN-¿) and IL-10 upon activation are expanded in individuals cured of visceral leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, K; Kemp, M; Kharazmi, A

    1999-01-01

    Peripheral blood mononuclear cells (PBMC) from patients who have recovered from visceral leishmaniasis often respond to Leishmania antigens in vitro by production of both IL-4, IFN-gamma and IL-10. In order to establish the cellular sources of these cytokines, we activated cells from individuals...... with a history of visceral leishmaniasis with Leishmania antigen for 6 days in culture, and identified cytokine production at the single-cell level by flow cytometry. The cytokines were only found in CD3+ cells and among these mainly within the CD4+ subset. The percentage of cytokine-producing cells was compared...... in Leishmania-activated PBMC cultures from the previous patients and from individuals living in a village where leishmaniasis does not occur. The percentage of IL-10- and IFN-gamma-containing cells was significantly higher in the previous patients than in the controls, indicating that Leishmania-specific T...

  16. Leishmania Interferes with Host Cell Signaling to Devise a Survival Strategy

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    Suvercha Bhardwaj

    2010-01-01

    Full Text Available The protozoan parasite Leishmania spp. exists as extracellular promastigotes in its vector whereas it resides and replicates as amastigotes within the macrophages of its mammalian host. As a survival strategy, Leishmania modulates macrophage functions directly or indirectly. The direct interference includes prevention of oxidative burst and the effector functions that lead to its elimination. The indirect effects include the antigen presentation and modulation of T cell functions in such a way that the effector T cells help the parasite survive by macrophage deactivation. Most of these direct and indirect effects are regulated by host cell receptor signaling that occurs through cycles of phosphorylation and dephosphorylation in cascades of kinases and phosphatases. This review highlights how Leishmania selectively manipulates the different signaling pathways to ensure its survival.

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

    Science.gov (United States)

    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.

  18. Longlasting insecticidal nets for prevention of Leishmania donovani infection in India and Nepal: paired cluster randomised trial

    DEFF Research Database (Denmark)

    Picado, Albert; Singh, Shri Prakash; Rijal, Suman

    2010-01-01

    To test the effectiveness of large scale distribution of longlasting nets treated with insecticide in reducing the incidence of visceral leishmaniasis in India and Nepal.......To test the effectiveness of large scale distribution of longlasting nets treated with insecticide in reducing the incidence of visceral leishmaniasis in India and Nepal....

  19. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

  20. Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins

    OpenAIRE

    Maria eAgallou; Evita eAthanasiou; Olga eKoutsoni; Eleni eDotsika; Evdokia eKaragouni

    2014-01-01

    Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4+ and CD8+ T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were ana...

  1. Plasmenylethanolamine synthesis in Leishmania major.

    Science.gov (United States)

    Pawlowic, Mattie C; Hsu, Fong-Fu; Moitra, Samrat; Biyani, Neha; Zhang, Kai

    2016-07-01

    Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT-null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI-anchored surface molecules. EPT-null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors. © 2016 John Wiley & Sons Ltd.

  2. Deciphering the Leishmania exoproteome: what we know and what we can learn.

    Science.gov (United States)

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

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    Irazú Contreras

    2014-09-01

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

  6. Leishmania spp. Epidemiology of Canine Leishmaniasis in the Yucatan Peninsula

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    A. López-Céspedes

    2012-01-01

    Full Text Available Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico. A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

  7. Interleukin-4 and interferon-¿ production by Leishmania stimulated peripheral blood mononuclear cells from nonexposed individuals

    DEFF Research Database (Denmark)

    Kurtzhals, J A; Kemp, M; Poulsen, L K

    1995-01-01

    Leishmania reactive CD4+ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-gamma and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-gamma and IL-4 is important for the clinical outcome. Our findings...

  8. Antibodies against Leishmania cross-react with Crithidia luciliae: indirect immunofluorescence and Dot-ELISA study in dogs.

    Science.gov (United States)

    Martinkovic, Franjo; Marinculic, Albert

    2006-03-01

    Leismaniasis is zoonotic parasitic disease distributed in many areas of Mediterranean basin. Because of its pathogenicity and difficulty to grow in vitro, we used Leishmania infantum as primary and Crithidia luciliae as secondary source of antigen. We compared 100 canine sera by indirect immunofluorescence and Dot-ELISA. Both atigens showed same results.

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

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    Olga Koutsoni

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

  10. Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

    Science.gov (United States)

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-11-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

  11. Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains▿

    Science.gov (United States)

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-01-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials. PMID:19726616

  12. Detection of Leishmania RNA virus in Leishmania parasites.

    Directory of Open Access Journals (Sweden)

    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.

  13. Detection of Leishmania RNA Virus in Leishmania Parasites

    Science.gov (United States)

    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

  14. Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World.

    Science.gov (United States)

    Salay, G; Dorta, M L; Santos, N M; Mortara, R A; Brodskyn, C; Oliveira, C I; Barbiéri, C L; Rodrigues, M M

    2007-09-01

    We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies.

  15. Testing of Four Leishmania Vaccine Candidates in a Mouse Model of Infection with Leishmania (Viannia) braziliensis, the Main Causative Agent of Cutaneous Leishmaniasis in the New World▿

    Science.gov (United States)

    Salay, G.; Dorta, M. L.; Santos, N. M.; Mortara, R. A.; Brodskyn, C.; Oliveira, C. I.; Barbiéri, C. L.; Rodrigues, M. M.

    2007-01-01

    We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies. PMID:17626159

  16. Field Evaluation of a Fluorogenic Probe-Based PCR Assay for Identification of a Visceral Leishmaniasis Gene Target

    Science.gov (United States)

    2004-06-01

    family loci and aligning homologous regions within Leishmania donovani complex species that excluded species from L. tropica , L. major, L. aethiopica...Genebank. Leishmania tropica , L. major, L. aethiopica, and L. mexicana complex species genomic sequences were aligned and visually evaluated to validate...Species LVL Leishmania donovani + Leishmania tropica - Leishmania major - Leishmania mexicana - Leishmania braziliensis - Leishmania guyanensis

  17. Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

    Science.gov (United States)

    Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

    2017-01-01

    BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis

  18. A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

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    Geraldine De Muylder

    2011-07-01

    Full Text Available The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

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

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    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

  20. Distribution of Blood Groups(ABO between Symptomatic & Asymptomatic Human Leishmania Infantum Infection in Human

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    S Molaie

    2013-09-01

    Full Text Available Abstract Background & aim: According to the hypothesis that leishmania parasites can be escaped from immune system covered by blood group antigens (ABO to prevent its recognition by the immune system. The aim of this study was to show the associated blood groups with symptomatic or asymptomatic visceral leishmaniasis due to Leishmania infantum in human. Methods: In this cross-sectional study the population was divided into two groups. The first group included 54 patients with kala-azar (antibody against Leishmania titers ≥1:3200 by TDA with clinical specificity and the second group consisted of 45 subjects infected with Leishmania infantum (Leishmania antibody titers of1: 800 and 1:1600 by DAT method and non-specific symptoms. The distribution of the 4 main blood groups ABO type, sex, age, presence or absence of symptoms, clinical signs, and response to Glucantim therapy and DAT results were evaluated. Data were analyzed by chi-square test. Results: Most of the patients in group 1 were blood group A (37% and the lowest number of blood group were B (12.8%. In the second group, most of the ABO blood group A (42.2% and lowest in the ABO blood group AB (8.9%.There was no significant association between blood groups and clinical symptoms (p>0.05. Conclusion: This study showed that there is no association between blood group and incidence of symptomatic and asymptomatic kala-azar. Key words: Leishmania Infantum, Kala-azar, Blood Group, Human

  1. The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis

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    Requena Jose M

    2010-01-01

    Full Text Available Abstract Background RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether Leishmania PUF proteins result antigenic during visceral leishmaniasis (VL. Findings Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in Leishmania infantum. Additionally, the 10 genes of the L. infantum PUF family were cloned and used to express the Leishmania PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The Leishmania PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients. Conclusion Our results suggest that Leishmania PUFs are targets of the humoral response during L. infantum infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as Trypanosoma cruzi.

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

    Science.gov (United States)

    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

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

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    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  6. Macrophage and T-cell gene expression in a model of early infection with the protozoan Leishmania chagasi.

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    Nicholas A Ettinger

    2008-06-01

    Full Text Available Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-naïve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA, respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-naïve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-gamma, IL-6, IL-1alpha, IL-1beta. There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-beta Signaling Pathway. We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T

  7. Gene deleted live attenuated Leishmania vaccine candidates against visceral leishmaniasis elicit pro-inflammatory cytokines response in human PBMCs

    Science.gov (United States)

    Avishek, Kumar; Kaushal, Himanshu; Gannavaram, Sreenivas; Dey, Ranadhir; Selvapandiyan, Angamuthu; Ramesh, V.; Negi, Narender Singh; Dubey, Uma S.; Nakhasi, Hira L.; Salotra, Poonam

    2016-01-01

    Currently no effective vaccine is available for human visceral leishmaniasis(VL) caused by Leishmania donovani. Previously, we showed that centrin1 and p27gene deleted live attenuated Leishmania parasites (LdCen1−/− and Ldp27−/−) are safe, immunogenic and protective in animal models. Here, to assess the correlates of protection, we evaluated immune responses induced by LdCen1−/− and Ldp27−/− in human blood samples obtained from healthy, healed VL (HVL), post kala-azar dermal leishmaniasis(PKDL) and VL subjects. Both parasites infected human macrophages, as effectively as the wild type parasites. Further, LdCen1−/− and Ldp27−/− strongly stimulated production of pro-inflammatory cytokines including, IL-12, IFN-γ, TNF-α, IL-2, IL-6 and IL-17 in the PBMCs obtained from individuals with a prior exposure to Leishmania (HVL and PKDL). There was no significant stimulation of anti-inflammatory cytokines (IL-4 and IL-10). Induction of Th1 biased immune responses was supported by a remarkable increase in IFN-γ secreting CD4+ and CD8+ T cells and IL-17 secreting CD4+ cells in PBMCs from HVL cases with no increase in IL-10 secreting T cells. Hence, LdCen1−/− and Ldp27−/− are promising as live vaccine candidates against VL since they elicit strong protective immune response in human PBMCs from HVL, similar to the wild type parasite infection, mimicking a naturally acquired protection following cure. PMID:27624408

  8. In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis.

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    Adriano C Coelho

    2014-07-01

    Full Text Available Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in

  9. PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil.

    Science.gov (United States)

    Oliveira, Fernanda S; Pirmez, Claude; Pires, Marize Q; Brazil, Reginaldo P; Pacheco, Raquel S

    2005-05-15

    The technique of polymerase chain reaction (PCR) associated to hybridization was used to screen 123 samples collected from wild and synanthropic rodents captured in a cutaneous and visceral leishmaniasis endemic area in the State of Minas Gerais, Brazil. The detection of Leishmania spp in naturally infected rodents is of fundamental importance for incriminating them as possible reservoir hosts of the diseases in Minas Gerais. A total of 62 specimens belonging to wild (Thrichomys apereoides, Oryzomys subflavus, Galea spixii, Bolomys lasiurus and Wiedomys pyrrhorhinos) and synanthropic (R. rattus) rodent species were captured in different ecotopes. Blood and skin samples were submitted for PCR analyses followed by molecular hybridization with specific probes for the three Leishmania-species complexes. Fifteen samples were found positive after PCR-hybridization and identified as follows: nine belonging to the L. mexicana complex, three to the L. braziliensis complex and three to the L. donovani complex. Positive PCR results were found in 11 out of the 61 (18%) blood samples and in four out of the 62 (6.4%) skin fragments screened. R. rattus and T. apereoides were the most abundant species in the area also presenting high prevalence of natural infection. The presence of parasite DNA belonging to L. braziliensis, L. mexicana and L. donovani complexes was confirmed in several individuals of a rodent species, R. rattus. This work is the first report of the detection of L. (L.) chagasi in a naturally infected T. apereoides. The utility of filter paper as a substrate for PCR analyses and the efficacy of the procedure associated to the hybridization is emphasized.

  10. Golden hamster (Mesocricetus auratus) as an experimental model for Leishmania (Viannia) braziliensis infection.

    Science.gov (United States)

    Gomes-Silva, Adriano; Valverde, Joanna Gardel; Ribeiro-Romão, Raquel Peralva; Plácido-Pereira, Rosa Maria; Da-Cruz, Alda Maria

    2013-05-01

    The lack of an adequate model for Leishmania (Viannia) braziliensis infection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropic Leishmania infection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, different Leishmania isolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility of L. braziliensis infection in this model. The natural history of L. braziliensis infection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-Leishmania IgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused by L. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

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

    Science.gov (United States)

    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.

  12. Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

    Directory of Open Access Journals (Sweden)

    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. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

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    Gareth D Westrop

    Full Text Available Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

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

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    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.

  15. Homologous recombination in Leishmania enriettii.

    Science.gov (United States)

    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.

  16. Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis

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    V.M.F. Lima

    2003-04-01

    Full Text Available Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF. Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance ± SD, 1.939 ± 0.405; negative control, N = 20, 0.154 ± 0.074 and CSF (1.571 ± 0.532; negative control, N = 10, 0.0195 ± 0.040 from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

  17. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

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    Felix Hugentobler

    Full Text Available BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H1 CD4(+ and CD8(+ T cells and a systemic LACK-specific T(H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

  18. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

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    Hugentobler, Felix; Yam, Karen K; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

  19. 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

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    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.

  20. Immunogenicity of the P-8 amastigote antigen in the experimental model of canine visceral leishmaniasis.

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    Carrillo, E; Ahmed, S; Goldsmith-Pestana, K; Nieto, J; Osorio, Y; Travi, B; Moreno, J; McMahon-Pratt, D

    2007-02-09

    The P-8 proteoglycolipid complex (P-8 PGLC), an amastigote antigen of Leishmania pifanoi, has been demonstrated to induce protection in mouse models, as well as to induce Tc1/Th1-like cellular responses in American cutaneous leishmaniasis patients. Because the immunization with P-8 PGLC in the murine model does not appear to be genetically restricted, we have studied the reactivity of the P-8 PGLC in Leishmania infantum infected dogs. In this study, it is shown that PBMC from experimentally infected dogs (asymptomatic, oligosymptomatic) significantly proliferated in response to soluble leishmanial antigen (SLA) or the P-8 PGLC. Further, quantification of the gene expression induced by the stimulation with P-8 in asymptomatically infected dogs showed an up-regulation of IFN-gamma and TNF-alpha, which were three to 4-fold higher than that induced by soluble Leishmania antigen (SLA). While no measurable induction of IL-10 was observed, low levels of IL-4 mRNA were observed in response to both P-8 and SLA antigens. Thus, our studies establish that P-8 is recognized by infected canines and elicits a potentially curative/protective Th1-like immune response. The identification of Leishmania antigens that elicit appropriate immune responses across different host species (humans, canine) and disease manifestations (cutaneous or visceral) could be an advantage in generating a general vaccine for leishmaniasis.

  1. Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

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    Felipe da Silva Krawczak

    2015-02-01

    Full Text Available INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT and by anti-Leishmania immunoglobulin G (IgG antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP (Bio Manguinhos(r/FIOCRUZ/MS and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios. RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2% were positive for Leishmania as determined by ELISA; 12 (12.5%, by IFAT; 14 (14.6% by rK39 RDT; and 20 (20.8%, by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9% and 30/96 (31.2% samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05. CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil; we also found that the serological tests that were used did not cross-react.

  2. Immunoinformatics Features Linked to Leishmania Vaccine Development: Data Integration of Experimental and In Silico Studies

    Science.gov (United States)

    Brito, Rory C. F.; Guimarães, Frederico G.; Velloso, João P. L.; Corrêa-Oliveira, Rodrigo; Ruiz, Jeronimo C.; Reis, Alexandre B.; Resende, Daniela M.

    2017-01-01

    Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. There is no human vaccine available and it is considered by many studies as apotential effective tool for disease control. To discover novel antigens, computational programs have been used in reverse vaccinology strategies. In this work, we developed a validation antigen approach that integrates prediction of B and T cell epitopes, analysis of Protein-Protein Interaction (PPI) networks and metabolic pathways. We selected twenty candidate proteins from Leishmania tested in murine model, with experimental outcome published in the literature. The predictions for CD4+ and CD8+ T cell epitopes were correlated with protection in experimental outcomes. We also mapped immunogenic proteins on PPI networks in order to find Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with them. Our results suggest that non-protective antigens have lowest frequency of predicted T CD4+ and T CD8+ epitopes, compared with protective ones. T CD4+ and T CD8+ cells are more related to leishmaniasis protection in experimental outcomes than B cell predicted epitopes. Considering KEGG analysis, the proteins considered protective are connected to nodes with few pathways, including those associated with ribosome biosynthesis and purine metabolism. PMID:28208616

  3. 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.

    Science.gov (United States)

    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.

  4. Genetic Structure and Evolution of the Leishmania Genus in Africa and Eurasia: What Does MLSA Tell Us

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    El Baidouri, Fouad; Diancourt, Laure; Berry, Vincent; Chevenet, François; Pratlong, Francine; Marty, Pierre; Ravel, Christophe

    2013-01-01

    Leishmaniasis is a complex parasitic disease from a taxonomic, clinical and epidemiological point of view. The role of genetic exchanges has been questioned for over twenty years and their recent experimental demonstration along with the identification of interspecific hybrids in natura has revived this debate. After arguing that genetic exchanges were exceptional and did not contribute to Leishmania evolution, it is currently proposed that interspecific exchanges could be a major driving force for rapid adaptation to new reservoirs and vectors, expansion into new parasitic cycles and adaptation to new life conditions. To assess the existence of gene flows between species during evolution we used MLSA-based (MultiLocus Sequence Analysis) approach to analyze 222 Leishmania strains from Africa and Eurasia to accurately represent the genetic diversity of this genus. We observed a remarkable congruence of the phylogenetic signal and identified seven genetic clusters that include mainly independent lineages which are accumulating divergences without any sign of recent interspecific recombination. From a taxonomic point of view, the strong genetic structuration of the different species does not question the current classification, except for species that cause visceral forms of leishmaniasis (L. donovani, L. infantum and L. archibaldi). Although these taxa cause specific clinical forms of the disease and are maintained through different parasitic cycles, they are not clearly distinct and form a continuum, in line with the concept of species complex already suggested for this group thirty years ago. These results should have practical consequences concerning the molecular identification of parasites and the subsequent therapeutic management of the disease. PMID:23785530

  5. Biomarkers of safety and immune protection for genetically modified live attenuated Leishmania vaccines against visceral leishmaniasis-Discovery and implications

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    Sreenivas eGannavaram

    2014-05-01

    Full Text Available Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, sub-unit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in L. donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen1-/- in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated

  6. Development of Cutaneous Leishmaniasis after Leishmania Skin Test

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    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

  7. Development of cutaneous leishmaniasis after leishmania skin test.

    Science.gov (United States)

    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. Mechanisms of pathogenesis: differences amongst Leishmania species.

    Science.gov (United States)

    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.

  9. Leishmania braziliensis-reactive T cells are down-regulated in long-term cured cutaneous Leishmaniasis, but the renewal capacity of T effector memory compartments is preserved.

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    Regina Pereira-Carvalho

    Full Text Available Leishmania (Viannia braziliensis control and tissue damage relate to the effector immune response, which in turn affects clinical outcome. Leishmania reactive CD4(+ and CD8(+ T cells are expanded in long-term healed cutaneous leishmaniasis (hCL patients but their functional characteristics remain to be determined. This study investigates antigen-specific recall in long-term healed CL caused by L. braziliensis infection. Healed CL subjects were grouped according to the time elapsed since the end of therapy: less than two years and two to five years. Activation phenotype (CD69(+ or CD25(+ and subpopulations of memory T cell phenotypes [central memory (Tcm: CD45RO(+ CCR7(+ or effector memory (Tem: CD45RO(+ CCR7(-] were quantified in ex vivo blood mononuclear cells and after Leishmania antigens stimuli. A reduction in the percentage of activated Leishmania-responder CD4(+ and CD8(+ T cells in hCL was associated with the time elapsed since clinical cure. Percentage of CD69(+ in TCD4(+ and TCD8(+ cells were negatively correlated with IL-10 levels. Ex vivo analyses showed contracted Tem CD4(+ and Tem CD8(+ compartments from hCL with long time elapsed since clinical cure, although renewal of these compartments was observed following in vitro exposure to leishmanial stimuli. Our results show that healed L. braziliensis infected patients exhibit a recall response to Leishmania antigens with evident expansion of effector memory T cells. Regulated leishmanial-specific response seems to emerge only about two years after initial contact with the parasite antigens.

  10. The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite.

    Science.gov (United States)

    Tschoeke, Diogo A; Nunes, Gisele L; Jardim, Rodrigo; Lima, Joana; Dumaresq, Aline Sr; Gomes, Monete R; de Mattos Pereira, Leandro; Loureiro, Daniel R; Stoco, Patricia H; de Matos Guedes, Herbert Leonel; de Miranda, Antonio Basilio; Ruiz, Jeronimo; Pitaluga, André; Silva, Floriano P; Probst, Christian M; Dickens, Nicholas J; Mottram, Jeremy C; Grisard, Edmundo C; Dávila, Alberto Mr

    2014-01-01

    Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the "Mexicana complex", reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.

  11. The Dialogue of the Host-Parasite Relationship: Leishmania spp. and Trypanosoma cruzi Infection

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    Carlos Gustavo Vieira de Morais

    2015-01-01

    Full Text Available The intracellular protozoa Leishmania spp. and Trypanosoma cruzi and the causative agents of Leishmaniasis and Chagas disease, respectively, belong to the Trypanosomatidae family. Together, these two neglected tropical diseases affect approximately 25 million people worldwide. Whether the host can control the infection or develops disease depends on the complex interaction between parasite and host. Parasite surface and secreted molecules are involved in triggering specific signaling pathways essential for parasite entry and intracellular survival. The recognition of the parasite antigens by host immune cells generates a specific immune response. Leishmania spp. and T. cruzi have a multifaceted repertoire of strategies to evade or subvert the immune system by interfering with a range of signal transduction pathways in host cells, which causes the inhibition of the protective response and contributes to their persistence in the host. The current therapeutic strategies in leishmaniasis and trypanosomiasis are very limited. Efficacy is variable, toxicity is high, and the emergence of resistance is increasingly common. In this review, we discuss the molecular basis of the host-parasite interaction of Leishmania and Trypanosoma cruzi infection and their mechanisms of subverting the immune response and how this knowledge can be used as a tool for the development of new drugs.

  12. CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

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    Ali, S A; Rezvan, H; McArdle, S E; Khodadadi, A; Asteal, F A; Rees, R C

    2009-07-01

    Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

  13. Innate Immunity to Leishmania Infection: Within Phagocytes

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    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.

  14. A novel role for Stat1 in phagosome acidification and natural host resistance to intracellular infection by Leishmania major.

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    Gerald F Späth

    2009-04-01

    Full Text Available Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma-mediated immune response. We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1 and derived macrophages. Unexpectedly, infection of Stat1-deficient macrophages in vitro with promastigotes from Leishmania major and attenuated LPG1 knockout mutants (lpg(- specifically lacking lipophosphoglycan (LPG resulted in a twofold increased intracellular growth, which was independent of IFNgamma and associated with a substantial increase in phagosomal pH. Phagosomes in Stat1-/- macrophages showed normal maturation as judged by the accumulation of the lysosomal marker protein rab7, and provided normal vATPase activity, but were defective in the anion conductive pathway required for full vesicular acidification. Our results suggest a role of acidic pH in the control of intracellular Leishmania growth early during infection and identify for the first time an unexpected role of Stat1 in natural anti-microbial resistance independent from its function as IFNgamma-induced signal transducer. This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

  15. Target oriented drugs against leishmania. Annual summary report no. 2, 1 May 1980-30 April 1981

    Energy Technology Data Exchange (ETDEWEB)

    Zehavi, U.; El-On, J.

    1981-01-31

    Excreted Factor (EF) is a carbohydrate-rich material released by different strains of Leishmania during growth. It has antigenic properties similar to those of the intact parasite and plays a role in the infective process. Isolation and purification of EF is necessary for study of its biological function, its use for diagnostic purposes, its use in immunization experiments, the study of its biosynthesis, and the preparation of inhibitors of particular biosynthetic steps. Purification of EF by affinity chromatography was markedly improved by introducing Ricinus lectin (specific for galactose) column. This enabled us to obtain more reliable amino acid and sugar analysis and will be instrumental in more advanced physical, chemical, and immunological studies. We have developed a radioimmunoassay for leishmaniasis utilizing purified EF. The assay can distinguish between Leishmania strains and once further developed, should prove most valuable for the diagnosis of the disease. EF plays a role in the infective process of Leishmania. We have now shown that surface carbohydrate, related to EF, plays a role in the initial attachment of Leishmania promastigots to macrophages - a stage that is a prelude to their engulfment by the macrophages followed by multiplication in their cells.

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

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    B Sarkari

    2006-04-01

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

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

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    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.

  18. 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

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    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

  19. Diverse modes of binding in structures of Leishmania majorN-myristoyltransferase with selective inhibitors

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    James A. Brannigan

    2014-07-01

    Full Text Available The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

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

    NARCIS (Netherlands)

    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

  1. Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia braziliensis Reatividade cruzada de anticorpos em pacientes com infecções pelos protozoários Trypanosoma cruzi, Leishmania chagasi e Leishmania (Viannia braziliensis

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    Ana de Cássia Vexenat

    1996-06-01

    Full Text Available We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.Foram detectados anticorpos, nos soros de pacientes com doença de Chagas, Calazar e Leishnnaniose cutâneo-mucosa, que se ligam a antígenos compartilhados pelos três agentes causais. Os pacientes chagásicos mostraram 98 a 100% de soropositividade pelo ELISA quando antígenos de Leishmania braziliensis e de Leishmania chagasi foram usados, respectivamente. Os soros de

  2. [Taxonomic problems of the Leishmania of reptiles].

    Science.gov (United States)

    Ovezmukhammedov, A; Saf'ianova, V M

    1989-01-01

    The history of description and state of knowledge of 17 species and 40 not identified to species forms of Leishmania, described from reptiles of the world, are traced. It is suggested to retain 10 species and 3 forms of Leishmania in the list of the subgenus Sauroleishmania as follows: L. (S) tarentolae, L.(S.) hemidactyli, L.(S.) ceramodactyli, L.(S.) nicollei, L.(S.) gymnodactyli, L.(S.) adleri, L.(S.) hoogstraali, L.(S.) senegalensis, L.(S.) gulikae, L.(S.) sp., L.(S.) sp. I, L.(S.) sp. II. 7 species and one form, L(S.) henrici, L.(S.) davidi, L.(S.) zmeevi, L.(S.) sofieffi, L.(S.) chameleonis, L.(S.) phrynocephali, L.(S.) helioscopi, L.(S.) sp. Markov e.a., 1964 must be excluded from the above subgenus since their description does not correspond to the development of the life cycle of Leishmania from reptiles. Flagellata Protozoa from the peripheral blood and intestine of reptiles, which were regarded by some authors as a "leptomonad stage of Leishmania", appear to belong to the genera Proteromonas, Monocercomonas and other Protozoa.

  3. Comorbidity of Leishmania major with cutaneous sarcoidosis

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    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.

  4. Developmentally regulated sphingolipid degradation in Leishmania major.

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    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.

  5. Antibodies to Leishmania spp. in domestic felines

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    Gabriela Capriogli Oliveira

    Full Text Available Abstract Leishmaniasis is a vector-borne zoonotic disease caused by protozoa in the genus Leishmania, typical of rural and peri-urban environments. The causative agent of American visceral leishmaniasis is Leishmania (Leishmania infantum chagasi and the main insect vector in Brazil is Lutzomyia longipalpis. Dogs (Canis familiaris are important in the transmission of the disease, as a reservoir closely related to humans and an infection source for phlebotomine vectors. Since 1990, an increasing number of feline leishmaniasis cases have been reported, suggesting that domestic cats (Felis catus might be involved in the epidemiology of the disease. The present study analyzed the prevalence of anti-Leishmania spp. antibodies in naturally infected domestic cats from various neighborhoods in the municipality of Belém, Pará, Brazil, using the indirect immunofluorescence assay (IFA and the direct agglutination test (DAT. Among the 443 samples tested, 18 (4.06% presented positive reactions in the IFA. The observed titers were 40 IU in 4.97% of the samples and 80 IU in 0.90%. In the DAT test, positive results were found in 25 (5.64% of the samples. The observed titers were also 40 IU (4.97% and 80 IU (0.68%. The agreement rate between the two tests was considered low (Kappa coefficient = 0.10.

  6. 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

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    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.

  7. Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces cross-protection against L. major infection.

    Science.gov (United States)

    Goto, Yasuyuki; Bhatia, Ajay; Raman, Vanitha S; Vidal, Silvia E Z; Bertholet, Sylvie; Coler, Rhea N; Howard, Randall F; Reed, Steven G

    2009-05-11

    The enzyme sterol 24-c-methyltranferase (SMT) is required for the biosynthesis of ergosterol, the major membrane sterol in Leishmania parasites. SMT and ergosterol are not found in mammals, so this protein may be an attractive target for anti-leishmanial vaccines and drugs. We have previously demonstrated that SMT from L. infantum, which causes visceral leishmaniasis, is a protective antigen against this parasite. Because this protein is highly conserved among Leishmania species, we evaluated the potential of SMT to cross-protect against a different form of leishmaniasis. Here, we show that immunization with L. infantum SMT, formulated with monophosphoryl lipid A in stable emulsion (MPL-SE), protects mice from cutaneous leishmaniasis caused by L. major. In BALB/c mice the vaccine preparation induced antigen-specific multi-functional CD4(+) T cells capable of producing IFN-gamma, IL-2, and/or TNF-alpha upon antigen re-exposure, and MPL-SE was indispensable to direct immune responses to SMT towards Th1. Mice immunized with the SMT/MPL-SE vaccine developed significantly smaller lesions following ear challenge with L. major. These results suggest that SMT is a promising vaccine antigen for multiple forms of leishmaniasis.

  8. 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

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    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

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

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    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.

  10. Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania major vaccines in a rhesus monkey (Macaca mulatta model of the human disease

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    VF Amaral

    2002-10-01

    Full Text Available We have compared the efficacy of two Leishmania (Leishmania major vaccines, one genetically attenuated (DHFR-TS deficient organisms, the other inactivated [autoclaved promastigotes (ALM with bacillus Calmete-Guérin (BCG], in protecting rhesus macaques (Macaca mulatta against infection with virulent L. (L. major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals, although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g production or positive delayed-type hypersensitivity (DTH response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05 between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

  11. Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India

    Science.gov (United States)

    Ejazi, Sarfaraz Ahmad; Bhattacharya, Pradyot; Bakhteyar, Md. Asjad Karim; Mumtaz, Aquil Ahmad; Pandey, Krishna; Das, Vidya Nand Ravi; Das, Pradeep; Rahaman, Mehebubar; Goswami, Rama Prosad; Ali, Nahid

    2016-01-01

    Background Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. Methodology/principal findings We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. Conclusions/significance ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response. It can therefore, be deployed widely for interventions in disease management of VL particularly in poor resource outskirts. PMID:27741241

  12. Downregulation of host tryptophan-aspartate containing coat (TACO) gene restricts the entry and survival of Leishmania donovani in human macrophage model

    National Research Council Canada - National Science Library

    Gogulamudi, Venkateswara Reddy; Dubey, Mohan Lal; Kaul, Deepak; Atluri, Venkata Subba Rao; Sehgal, Rakesh

    2015-01-01

    .... Recently, tryptophan-aspartate containing coat (TACO) gene has been recognized as playing a central role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process...

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

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    Elizaide L. A. Yoshida

    1993-09-01

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

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

    Science.gov (United States)

    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

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

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    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.

  16. Leishmania major: Parasite Interactions Suggesting Sexuality

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    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

  17. The genetic toolbox for Leishmania parasites.

    Science.gov (United States)

    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.

  18. Herbal extract targets in Leishmania tropica.

    Science.gov (United States)

    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.

  19. TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA enhances CD8+ T Cell responses providing protection against Leishmania (Viannia.

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    Asha Jayakumar

    2011-06-01

    Full Text Available Leishmania (Viannia parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V. panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that

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

    Science.gov (United States)

    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

  1. Testing of Experimental Compounds for Efficacy against Leishmania.

    Science.gov (United States)

    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

  2. Vaccinations against cutaneous Leishmania infection.

    Science.gov (United States)

    Kronenberg, K; Brosch, S; Von Stebut, E

    2008-04-01

    Cutaneous leishmaniasis is an endemic disease with increasing incidence, even in Europe. Recently, it has attracted more attention due to reactivation in immunocompromised hosts, e.g. in the context of HIV. Therapeutic options range from topical treatment to systemic therapy for more complex cases. A vaccine does not exist at present. Despite of several attempts, vaccine generation has proven to be difficult even though protective immunity against this obligate intracellular protozoan parasite is dependent on the development of antigen-specific CD4+ and CD8+ T cells capable of releasing IFN?. IFN?, in turn, activates phagocytic host cells to generate oxidative radicals and to eliminate the parasite. This review will describe the basic immunology leading to the development of protective immunity in infected individuals. In addition, the authors will focus on highlighting the different approaches utilized for vaccine development and describe what a efficient vaccine may consist of. Combined intensive research in the fields of basic parasitology and immunology may allow for the generation of an efficacious vaccine against this important human pathogen in the near future.

  3. Immunization against leishmaniasis by PLGA nanospheres loaded with an experimental autoclaved Leishmania major (ALM) and Quillaja saponins.

    Science.gov (United States)

    Tafaghodi, M; Eskandari, M; Kharazizadeh, M; Khamesipour, A; Jaafari, M R

    2010-12-01

    Immune responses against the Leishmania antigens are not sufficient to protect against a leishmania challenge. Therefore these antigens need to be potentiated by various adjuvants and delivery systems. In this study, Poly (d,l-lactide-co-glycolide (PLGA) nanospheres as antigen delivery system and Quillaja saponins (QS) as immunoadjuvant have been used to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter for nanospheres loaded with ALM+QS was 294 ± 106 nm. BALB/c mice were immunized three times in 3-weeks intervals using ALM plus QS loaded nanospheres [(ALM+QS)PLGA], ALM encapsulated with PLGA nanospheres [(ALM)PLGA], (ALM)PLGA + QS, ALM + QS, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P < 0.05) smaller footpad, were observed in mice immunized with (ALM)PLGA. The (ALM+QS)PLGA group showed the least protection and highest swelling, while the (ALM)PLGA+QS, ALM+QS and ALM showed an intermediate protection with no significant difference. The mice immunized with ALM and ALM+QS showed the highest IgG2a/IgG1 ratio (P < 0.01), followed by (ALM)PLGA+QS. The highest IFN-γ and lowest IL-4 production was seen in (ALM)PLGA+QS, ALM+QS groups. The highest parasite burden was observed in (ALM)PLGA+QS and (ALM+QS)PLGA groups. It is concluded that PLGA nanospheres as a vaccine delivery system could increase the protective immune responses, but QS adjuvant has a reverse effect on protective immune responses and the least protective responses were seen in the presence of this adjuvant.

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

    Science.gov (United States)

    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.

  5. Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

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    Bruno Guedes Alcoforado Aguiar

    2014-10-01

    Full Text Available Introduction Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2 and N-acetylglucosamine-1-phosphotransferase (NAGT, which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR, the fragments were sequenced, followed by polymorphism identification. Results The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

  6. KMP11-HASPB fusion protein-expressing lentiviral vaccine protects BALB/c mice against Leishmania major infection

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    Nahid Mortazavidehkordi

    2016-12-01

    Full Text Available Hydrophilic acylated surface protein B (HASPB is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11 is another Leishmania antigen and considered as suitable candidate for vaccine development leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFPlentiviral vector as a fusion protein to be used as a DNA vaccine against L.major. KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L.major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and liver of vaccinated mice after challenge was significantly lower than that of controls.

  7. Serological and Molecular Evaluation of Leishmania infantum Infection in Stray Cats in a Nonendemic Area in Northern Italy

    Science.gov (United States)

    Spada, Eva; Proverbio, Daniela; Migliazzo, Antonella; Della Pepa, Alessandra; Perego, Roberta; Bagnagatti De Giorgi, Giada

    2013-01-01

    Infection by Leishmania species is increasing worldwide. It was hypothesized recently that cats act as a secondary reservoir for Leishmania infection. The aim of the present study was to assess the prevalence of Leishmania infantum antibodies and DNA in blood samples collected in a sample of stray cats in metropolitan area of Milan in northern Italy, which is a nonendemic area for leishmaniasis. An indirect immunofluorescence antibody test for L. infantum showed that 59 of 233 cats (25.3%) were seroreactive, 38 samples (16.3%) had antibody titers of 1 : 40, 15 (6.4%) had antibody titers of 1 : 80, and 6 (2.6%) had antibody titers of 1 : 160. Feline immunodeficiency virus (FIV) seropositive status was statistically associated with seroreactivity to L. infantum (P = 0.01) as shown by univariate and multivariate logistic regression (P = 0.0098; OR = 7.34). All blood samples that were tested using real-time PCR were negative for parasite DNA. These results were surprising, since no autochthonous human or canine cases of leishmaniasis have ever been reported in this region of northern Italy. It is possible that this high seroreactivity to L. infantum could be due to cross-reaction with antigens from other parasites. Additional studies that include parasite isolation are needed to clarify our findings on feline leishmaniasis in this region. PMID:27335864

  8. Transcriptome patterns from primary cutaneous Leishmania braziliensis infections associate with eventual development of mucosal disease in humans.

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    Ana Claudia Maretti-Mira

    Full Text Available INTRODUCTION: Localized Cutaneous Leishmaniasis (LCL and Mucosal Leishmaniasis (ML are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not. METHODS: Using RNA-seq, we analyzed both the human and Leishmania transcriptomes in primary cutaneous lesions. RESULTS: Limited number of reads mapping to Leishmania transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome. CONCLUSIONS: We were able to simultaneously sequence both human and Leishmania mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of L. braziliensis infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity.

  9. Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge

    Science.gov (United States)

    Resende, Lucilene Aparecida; Aguiar-Soares, Rodrigo Dian de Oliveira; Gama-Ker, Henrique; Roatt, Bruno Mendes; de Mendonça, Ludmila Zanandreis; Alves, Marina Luiza Rodrigues; da Silveira-Lemos, Denise; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Araújo, Márcio Sobreira Silva; Fujiwara, Ricardo Toshio; Gontijo, Nelder Figueiredo; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2016-01-01

    Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the “LbSapSal” vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with “LbSapSal” is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-β), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after “LbSapSal” immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the “LbSapSal” vaccination is a potential tool to control the Leishmania chagasi infection. PMID:27556586

  10. 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.

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    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.

  11. Augmented Oxygen-Dependent Killing of Leishmania.

    Science.gov (United States)

    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

  12. Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis.

    Science.gov (United States)

    Agallou, Maria; Smirlis, Despina; Soteriadou, Ketty P; Karagouni, Evdokia

    2012-07-20

    Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis.

  13. Synthesis, biological evaluation, and structure-activity relationships of N-benzoyl-2-hydroxybenzamides as agents active against P. falciparum (K1 strain), Trypanosomes, and Leishmania.

    Science.gov (United States)

    Stec, Jozef; Huang, Qingqing; Pieroni, Marco; Kaiser, Marcel; Fomovska, Alina; Mui, Ernest; Witola, William H; Bettis, Samuel; McLeod, Rima; Brun, Reto; Kozikowski, Alan P

    2012-04-12

    In our efforts to identify novel chemical scaffolds for the development of new antiprotozoal drugs, a compound library was screened against Toxoplasma gondii tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2-hydroxybenzamide 1a against T. gondii as described elsewhere. Synthesis of a compound set was guided by T. gondii SAR with 1r found to be superior for T. gondii , also active against Thai and Sierra Leone strains of Plasmodium falciparum , and with superior ADMET properties as described elsewhere. Herein, synthesis methods and details of the chemical analysis of the compounds in this series are described. Further, this series of N-benzoyl-2-hydroxybenzamides was repurposed for testing against four other protozoan parasites: Trypanosoma brucei rhodesiense , Trypanosoma cruzi , Leishmania donovani , and P. falciparum (K1 isolate). Structure-activity analyses led to the identification of compounds in this set with excellent antileishmanial activity (compound 1d). Overall, compound 1r was the best and had activity 21-fold superior to that of the standard antimalarial drug chloroquine against the K1 P. falciparum isolate. © 2012 American Chemical Society

  14. Investigation and Comparison of Leishmania major Promastigote and Amastigote Protein Content by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

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    S. Soleimanifard

    2013-04-01

    Full Text Available ntroduction & Objective: Leishmania is a protozoan of the trypanosomatidae family. This pro-tozoan has two stages in its life cycle, promastigote form in sand flies and amastigote form in macrophage of mammalian hosts. The purpose of this study was identification and compari-son of proteins of Leishmania amastigote and promastigote stages. Materials & Methods: The present study is a cross sectional study of two forms of Leishmania major. To culture promastigotes , L.major (MRHO/IR/75/ER from previously infected Balb/c mice was transferred to modified N.N.N medium with overlay of liquid BHI and then transferred to RPMI-1640 at 26oc ± 1 for mass production. After isolation and growth, pro-mastigotes were transferred to liquid cell culture medium RPMI-1640 with pH 5.5 and incu-bated at 5% CO2 at 37oc for 72 hours until promastigote to amastigote transformation. Elec-trophoresis was performed with SDS-PAGE method to find and compare the molecular weight of the antigens of two stages. Results: The molecular weights of the bands observed in both forms were as follows: 19, 36, 50, 63, 65, 80, 90, 94, 96, 110- 130 KDa. The proteins in the surface of only promastigote were 22, 28 and 46 KDa and special proteins in the surface of amastigote were 12 and 32 KDa. Conclusion : According to this study Leishmania parasite has stage specific proteins. Various studies have shown that axenic amastigotes and tissue amastigotes are similar in their protein content. Therefore, based on stage specific proteins ,effective drugs and vaccines can be de-signed against leishmaniasis. (Sci J Hamadan Univ Med Sci 2013; 20 (1:1-8

  15. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

    Science.gov (United States)

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4+ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis. PMID:25801301

  16. Comparison of molecular markers for strain typing of Leishmania infantum.

    Science.gov (United States)

    Botilde, Yanick; Laurent, Thierry; Quispe Tintaya, Wilber; Chicharro, Carmen; Cañavate, Carmen; Cruz, Israel; Kuhls, Katrin; Schönian, Gabriele; Dujardin, Jean-Claude

    2006-11-01

    The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles (kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV- patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.

  17. Cloning of Leishmania Major P4 Gene

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    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.

  18. The leishmania ARL-1 and Golgi traffic.

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    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.

  19. Effects of Brazilian propolis on Leishmania amazonensis.

    Science.gov (United States)

    Ayres, Diana Copi; Marcucci, Maria Cristina; Giorgio, Selma

    2007-05-01

    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 microg/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.

  20. Effects of Brazilian propolis on Leishmania amazonensis

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    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.

  1. 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.

    Science.gov (United States)

    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.

  2. Cyclic nucleotide specific phosphodiesterases of Leishmania major

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  5. Synthetic peptides derived from ribosomal proteins of Leishmania spp. in mucocutaneous leishmaniasis: Diagnostic usefulness.

    Science.gov (United States)

    Florez, Magda Melissa; de Oliveira, Camila Indiani; Puerta, Concepción; Guzmán, Fanny; Ayala, Martha; Montoya, Gladis; Delgado, Gabriela

    2017-07-28

    The serological diagnostic methods currently available for mucocutaneous leishmaniasis (MCL) lack specificity when complete parasites are used; however, such specificity increases when protein fractions are used. Ribosomal proteins have been reported to induce antibodies in animal and humans infected with the parasite, making them a worth candidate to assess its diagnosis potential. This study was thus aimed at evaluating synthetic peptides derived from Leishmania braziliensis ribosomal proteins S25 and S5 as antigen candidates for diagnosing MCL by ELISA; 4 of these 21 peptides (P4, P6, P19 and P21) had the greatest sensitivity (21.7%, 13.04%, 20% and 20%, respectively) as well as having 95%, 100%, 100% and 82.5% specificity, respectively. The study revealed the limited usefulness of the peptides being studied as a diagnostic tool in the conditions used here, because its low sensitivity, but it is worth highlighting that the use of peptides as antigen in the serodiagnosis of MCL may overcome the cross reaction presented with other antigens, thus avoiding false positives. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Proteins of Leishmania (Viannia shawi confer protection associated with Th1 immune response and memory generation

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    Passero Luiz Felipe D

    2012-03-01

    Full Text Available Abstract Background Leishmania (Viannia shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA from L. (V. shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods F1 fraction was purified from L. (V. shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V. shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8+T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4+ central memory T lymphocytes and activation of both CD4+ and CD8+ T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

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    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  11. Feline leishmaniasis due to Leishmania (Leishmania) amazonensis in Mato Grosso do Sul State, Brazil.

    Science.gov (United States)

    de Souza, Alda Izabel; Barros, Evânia Marcia Silva; Ishikawa, Edna; Ilha, Iêda Maria Novaes; Marin, Glaucia Rosely Barbosa; Nunes, Vânia Lúcia Brandão

    2005-03-10

    A case of leishmaniasis in a domestic cat (Felis domesticus) is described. The animal showed a single, nodular lesion on the nose and many nodules of different size on the ears and digital regions of all the paws. Diagnosis was made by microscopic detection of amastigotes in Giemsa-stained smears from the lesions. By monoclonal antibodies the aetiological agent was identified as Leishmania (Leishmania) amazonensis, one of the seven species implicated in human leishmaniasis in Brazil. The clinical signs in feline leishmaniasis are unspecific and similar to those observed in other diseases such as cryptococcosis and in sporotrichosis, commonly found in cats. Leishmaniasis should therefore, be added to the differential diagnosis by feline veterinary practitioners and adequate investigations should carried out for dermal leishmaniasis in the area where the feline infection is detected.

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

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    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.

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

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

    OpenAIRE

    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.

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

    OpenAIRE

    Prisco, Michele

    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.

  17. Implications of a Neotropical Origin of the Genus Leishmania

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    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.

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

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    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.

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

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    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.

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

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    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.

  1. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    OpenAIRE

    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...

  2. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  4. The genome of the kinetoplastid parasite, Leishmania major

    OpenAIRE

    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...

  5. Canine inflammatory myopathy associated with Leishmania Infantum infection.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Leishmaniasis cutánea zosteriforme causada por Leishmania (Viannia panamensis y Leishmania (Viannia braziliensis: reporte de tres casos

    Directory of Open Access Journals (Sweden)

    Camilo Andrés Morales

    2014-09-01

    Se presentan tres casos de leishmaniasis cutánea zosteriforme en los que se identificaron Leishmania panamensis y Leishmania braziliensis como especies infectantes. La sospecha epidemiológica derivada de la procedencia de los pacientes, así como la sospecha clínica a partir del reconocimiento de una presentación infrecuente de la enfermedad, permitieron hacer el diagnóstico.

  8. Delayed culture of Leishmania in skin biopsies.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 h