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Sample records for leishmania donovani infected

  1. 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.

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

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

    1994-01-01

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

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

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    Rijal, Suman; Uranw, Surendra; Chappuis, François

    2010-01-01

    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...

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

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    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...

  5. rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donovani infection

    NARCIS (Netherlands)

    Zijlstra, E. E.; Daifalla, N. S.; Kager, P. A.; Khalil, E. A.; El-Hassan, A. M.; Reed, S. G.; Ghalib, H. W.

    1998-01-01

    The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months

  6. 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

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

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

    1993-01-01

    Clinical and immunological similarities between Leishmania donovani infections in humans and L. major infections in mice suggest that some of the pathophysiological mechanisms are the same in the two conditions. Both infections can result either in a fatal systemic disease or in a self-limiting i......Clinical and immunological similarities between Leishmania donovani infections in humans and L. major infections in mice suggest that some of the pathophysiological mechanisms are the same in the two conditions. Both infections can result either in a fatal systemic disease or in a self......-limiting infection with few and mild symptoms. In the murine model the outcome of the infection is critically related to the cytokines produced by T lymphocytes activated by leishmanial antigens. Activation of the IFN-gamma producing Th1 subset of CD4 positive T cells results in cure and survival, whereas activation...... of the IL-4 secreting Th2 subset results in a progressive disease with fatal outcome. A similar Th1/Th2 dichotomy in the cytokine response to L. donovani may exist in humans, and may have influence on the outcome of infection. In murine leishmaniasis the levels of IL-4 and IFN-gamma at the time of infection...

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

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    Epco Hasker

    Full Text Available 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.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.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.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.

  9. 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

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    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...... for malaria but free of leishmaniasis was negative in both assays....

  10. IgG and IgG subclasses antibody responses to rK39 in Leishmania donovani infections

    International Nuclear Information System (INIS)

    Daifalla, N.S.; El Hassan, A.M.

    1998-01-01

    Leishmania donovani infection cause a wide spectrum of human diseases ranging from self-healing subclinical infections to severe visceral leishmaniasis, post kal-azar dermal leishmaiasis, and mucosal leishmaiasis. The infection associated with high levels of anti-leishmania antibodies which offer a potential parameter for the serological diagnosis of L. donovani infection replacing the invasive parasitological methods. rK39, a cloned antigen of L. chagasis was reported to have high levels of anti-leishmania antibodies in Sudanese and American visceral leishmaniasis patients. In an assessment of rK39-ELISA in detecting L. donovani infection we found that the antigen detected visceral leishmaniasis, post kala-azar dermal leishmaniasis, and mucosal leismaniasis with the sensitives of 96.6%, 95.91% and 90.91% respectively. The test has the specificity of 96.7%. Further investigation of 25 visceral leishmaniasis patients showed elevated anti-rK39 antibody responses of IgG subclasses with IgG1 and IgG3 significantly higher than IgG4. igG3 showed the highest sensitivity (84.00%) whereas IgG1 showed the highest sensitivity (100%). The dynamics of the serological reactivity to rK39 in l.donovani infections will be discussed in relation to exposure, infection, cure and relapse.(Author)

  11. Seasonal variation in the prevalence of sand flies infected with Leishmania donovani.

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    Tiwary, Puja; Kumar, Dinesh; Mishra, Mukesh; Singh, Rudra Pratap; Rai, Madhukar; Sundar, Shyam

    2013-01-01

    Visceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures. We collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR. Though sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.

  12. Vaccine development against Leishmania donovani

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    Amrita eDas

    2012-05-01

    Full Text Available Visceral leishmaniasis (VL caused by Leishmania donovani and Leishmania infantum/ chagasi represents the second most challenging infectious disease worldwide, affecting nearly 500,000 people and 60,000 deaths annually. Zoonotic VL (ZVL caused by L. infantum is re-emergent canid zoonoses which represents a complex epidemiological cycle in New world where domestic dogs serve as reservoir host responsible for potentially fatal human infection where dog culling is the only control measure for eliminating reservoir host. Lifelong immunity in human against reinfection has motivated several attempts in developing prophylactic vaccines against the disease but very few have progressed beyond experimental stage. Absence of any licensed vaccine along with high toxicity and increasing resistance to the current chemotherapeutic drugs has further complicated the situation in endemic regions of the world. Advances in vaccinology, including recombinant proteins, novel antigen-delivery systems/adjuvants, heterologous prime-boost regimens and strategies for intracellular antigen presentation, have contributed to recent advances in vaccine development against VL. Attempts to develop an effective vaccine for use in domestic dogs in areas of canine VL should be pursued for preventing human infection. Studies in animal models and human patients have revealed the pathogenic mechanisms of disease progression and features of protective immunity. This review will summarize the accumulated knowledge about pathogenesis, immune response and prerequisites for protective immunity against human VL. Authors will discuss promising vaccine targets, their developmental status and future prospects in a quest for rational vaccine development against VL. In addition, several challenges such as safety issues, a renewed and coordinated commitment to basic research, preclinical studies and trial design will be addressed to overcome the problems faced in developing effective vaccines

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

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    Nevien Ismail

    2017-12-01

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

  14. The mitochondrial SIR2 related protein 2 (SIR2RP2 impacts Leishmania donovani growth and infectivity.

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    Nimisha Mittal

    2017-05-01

    Full Text Available Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2 of L. donovani.Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors.Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.

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

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    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.

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

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    Crocker, P.R.; Blackwell, J.M.; Bradley, D.J.

    1984-01-01

    Reciprocal radiation bone marrow chimaeras mere 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. (author)

  17. 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.

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

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

    2017-01-01

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

  19. 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.

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

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    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

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

    Indian Academy of Sciences (India)

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

  2. Critical roles for LIGHT and its receptors in generating T cell-mediated immunity during Leishmania donovani infection.

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    Amanda C Stanley

    2011-10-01

    Full Text Available LIGHT (TNFSF14 is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM and lymphotoxin-beta receptor (LTβR. We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTβR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4(+ T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.

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

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    Chakrabarti, Saikat; Roy, Syamal

    2016-01-01

    Background 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. Methodology/Principal Findings 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. Conclusion 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

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

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

    2017-01-01

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

  5. Adenine phosphoribosyltransferase-deficient Leishmania donovani

    International Nuclear Information System (INIS)

    Kaur, K.; Iovannisci, D.M.; Ullman, B.

    1986-01-01

    To elucidate the relative roles of two routes for adenine salvage, the authors use biochemical genetic approaches to isolate clonal strains of Leishmania donovani promasatigotes genetically deficient in APRTase activity. The studies suggest that the metabolic rate of adenine in these organisms is initiated by deamination. The radiolabel incorporation experiments and biochemical experiments are described in which the rate of uptake of radiolabelled purine nucleobases (C 14) was determined. Results are presented

  6. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

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    Zhang, Wen-Wei; Matlashewski, Greg

    2015-07-21

    The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this

  7. Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

    NARCIS (Netherlands)

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

    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

  8. In vitro permissiveness of bovine neutrophils and monocyte derived macrophages to Leishmania donovani of Ethiopian isolate.

    Science.gov (United States)

    Tasew, Geremew; Gadisa, Endalamaw; Abera, Adugna; Zewude, Aboma; Chanyalew, Menberework; Aseffa, Abraham; Abebe, Markos; Ritter, Uwe; van Zandbergen, Ger; Laskay, Tamás; Tafess, Ketema

    2016-04-18

    Epidemiological studies in Ethiopia have documented that the risk of visceral leishmaniasis (VL, Kala-azar) is higher among people living with domestic animals. The recent report on isolation of Leishmania donovani complex DNA and the detected high prevalence of anti-leishmanial antibodies in the blood of domestic animals further strengthen the potential role of domestic animals in the epidemiology of VL in Ethiopia. In mammalian hosts polymorphonuclear cells (PMN) and macrophages are the key immune cells influencing susceptibility or control of Leishmania infection. Thus to substantiate the possible role of cattle in VL transmission we investigate the permissiveness of bovine PMN and monocyte derived macrophages (MDM) for Leishmania (L.) donovani infection. Whole blood was collected from pure Zebu (Boss indicus) and their cross with Holstein Friesian cattle. L. donovani (MHOM/ET/67/HU3) wild and episomal green fluorescent protein (eGFP) labelled stationary stage promastigotes were co-incubated with whole blood and MDM to determine infection of these cells. Engulfment of promastigotes by the cells and their transformation to amastigote forms in MDM was studied with direct microscopy. Microscopy and flow cytometry were used to measure the infection rate while PCR-RLFP was used to confirm the infecting parasite. L. donovani infected bovine whole blood PMN in the presence of plasma factors and all cellular elements. Morphological examinations of stained cytospin smears revealed that PMN engulfed promastigotes. Similarly, we were able to show that bovine MDM can be infected by L. donovani, which transformed to amastigote forms in the cells. The in vitro infection of bovine PMN and MDM by L. donovani further strengthens the possibility that cattle might serve as source of L. donovani infection for humans.

  9. 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

  10. Studies on Stibanate unresponsive isolates of Leishmania donovani

    Indian Academy of Sciences (India)

    Unknown

    503. Keywords. Drug resistance; Leishmania donovani; sodium stibogluconate; visceral leishmaniasis ... cases to less than 5% in treated ones (Zuckerman and. Lainson 1977). Later on .... Effect of Stibanate on in vitro growth of Ag83 promastigotes. (A) Virulent .... In rural areas where health care facilities are not adequate ...

  11. Seroepidemiology and molecular diversity of Leishmania donovani complex in Georgia.

    Science.gov (United States)

    Babuadze, Giorgi; Farlow, Jason; de Koning, Harry P; Carrillo, Eugenia; Chakhunashvili, Giorgi; Murskvaladze, Mari; Kekelidze, Merab; Karseladze, Irakli; Kokaia, Nora; Kalandadze, Irine; Tsereteli, David; Markhvashvili, Ivane; Sidamonidze, Ketevan; Chanturia, Gvantsa; Adeishvili, Ekaterine; Imnadze, Paata

    2016-05-13

    Leishmaniasis includes multiple clinical syndromes, most notably visceral, cutaneous, and mucosal forms. Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease endemic to large parts of Africa and Asia, and in South-Eastern Europe (Greece, Turkey, Georgia). Visceral leishmaniasis is a parasitic zoonosis caused by species of the L. donovani complex. In the classical epidemiological model the main reservoir for VL are canines. The study included a cohort of 513 individuals of both genders (190 males and 323 females) from the ages of 1 to 70 years that were screened in ten villages across two districts in Kakheti using the Kalazar Detect™ rK39 rapid diagnostic test. The phylogenetic diversity patterns of local strains, based on the rDNA internal transcribed spacer (ITS) sequences, were assessed for samples obtained from patients with suspected L. donovani infection, from canine reservoirs and from Phlebotomus sand flies obtained from different geographical areas of Georgia and from Azerbaijan. Out of a total of 600 domestic dog blood samples 95 (15.8 %) were positive by rK39 rapid diagnostic tests. For symptomatic domestic dogs, the testing of conjunctival swabs or bone marrow aspirates revealed a higher VL incidence in Kvareli District (Kvareli; 19.4 %, n = 329) compared with that observed for Sagarejo District (Sagarejo; 11.4 %, n = 271). A total of 231 sand flies of both genders were collected during the 2-month period; of the 114 females, 1.75 % were PCR positive for the presence of Leishmania spp. VL infection rates remain high in both canines and humans in Georgia, with disease in several known natural foci. The genetic relationships derived from rDNA internal transcribed spacer (ITS) sequence comparisons identified genetic subgroups, revealing preliminary insights into the genetic structure of L. donovani complex members currently circulating in the South Caucasus and demonstrates the utility of ITS-based genotyping

  12. 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

    The recently cloned glucose regulated protein 78 (GRP78) of Leishmania donovani has been suggested as a new and promising Leishmania vaccine candidate. We assessed antibody and T-cell reactivity to GRP78 in an enzyme-linked immunosorbent assay (ELISA) and in lymphoproliferative assays. Serological...... with a positive leishmanin skin test showed antibody reactivity to recombinant GRP78 (rGRP78). In lymphoproliferative assays, 9 of 13 isolates of peripheral blood mononuclear cells (PBMC) from individuals previously infected with L. donovani and one of three individuals previously infected with L. major showed...... 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...

  13. Evidence for Seroprevalence in Human Localized Cutaneous Leishmaniasis Caused by Leishmania donovani in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Yamuna Deepani Siriwardana

    2018-01-01

    Full Text Available Visceral leishmaniasis (VL is considered as a major health threat in the Indian subcontinent. Leishmania donovani, a usually visceralizing species, causes cutaneous leishmaniasis (CL in Sri Lanka. However, visceralizing potential of the local L. donovani is not yet fully understood. This project studied the seroprevalence of local CL by using an in-house ELISA. An IgG-based ELISA using crude Leishmania antigen (Ag was developed and validated. A total of 50 laboratory confirmed cases of locally acquired CL were examined using the newly developed ELISA. According to the optimized ELISA, seroprevalence of anti-Leishmania IgG antibodies in the study group was 34.0% (n=17/50. Majority of seropositive individuals were males (n=13/17, representing 76%. Nearly half of the seropositive individuals were young adults (20–40 years, n=9/17, 53%. Higher proportions of single lesions, large lesions, and nodular lesions were associated with a seroconversion. A proportion of local L. donovani infections leading to CL have the ability to raise an antibody response in the host. This may indicate early systemic involvement as one possibility. Study of a large number of patients with adequate follow-up would be useful.

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  15. Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector.

    Science.gov (United States)

    Sadlova, Jovana; Yeo, Matthew; Seblova, Veronika; Lewis, Michael D; Mauricio, Isabel; Volf, Petr; Miles, Michael A

    2011-01-01

    The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described. Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro. For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Abul Hasan Sardar

    2016-03-01

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

  18. 2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani.

    Science.gov (United States)

    Carballeira, Néstor M; Cartagena, Michelle; Sanabria, David; Tasdemir, Deniz; Prada, Christopher F; Reguera, Rosa M; Balaña-Fouce, Rafael

    2012-10-01

    2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7μM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1μM) and Trypanosoma brucei rhodesiense (IC(50)=64.5μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Comparison of Bloodmeal Digestion and the Peritrophic Matrix in Four Sand Fly Species Differing in Susceptibility to Leishmania donovani.

    Science.gov (United States)

    Pruzinova, Katerina; Sadlova, Jovana; Seblova, Veronika; Homola, Miroslav; Votypka, Jan; Volf, Petr

    2015-01-01

    The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1-2 parasites was enough to initiate mature infections.

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

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    Raquel García-Hernández

    2015-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Debarati Mukherjee

    2016-01-01

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

  2. Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

    Science.gov (United States)

    Valverde, Joanna G; Paun, Andrea; Inbar, Ehud; Romano, Audrey; Lewis, Michael; Ghosh, Kashinath; Sacks, David

    2017-04-15

    Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed. L. donovani-infected hamsters underwent xenodiagnoses with Lutzomyia longipalpis on the same or different sites on the abdomen on 2 consecutive days or by artificial feeding on the skin or blood. The transmission of L. donovani from sick hamsters to flies was surprisingly low (mean, 24% of fed flies). New flies fed on the same site acquired significantly more infections (mean, 61%; P Leishmania donovani. Using the hamster model of visceral disease, we demonstrate that prior exposure to bites of uninfected sand flies potentiates their ability to transmit infection to the vector. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  3. Leishmaniasis in Turkey: Visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey

    NARCIS (Netherlands)

    Özbilgin, Ahmet; Harman, Mehmet; Karakuş, Mehmet; Bart, Aldert; Töz, Seray; Kurt, Özgür; Çavuş, İbrahim; Polat, Erdal; Gündüz, Cumhur; van Gool, Tom; Özbel, Yusuf

    2017-01-01

    In Turkey, the main causative agents are Leishmania tropica (L. tropica) and Leishmania infantum (L. infantum) for cutaneous leishmaniasis (CL) and L. infantum for visceral leishmaniasis (VL). In this study, we investigated leishmaniasis cases caused by L. donovani and established animal models for

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

    Directory of Open Access Journals (Sweden)

    Schallig Henk DFH

    2009-06-01

    Full Text Available Abstract 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 reservoir host of Leishmania donovani. In this study, 87 dogs were screened for infection by Leishmania donovani. Blood and lymph node samples were taken from 87 and 33 dogs respectively and subsequently screened by the Polymerase Chain Reaction (PCR and Direct Agglutination Test (DAT test. Additional lymph node smears were processed for microscopy and parasite culture. Host preference of the visceral leishmaniasis (VL vector in the area, Phlebotomus orientalis, and other sandflies for the Nile rat (Arvicanthis niloticus, É. Geoffrey, 1803, the genet (Genetta genetta, Linnaeus, 1758, the mongoose (Herpeistes ichneumon, Linnaeus, 1758, and the domestic dog were determined by counting numbers of sand flies attracted to CDC traps that were baited by these animals. Results DAT on blood samples detected anti-Leishmania antibodies in 6 samples (6.9%. Two out of 87 (2.3% blood samples tested were PCR positive, giving an amplification product of 560 bp. The two positive samples by PCR were also positive by DAT. However, none of the 33 lymph nodes aspirates were Leishmania positive when screened by microscopy, culture and genus-specific PCR. The dog-baited trap significantly attracted the highest number of P. orientalis and sand fly species (P Conclusion It is concluded that the results obtained from host attraction studies indicate that dog is more attractive for P. orientalis than Egyptian mongoose, common genet and Nile rat.

  5. IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.

    Science.gov (United States)

    Terrazas, Cesar; Varikuti, Sanjay; Kimble, Jennifer; Moretti, Ellen; Boyaka, Prosper N; Satoskar, Abhay R

    2016-03-01

    Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A(-/-) mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A(+) cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation. © FASEB.

  6. Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani

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    Aditya Verma

    2017-12-01

    Full Text Available Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL in the Indian subcontinent. Paromomycin (PMM, an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R and the corresponding PMM sensitive (PMM-S parasites revealed modulated expression of 500 genes (1.5 fold cut off in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a reduced oxidative phosphorylation; b increased glycosomal succinate fermentation and substrate level phosphorylation; c dependency on lipids and amino acids for energy generation; d reduced DNA synthesis and increased DNA damage repair and e decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO levels while reactive oxygen species (ROS level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania. Keywords: Leishmania donovani, Drug resistance, Paromomycin, Transcriptome, ABC transporters, Nitric oxide, Visceral

  7. Detection of Leishmania donovani and L. tropica in ethiopian wild rodents

    Czech Academy of Sciences Publication Activity Database

    Kassahun, A.; Sádlová, J.; Dvořák, V.; Košťálová, T.; Rohoušová, I.; Frynta, D.; Aghová, Tatiana; Yasur-Landau, D.; Lemma, W.; Hailu, A.; Baneth, G.; Warburg, A.; Volf, P.; Votýpka, J.

    2015-01-01

    Roč. 145, May 2015 (2015), s. 39-44 ISSN 0001-706X R&D Projects: GA ČR GAP506/10/0983 EU Projects: European Commission(XE) 261504 - EDENEXT Institutional support: RVO:68081766 Keywords : Leishmania donovani * Leishmania tropica * Phlebotomine sand fly * Rodents * kDNA * ITS1 Subject RIV: EG - Zoology Impact factor: 2.380, year: 2015

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

    Directory of Open Access Journals (Sweden)

    Venkateswara Reddy Gogulamudi

    2015-10-01

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

  9. Leishmania donovani: an in vitro study of antimony-resistant amphotericin B-sensitive isolates

    DEFF Research Database (Denmark)

    Sharief, Abdalla Hassan; Gasim Khalil, Eltahir Awad; Theander, Thor G

    2006-01-01

    Drug sensitivity of clinically antimony-unresponsive Leishmania donovani isolates from Eastern Sudan was evaluated in an in vitro culture system against sodium stibogluconate (Pentostam) and Amphotericin B. Eight isolates, six from antimony-resistant and two from clinically responsive patients were...

  10. Genetic polymorphism within the Leishmania donovani complex: correlation with geographic origin

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Miles, M. A.; Lukeš, Julius

    2004-01-01

    Roč. 70, č. 6 (2004), s. 613-617 ISSN 0002-9637 Grant - others:European Community(XE) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z6022909 Keywords : genetic polymorphism * Leishmania donovani * RAPD Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.013, year: 2004

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

    Science.gov (United States)

    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.

  12. Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

    International Nuclear Information System (INIS)

    Beck, J.T.; Ullman, B.

    1989-01-01

    An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 μM concentration of either activated [ 3 H]folate or activated [ 3 H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms

  13. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

    Science.gov (United States)

    Abbasi, Ibrahim; Aramin, Samar; Hailu, Asrat; Shiferaw, Welelta; Kassahun, Aysheshm; Belay, Shewaye; Jaffe, Charles; Warburg, Alon

    2013-03-27

    Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

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

    Directory of Open Access Journals (Sweden)

    Abhishek Mandal

    2017-07-01

    Full Text Available 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

  15. Immunotherapy against visceral leishmaniasis with the nucleoside hydrolase-DNA vaccine of Leishmania donovani.

    Science.gov (United States)

    Gamboa-León, R; Paraguai de Souza, E; Borja-Cabrera, G P; Santos, F N; Myashiro, L M; Pinheiro, R O; Dumonteil, E; Palatnik-de-Sousa, C B

    2006-05-29

    The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using two doses of 100 or 20 microg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 microg (91%) and 20 microg (77%) of the DNA vaccine, and by 20 microg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 microg vaccine, and 83 and 67% for the 20 microg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-gamma secretion.

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

    Directory of Open Access Journals (Sweden)

    Anjali Kumari

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

  17. L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

    Directory of Open Access Journals (Sweden)

    Jasdeep Singh

    2017-12-01

    Full Text Available Emergence of Amphotericin B (AmB resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania. Keywords: Leishmania donovani, L-asparaginase, Amphotericin B resistance, Metabolic target

  18. Solute carrier protein family 11 member 1 (Slc11a1) activation efficiently inhibits Leishmania donovani survival in host macrophages.

    Science.gov (United States)

    Singh, Nisha; Gedda, Mallikarjuna Rao; Tiwari, Neeraj; Singh, Suya P; Bajpai, Surabhi; Singh, Rakesh K

    2017-09-01

    Visceral leishmaniasis (kala-azar), a life threatening disease caused by L. donovani , is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The therapeutic option to control leishmanial infections are very limited, and at present comprise only two drugs, an antifungal amphotericin B and an antitumor miltefosine, which are also highly vulnerable for parasitic resistance. Therefore, identification and development of alternate control measures is an exigent requirement to control leishmanial infections. In this study, we report that functionally induced expression of solute carrier protein family 11 member 1 ( Slc11a1), a transmembrane divalent cationic transporter recruited on the surface of phagolysosomes after phagocytosis of parasites, effectively inhibits Leishmania donovani growth in host macrophages. Further, the increased Slc11a1 functionality also resulted in increased production of NOx, TNF-α and IL-12 by activated macrophages. The findings of this study signify the importance of interplay between Slc11a1 expression and macrophages activation that can be effectively used to control of Leishmania growth and survival.

  19. Natural hybrid of Leishmania infantum/L. donovani: development in Phlebotomus tobbi, P. perniciosus and Lutzomyia longipalpis and comparison with non-hybrid strains differing in tissue tropism.

    Science.gov (United States)

    Seblova, Veronika; Myskova, Jitka; Hlavacova, Jana; Votypka, Jan; Antoniou, Maria; Volf, Petr

    2015-11-25

    Infection caused by parasites from L. donovani complex can manifest as a serious visceral disease or a self-healing milder cutaneous form. The different tropism and pathology in humans is caused by the interaction between parasites, host and vector determinants but the mechanisms are not well understood. In Cukurova region in Turkey we previously identified a major focus of cutaneous leishmaniasis caused by L. donovani/infantum hybrids (CUK strain) and isolated this parasite from the locally abundant sand fly, Phlebotomus tobbi. Here, we present the first experimental study with P. tobbi. We tested the susceptibility of this species to various Leishmania under laboratory conditions, characterized glycoproteins in the P. tobbi midgut putatively involved in parasite-vector interaction and compared the development of the CUK strain in the sand fly with one other dermotropic and three viscerotropic strains belonging to the L. donovani complex. Females of laboratory reared P. tobbi, P. perniciosus and Lutzomyia longipalpis were infected using membrane feeding on rabbit blood containing promastigotes of various Leishmania species with different tropisms. The individual guts were checked microscopically for presence and localization of Leishmania parasites; the number of parasites was assessed more precisely by qPCR. In addition, glycosylation of midgut proteins of P. tobbi was studied by lectin blotting of midgut lysate with lectins specific for terminal sugars of N-type and O-type glycans. High infection rates, heavy parasite loads and late-stage infection with colonization of the stomodeal valve were observed in P. tobbi infected by Leishmania major or L. infantum CUK hybrid. In parallel, lectin blotting revealed the presence of O-glycosylated proteins in the P. tobbi midgut. In P. perniciosus and L. longipalpis all five Leishmania strains tested developed well. In both vectors, significantly higher parasite numbers were detected by qPCR for dermotropic L. donovani

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

    Directory of Open Access Journals (Sweden)

    Mary Dan-Goor

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

  1. Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms.

    Science.gov (United States)

    Mondal, Subhasish; Roy, Jay Jyoti; Bera, Tanmoy

    2014-03-01

    Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.

  2. The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

    Science.gov (United States)

    Chowdhury, Sayan; Mukherjee, Tulika; Mukhopadhyay, Rupkatha; Mukherjee, Budhaditya; Sengupta, Souvik; Chattopadhyay, Sharmila; Jaisankar, Parasuraman; Roy, Syamal; Majumder, Hemanta K

    2012-01-01

    Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topoisomerase of L. donovani and acts as a non-competitive inhibitor interacting with both subunits of the enzyme. Niranthin interacts with DNA–protein binary complexes and thus stabilizes the ‘cleavable complex’ formation and subsequently inhibits the religation of cleaved strand. The compound inhibits the proliferation of Leishmania amastigotes in infected cultured murine macrophages with limited cytotoxicity to the host cells and is effective against antimony-resistant Leishmania parasites by modulating upregulated P-glycoprotein on host macrophages. Importantly, besides its in vitro efficacy, niranthin treatment leads to a switch from a Th2- to a Th1-type immune response in infected BALB/c mice. The immune response causes production of nitric oxide, which results in almost complete clearance of the liver and splenic parasite burden after intraperitoneal or intramuscular administration of the drug. These findings can be exploited to develop niranthin as a new drug candidate against drug-resistant leishmaniasis. PMID:23027614

  3. 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

  4. Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Smita Rai

    Full Text Available BACKGROUND: In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s and expression profiles of known genes involved in transport and thiol based redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We selected 7 clinical isolates (2 sensitive and 5 resistant in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. CONCLUSIONS/SIGNIFICANCE: Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

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

    Directory of Open Access Journals (Sweden)

    Sreenivas Gannavaram

    2011-01-01

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

  6. F1 Domain of the Leishmania (Leishmania donovani Nucleoside Hydrolase Promotes a Th1 Response in Leishmania (Leishmania infantum Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Eugenia Carrillo

    2017-07-01

    Full Text Available The Leishmania (Leishmania donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1, the central (F2, or the C-terminal recombinant domain (F3 of NH36 was evaluated using peripheral blood mononuclear cells (PBMC from individuals infected with L. (L. infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42–43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17 secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L. infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L. donovani (Ethiopia and L. (L. infantum (Spain from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L. donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L. infantum chagasi, may

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  8. Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

    Science.gov (United States)

    Verma, Jitender Kumar; Rastogi, Ruchir

    2017-01-01

    Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

  9. 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.

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

    OpenAIRE

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

    2001-01-01

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

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

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    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.

  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. Homology modeling of Leishmania donovani enolase and its molecular interaction with novel inhibitors

    Directory of Open Access Journals (Sweden)

    Jay Prakash Mahato

    2017-01-01

    Full Text Available Introduction: The treatment of Indian tropical disease such as kala-azar is likely to be troublesome to the clinicians as AmpB- and miltefosine-resistant Leishmania donovani has been reported. The rationale behind designed a novel inhibitors of model of L. donovani enolase and performing a binding study with its inhibitors to gain details of the interaction between protein residues and ligand molecules. Methods and Materials: The L. donovani enolase model consists of two typical domains. The N-terminal one contains three-stranded antiparallel β-sheets, followed by six α-helices. The C-terminal domain composes of eleven-stranded mixed α/β-barrel with connectivity. The first α-helix within the C-terminal domain, H7, and the second β-strand, S7, of the barrel domain was arranged in an antiparallel fashion compared to all other α-helices and β-strands. The root-mean-square deviation between predicted model and template is 0.4 Å. The overall conformation of L. donovani enolase model is similar to those of Trypanosoma cruzi enolase and Streptococcus pneumoniae enolase crystal structures. Result: The key amino acid residues within the docking complex model involved in the interaction between model enolase structure and ligand molecule are Lys70, Asn165, Ala168, Asp17, and Asn213. Conclusion: Our theoretical prediction may lead to the establishment of prophylactic and therapeutic approaches for the treatment of kala-azar. This biomedical informatics analysis will help us to combat future kala-azar.

  14. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

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    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  15. 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.

  16. 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.

  17. Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

    Czech Academy of Sciences Publication Activity Database

    Faber, W. R.; Wonders, J.; Jensema, A. J.; Chocholová, Eva; Kager, P. A.

    2009-01-01

    Roč. 34, č. 5 (2009), E196-E198 ISSN 0307-6938 Institutional research plan: CEZ:AV0Z60220518 Keywords : LYMPH -NODE INVOLVEMENT * BRAZILIENSIS * INFECTION Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 1.550, year: 2009

  18. Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

    Directory of Open Access Journals (Sweden)

    Kuljit Singh

    2017-08-01

    Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

  19. Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani.

    Science.gov (United States)

    Soysa, Radika; Tran, Khoa D; Ullman, Buddy; Yates, Phillip A

    2015-12-01

    We have designed a novel series of integrating ribosomal RNA promoter vectors with five incrementally different constitutive expression profiles, covering a 250-fold range. Differential expression was achieved by placing different combinations of synthetic or leishmanial DNA sequences upstream and downstream of the transgene coding sequence in order to modulate pre-mRNA processing efficiency and mRNA stability, respectively. All of the vectors have extensive multiple cloning sites, and versions are available for producing N- or C- terminal GFP fusions at each of the possible relative expression levels. In addition, the modular configuration of the vectors allows drug resistance cassettes and other components to be readily exchanged. In toto, these vectors should be useful additions to the toolkit available for molecular and genetic studies of Leishmania donovani. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Glycyrrhizic acid attenuates growth of Leishmania donovani by depleting ergosterol levels.

    Science.gov (United States)

    Dinesh, Neeradi; Neelagiri, Soumya; Kumar, Vinay; Singh, Sushma

    2017-05-01

    In the present study, glycyrrhizic acid (GA) the main component of Glycyrrhiza glabra was evaluated for its efficacy as antileishmanial agent and its mode of action explored. GA inhibits promastigotes and intracellular amastigotes in a dose dependent manner at an IC 50 value of 34 ± 3.0 μM and 20 ± 4.2 μM respectively. GA was non-toxic against THP-1 macrophage host cell line. GA was found to inhibit recombinant Leishmania donovani HMG-CoA reductase (LdHMGR) enzyme at the half-maximum inhibitory concentration of 24 ± 4.3 μM indicating the sensitivity and specificity of GA towards the enzyme. However, GA could cause only 30% reduction in HMGR activity when measured in Leishmania promastigotes treated with 34 μM of GA. Interestingly western blot analysis revealed fivefold reduced HMGR expression in GLA treated promastigotes. To further study the mode of action of GA, we used transgenic parasites overexpressing LdHMGR. Results indicated that ∼2 fold resistance was exhibited by LdHMGR overexpressing promastigotes to GA with an IC 50 value of 74 μM compared to the wild type parasite. This explained the specific binding of GA to LdHMGR enzyme. There was ∼2 fold depletion in ergosterol levels in wild type promastigotes compared to the HMGR overexpressors. This data was further validated by exogenous supplementation of GA treated cells with ergosterol and 40% reversal of growth inhibition was observed. The results obtained suggested that GA kills the parasite by affecting sterol biosynthetic pathway, especially by inhibiting the L. donovani HMGR and altering ergosterol levels. The finding from the current study shows that GA is a potential antileishmanial chemotherapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2009-01-01

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

  2. Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+-epitopes of Leishmania donovani Nucleoside Hydrolase terminal domains

    Directory of Open Access Journals (Sweden)

    Dirlei eNico

    2014-05-01

    Full Text Available The Nucleoside hydrolase of Leishmania donovani (NH36 is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3. The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1-103 and F3 peptide (amino acids 199-314 vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions. Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing in a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonesis infection represents a basis for the rationale development of a bivalent vaccine

  3. A diagnostic assay based on variable intergenic region distinguishes between Leishmania donovani and Leishmania infantum

    Czech Academy of Sciences Publication Activity Database

    Chocholová, Eva; Jirků, Milan; Lukeš, Julius

    2008-01-01

    Roč. 55, č. 1 (2008), s. 75-78 ISSN 0015-5683 R&D Projects: GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : Leishmania * assay * diagnosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.307, year: 2008

  4. Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

    OpenAIRE

    Mutiso,Joshua M.; Macharia,John C.; Taracha,Evans; Wafula,Kellern; Rikoi,Hitler; Gicheru,Michael M.

    2012-01-01

    In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels ...

  5. Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent.

    Science.gov (United States)

    Dumetz, F; Cuypers, B; Imamura, H; Zander, D; D'Haenens, E; Maes, I; Domagalska, M A; Clos, J; Dujardin, J-C; De Muylder, G

    2018-04-25

    Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani : the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (Sb III ) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline Sb III susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to Sb III resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to Sb III resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of Sb III The main driver of this preadaptation was shown to be MRPA , a gene involved in Sb III sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence. IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti- Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious

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

    Czech Academy of Sciences Publication Activity Database

    Mauricio, I. L.; Yeo, M.; Baghaei, M.; Doto, D.; Pratlong, F.; Zemanová, Eva; Dedet, J.-P.; Lukeš, Julius; Miles, M. A.

    2006-01-01

    Roč. 36, č. 7 (2006), s. 757-769 ISSN 0020-7519 Grant - others:European Comission(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Keywords : Leishmania donovani * Leishmania infantum * multilocus sequence typing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.337, year: 2006

  7. 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.

  8. The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells

    DEFF Research Database (Denmark)

    Hey, A S; Theander, T G; Hviid, L

    1994-01-01

    The effect of Leishmania major and L. donovani surface protease gp63 on surface markers on human T cells was studied using fluorescence-activated flow cytometry. Purified gp63 (63,000 m.w. glycoprotein) at concentrations above 10 micrograms/ml completely inhibited binding of six different anti-CD4......-expression of CD4, reaching 50% of the initial level after 72 h of incubation in medium. Preincubation of cells with live promastigotes showed an inhibitory effect on CD4 comparable to that seen with purified gp63. The binding of Abs directed against other surface markers present on human T-cells--CD2, CD3, CD5......, CD8, CD11A, CD25, CD45RO, CD45RA, CD58, TCR-alpha, TCR-gamma, and HLA DQ--was not inhibited by gp63. These data suggest that gp63, both in its purified form and in the form anchored to the parasite membrane, cleaves CD4 on human T cells. The cleavage of CD4 by the protease might play a role...

  9. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

    Science.gov (United States)

    Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

    2017-01-01

    Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L

  10. Prevalence and risk factors associated with Leishmania infection in Trang Province, southern Thailand.

    Science.gov (United States)

    Manomat, Jipada; Leelayoova, Saovanee; Bualert, Lertwut; Tan-Ariya, Peerapan; Siripattanapipong, Suradej; Mungthin, Mathirut; Naaglor, Tawee; Piyaraj, Phunlerd

    2017-11-01

    Autochthonous cutaneous and visceral leishmaniasis (VL) caused by Leishmania martiniquensis and Leishmania siamensis have been considered emerging infectious diseases in Thailand. The disease burden is significantly underestimated, especially the prevalence of Leishmania infection among HIV-positive patients. A cross-sectional study was conducted to determine the prevalence and risk factors associated with Leishmania infection among patients with HIV/AIDS living in Trang province, southern Thailand, between 2015 and 2016. Antibodies against Leishmania infection were assayed using the direct agglutination test (DAT). DNA of Leishmania was detected by ITS1-PCR using the buffy coat. Species of Leishmania were also identified. Of 724 participants, the prevalence of Leishmania infection was 25.1% (182/724) using either DAT or PCR assays. Seroprevalence of Leishmania infection was 18.5% (134/724), while Leishmania DNA detected by the PCR method was 8.4% (61/724). Of these, 24.9% (180/724) were asymptomatic, whereas 0.3% (2/724) were symptomatic VL and VL/CL (cutaneous leishmaniasis). At least five species were identified: L. siamensis, L. martiniquensis, L. donovani complex, L. lainsoni, and L. major. Multivariate analysis showed that CD4+ levels Leishmania infection. Those who were PCR positive for Leishmania DNA were significantly associated with a detectable viral load, whereas non-injection drug use (NIDU) and CD4+ levels Leishmania seropositivity. A magnitude of the prevalence of underreporting Leishmania infection among Thai patients with HIV was revealed in this study. Effective public health policy to prevent and control disease transmission is urgently needed.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ines Lakhal-Naouar

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Intracellular pH homeostasis in Leishmania donovani amastigotes and promastigotes

    International Nuclear Information System (INIS)

    Glaser, T.A.; Baatz, J.E.; Kreishman, G.P.; Mukkada, A.J.

    1988-01-01

    Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31 P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment

  15. Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh, E-mail: jvpratap@cdri.res.in

    2015-01-09

    Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

  16. Characterization of dipeptidylcarboxypeptidase of Leishmania donovani: a molecular model for structure based design of antileishmanials

    Science.gov (United States)

    Baig, Mirza Saqib; Kumar, Ashutosh; Siddiqi, Mohammad Imran; Goyal, Neena

    2010-01-01

    Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 μmole/ml/min. Inhibition studies revealed that known ACE inhibitors (captopril and bradykinin potentiating peptide; BPP1) were weak inhibitors for LdDCP as compared to human testicular ACE (htACE) with Ki values of 35.8 nM and 3.9 μM, respectively. Three dimensional model of LdDCP was generated based on crystal structure of Escherichia coli DCP (EcDCP) by means of comparative modeling and assessed using PROSAII, PROCHECK and WHATIF. Captopril docking with htACE, LdDCP and EcDCP and analysis of molecular electrostatic potentials (MEP) suggested that the active site domain of three enzymes has several minor but potentially important structural differences. These differences could be exploited for designing selective inhibitor of LdDCP thereby antileishmanial compounds either by denovo drug design or virtual screening of small molecule databases.

  17. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  18. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  19. Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs.

    Science.gov (United States)

    Rastrojo, Alberto; García-Hernández, Raquel; Vargas, Paola; Camacho, Esther; Corvo, Laura; Imamura, Hideo; Dujardin, Jean-Claude; Castanys, Santiago; Aguado, Begoña; Gamarro, Francisco; Requena, Jose M

    2018-04-13

    Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (Sb III , S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. Novel Arsenic Nanoparticles Are More Effective and Less Toxic than As (III) to Inhibit Extracellular and Intracellular Proliferation of Leishmania donovani

    Science.gov (United States)

    Chakraborty, Sudipta; Bhar, Kaushik; Saha, Sandip; Chakrabarti, Rajarshi; Pal, Anjali

    2014-01-01

    Visceral leishmaniasis, a vector-borne tropical disease that is threatening about 350 million people worldwide, is caused by the protozoan parasite Leishmania donovani. Metalloids like arsenic and antimony have been used to treat diseases like leishmaniasis caused by the kinetoplastid parasites. Arsenic (III) at a relatively higher concentration (30 μg/mL) has been shown to have antileishmanial activity, but this concentration is reported to be toxic in several experimental mammalian systems. Nanosized metal (0) particles have been shown to be more effective than their higher oxidation state forms. There is no information so far regarding arsenic nanoparticles (As-NPs) as an antileishmanial agent. We have tested the antileishmanial properties of the As-NPs, developed for the first time in our laboratory. As-NPs inhibited the in vitro growth, oxygen consumption, infectivity, and intramacrophage proliferation of L. donovani parasites at a concentration which is about several fold lower than that of As (III). Moreover, this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line. It is evident from our findings that As-NPs have more potential than As (III) to be used as an antileishmanial agent. PMID:25614827

  3. The Leishmania donovani complex: Genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH and FH), new targets for multilocus sequence typing

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Horák, Aleš; Miles, M. A.; Lukeš, Julius

    2007-01-01

    Roč. 37, č. 2 (2007), s. 149-160 ISSN 0020-7519 R&D Projects: GA MŠk 2B06129 Grant - others:EU(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Source of funding: R - rámcový projekt EK Keywords : Leishmania donovani complex * zymodeme * multilocus sequence typing * Leishmania * phylogenetic network Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.392, year: 2007

  4. Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

    Directory of Open Access Journals (Sweden)

    Joshua M. Mutiso

    2012-02-01

    Full Text Available In this study, we report on the safety and skin delayed-type hypersensitivity (DTH, responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG, Montanide ISA 720 (MISA or Monophosphoryl lipid A (MPLA in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α and interferon gamma (IFN-γ levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001. The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001. Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Leishmania donovani isolates with antimony-resistant but not -sensitive phenotype inhibit sodium antimony gluconate-induced dendritic cell activation.

    Directory of Open Access Journals (Sweden)

    Arun Kumar Haldar

    2010-05-01

    Full Text Available The inability of sodium antimony gluconate (SAG-unresponsive kala-azar patients to clear Leishmania donovani (LD infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(RLD and antimony-sensitive (Sb(SLD was compared in vitro. Unlike Sb(SLD, infection of DCs with Sb(RLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(RLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(SLD failed to block activation of SAG (20 microg/ml-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(SLD also inhibited SAG (20 microg/ml-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(RLD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(RLD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(SLD expressing ATP-binding cassette (ABC transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(RLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc gene, presumably an important regulator of antimony resistance. Importantly, Sb(RLD but not Sb(SLD blocked SAG-induced mgammaGCS expression in DCs by

  7. 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...

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

    OpenAIRE

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

    1999-01-01

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

  9. AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

    Science.gov (United States)

    Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

    2015-04-01

    An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Wai-Lok Yau

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

  11. Zinc depletion promotes apoptosis-like death in drug-sensitive and antimony-resistance Leishmania donovani.

    Science.gov (United States)

    Saini, Shalini; Bharati, Kavita; Shaha, Chandrima; Mukhopadhyay, Chinmay K

    2017-09-05

    Micronutrients are essential for survival and growth for all the organisms including pathogens. In this manuscript, we report that zinc (Zn) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethylenediamine (TPEN) affects growth and viability of intracellular pathogen Leishmania donovani (LD) by a concentration and time dependent manner. Simultaneous addition of zinc salt reverses the effect of TPEN. Further experiments provide evidence of apoptosis-like death of the parasite due to Zn-depletion. TPEN treatment enhances caspase-like activity suggesting increase in apoptosis-like events in LD. Specific inhibitors of cathepsin B and Endoclease G block TPEN-induced leishmanial death. Evidences show involvement of reactive oxygen species (ROS) potentially of extra-mitochondrial origin in TPEN-induced LD death. Pentavalent antimonials remained the prime source of treatment against leishmaniasis for several decades; however, antimony-resistant Leishmania is now common source of the disease. We also reveal that Zn-depletion can promote apoptosis-like death in antimony-resistant parasites. In summary, we present a new finding about the role of zinc in the survival of drug sensitive and antimony-resistant LD.

  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. 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

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

    International Nuclear Information System (INIS)

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-01-01

    Highlights: ► The study presents cloning and characterization of TCP1γ gene from L. donovani. ► TCP1γ is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. ► LdTCPγ exhibited differential expression in different stages of promastigotes. ► LdTCPγ co-localized with actin, a cytoskeleton protein. ► The data suggests that this gene may have a role in differentiation/biogenesis. ► 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 (TCP1γ), 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 TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ 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 LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

  15. 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 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.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.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.

  16. In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

    Science.gov (United States)

    Shaw, C D; Lonchamp, J; Downing, T; Imamura, H; Freeman, T M; Cotton, J A; Sanders, M; Blackburn, G; Dujardin, J C; Rijal, S; Khanal, B; Illingworth, C J R; Coombs, G H; Carter, K C

    2016-03-01

    In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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 ...

  19. Innate Immunity to Leishmania Infection: Within Phagocytes

    Directory of Open Access Journals (Sweden)

    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.

  20. Innate Immunity against Leishmania Infections

    Science.gov (United States)

    Gurung, Prajwal; Kanneganti, Thirumala-Devi

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jacqueline Araújo Fiuza

    2016-01-01

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

  2. Experimental mixed infection of Leishmania (Leishmania) amazonensis and Leishmania (L.) infantum in hamsters (Mesocricetus auratus).

    Science.gov (United States)

    DE Lima Celeste, Jordanna Luíza; Venuto Moura, Ana Paula; França-Silva, João Carlos; Matos DE Sousa, Gabriela; Oliveira Silva, Soraia; Norma Melo, Maria; Luiz Tafuri, Wagner; Carvalho Souza, Carolina; Monteiro DE Andrade, Hélida

    2017-08-01

    In South America, visceral leishmaniasis is frequently caused by Leishmania infantum and, at an unknown frequency, by Leishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections with L. amazonensis and L. infantum in a hamster model. We show that mixed infections are associated with more severe clinical disease than infection with L. amazonensis or L. infantum alone. In spleens with mixed infections, L. infantum outcompeted L. amazonensis in the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected with L. infantum. Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected with L. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected with L. infantum. This finding has important implications regarding the biology of Leishmania and humoral immune responses to infections with these organisms.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Joana Faria

    2016-01-01

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

  5. Leishmania infections: Molecular targets and diagnosis.

    Science.gov (United States)

    Akhoundi, Mohammad; Downing, Tim; Votýpka, Jan; Kuhls, Katrin; Lukeš, Julius; Cannet, Arnaud; Ravel, Christophe; Marty, Pierre; Delaunay, Pascal; Kasbari, Mohamed; Granouillac, Bruno; Gradoni, Luigi; Sereno, Denis

    2017-10-01

    Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  7. Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

    Science.gov (United States)

    Bakhshi, H; Oshaghi, M A; Abai, M R; Rassi, Y; Akhavan, A A; Sheikh, Z; Mohtarami, F; Saidi, Z; Mirzajani, H; Anjomruz, M

    2013-10-01

    A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. A study of a self diagnostic platform for the detection of A2 biomarker for Leishmania donovani

    Science.gov (United States)

    Roche, Philip J. R.; Cheung, Maurice C.; Najih, Mohamed; McCall, Laura-Isobel; Fakih, Ibrahim; Chodavarapu, Vamsy P.; Ward, Brian; Ndao, Momar; Kirk, Andrew G.

    2012-03-01

    Visceral leishmaniasis (L.donovani) is a protozoan infection that attacks mononuclear phagocytes and causes the liver and spleen damage that can cause death. The investigation presented is a proof of concept development applying a plasmonic diagnostic platform with simple microfluidic sample delivery and optical readout. An immune-assay method is applied to the quantification of A2 protein, a highly immunogenic biomarker for the pathogen. Quantification of A2 was performed in the ng/ml range, analysis by ELISA suggested that a limit of 0.1ng/ml of A2 is approximate to 1 pathogen per ml and the sensing system shows the potential to deliver a similar level of quantification. Significant reduction in assay complexity as further enzyme linked enhancement is not required when applying a plasmonic methodology to an immunoassay. The basic instrumentation required for a portable device and potential dual optical readout where both plasmonic and photoluminescent response are assessed and investigated including consideration of the application of the device to testing where non-literate communication of results is considered and issues of performance are addressed.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    was assessed in mice vaccine experiments. A GRP78 DNA vaccine primed for an immune response that protected C57Bl/6 and C3H/He mice against infection with L. major. Similarly vaccination with a recombinant form of GRP78 purified from Escherichia coli and administered with Freund's as adjuvant induced protective...

  10. Arginine and Polyamines Fate in Leishmania Infection

    Science.gov (United States)

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

    2018-01-01

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

  11. 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.

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

    NARCIS (Netherlands)

    van Thiel, Pieter-Paul A. M.; van Gool, Tom; Kager, Piet A.; Bart, Aldert

    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

  13. 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...

  14. Novel agmatine analogue, γ-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels

    International Nuclear Information System (INIS)

    Singh, Sushma; Jhingran, Anupam; Sharma, Ankur; Simonian, Alina R.; Soininen, Pasi; Vepsalainen, Jouko; Khomutov, Alex R.; Madhubala, Rentala

    2008-01-01

    The efficacy of γ-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K i value of ∼60 μM. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK a 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels

  15. Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

    Science.gov (United States)

    Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

    2017-03-23

    Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid

  16. 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

    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...... 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...... only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may...

  17. In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

    Science.gov (United States)

    Kant, Vishnu; Vijayakumar, Saravanan; Sahoo, Ganesh Chandra; Chaudhery, Shailendra S; Das, Pradeep

    2018-02-07

    OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS.

  18. 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 production...... was infrequently observed. The results show that T cells from individuals who have been cured of visceral leishmaniasis recognize and respond to a wide range of leishmanial antigens. There was no evidence of particular fractions constantly giving either IFN-gamma or IL-4-producing responses....

  19. Natural infection of bats with Leishmania in Ethiopia.

    Science.gov (United States)

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

    2015-10-01

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

  20. The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Yates, Phillip A.; Soysa, D.Radika; Boitz, Jan M.; Carter, Nicola S.; Chang, Bailey; Ullman, Buddy; Ealick, Steven E. (Oregon HSU); (Cornell)

    2011-08-09

    The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

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

    Directory of Open Access Journals (Sweden)

    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. The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

    OpenAIRE

    Chowdhury, Sayan; Mukherjee, Tulika; Mukhopadhyay, Rupkatha; Mukherjee, Budhaditya; Sengupta, Souvik; Chattopadhyay, Sharmila; Jaisankar, Parasuraman; Roy, Syamal; Majumder, Hemanta K

    2012-01-01

    Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topois...

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

    Science.gov (United States)

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

    2013-11-08

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

  4. The Malnutrition-Related Increase in Early Visceralization of Leishmania donovani Is Associated with a Reduced Number of Lymph Node Phagocytes and Altered Conduit System Flow

    Science.gov (United States)

    Ibrahim, Marwa K.; Barnes, Jeffrey L.; Anstead, Gregory M.; Jimenez, Fabio; Travi, Bruno L.; Peniche, Alex G.; Osorio, E. Yaneth; Ahuja, Seema S.; Melby, Peter C.

    2013-01-01

    In a murine model of moderate childhood malnutrition we found that polynutrient deficiency led to a 4–5-fold increase in early visceralization of L. donovani (3 days post-infection) following cutaneous infection and a 16-fold decrease in lymph node barrier function (pmalnutrition-related parasite dissemination we analyzed the cellularity, architecture, and function of the skin-draining lymph node. There was no difference in the localization of multiple cell populations in the lymph node of polynutrient deficient (PND) mice, but there was reduced cellularity with fewer CD11c+dendritic cells (DCs), fibroblastic reticular cells (FRCs), MOMA-2+ macrophages, and CD169+ subcapsular sinus macrophage (p<0.05 for all) compared to the well-nourished (WN) mice. The parasites were equally co-localized with DCs associated with the lymph node conduit network in the WN and PND mice, and were found in the high endothelial venule into which the conduits drain. When a fluorescent low molecular weight (10 kD) dextran was delivered in the skin, there was greater efflux of the marker from the lymph node conduit system to the spleens of PND mice (p<0.04), indicating that flow through the conduit system was altered. There was no evidence of disruption of the conduit or subcapsular sinus architecture, indicating that the movement of parasites into the subcortical conduit region was due to an active process and not from passive movement through a leaking barrier. These results indicate that the impaired capacity of the lymph node to act as a barrier to dissemination of L. donovani infection is associated with a reduced number of lymph node phagocytes, which most likely leads to reduced capture of parasites as they transit through the sinuses and conduit system. PMID:23967356

  5. Dihydrotestosterone enhances growth and infectivity of Leishmania Mexicana.

    Science.gov (United States)

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

    2018-03-01

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

  6. 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

    2017-02-01

    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. 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). 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. 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 infection in outbreak areas where several species of Leishmania

  7. 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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Miriam Berzunza-Cruz

    2015-01-01

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

  10. 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.

  11. Leishmania major Infection Activates NF-κB and Interferon Regulatory Factors 1 and 8 in Human Dendritic Cells▿

    Science.gov (United States)

    Jayakumar, Asha; Donovan, Michael J.; Tripathi, Vinita; Ramalho-Ortigao, Marcelo; McDowell, Mary Ann

    2008-01-01

    The salient feature of dendritic cells (DC) is the initiation of appropriate adaptive immune responses by discriminating between pathogens. Using a prototypic model of intracellular infection, we previously showed that Leishmania major parasites prime human DC for efficient interleukin-12 (IL-12) secretion. L. major infection is associated with self-limiting cutaneous disease and powerful immunity. In stark contrast, the causative agent of visceral leishmaniasis, Leishmania donovani, does not prime human DC for IL-12 production. Here, we report that DC priming by L. major infection results in the early activation of NF-κB transcription factors and the up-regulation and nuclear translocation of interferon regulatory factor 1 (IRF-1) and IRF-8. The inhibition of NF-κB activation by the pretreatment of DC with caffeic acid phenethyl ester blocks L. major-induced IRF-1 and IRF-8 activation and IL-12 expression. We further demonstrate that IRF-1 and IRF-8 obtained from L. major-infected human DC specifically bind to their consensus binding sites on the IL-12p35 promoter, indicating that L. major infection either directly stimulates a signaling cascade or induces an autocrine pathway that activates IRF-1 and IRF-8, ultimately resulting in IL-12 transcription. PMID:18316378

  12. 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....... donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jarina Pena DaMata

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

  15. 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

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Erico Vinícius de Souza Carmo

    2010-11-01

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

  18. In Vitro Susceptibilities of Wild and Drug Resistant Leishmania donovani Amastigote Stages to Andrographolide Nanoparticle: Role of Vitamin E Derivative TPGS for Nanoparticle Efficacy

    Science.gov (United States)

    Mondal, Subhasish; Roy, Partha; Das, Suvadra; Halder, Asim; Mukherjee, Arup; Bera, Tanmoy

    2013-01-01

    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 improved in

  19. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Kárita Cláudia Freitas-Lidani

    2014-07-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  3. Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

    Science.gov (United States)

    Salotra, Poonam; Sreenivas, G.; Pogue, Gregory P.; Lee, Nancy; Nakhasi, Hira L.; Ramesh, V.; Negi, N. S.

    2001-01-01

    We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples. PMID:11230394

  4. An agent-based model for Leishmania major infection

    Science.gov (United States)

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

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

  5. Leishmania major infection in a dog with cutaneous manifestations

    OpenAIRE

    Baneth, Gad; Nachum-Biala, Yaarit; Shabat Simon, Maytal; Brenner, Ori; Gaier, Sarit; Rojas, Alicia; Yasur-Landau, Daniel

    2016-01-01

    Background Leishmania major is a main cause of cutaneous leishmaniasis in humans in an area that stretches from India through Central Asia, the Middle East, to North and West Africa. In Israel, it is a common infection of humans with rodents as the reservoir hosts and Phlebotomus papatasi as its sand fly vector. Findings A 6?months old spayed female mixed breed dog was referred to the Hebrew University Veterinary Teaching Hospital with a large ulcerative dermal lesion on the muzzle, and lesio...

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

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    Alessandro Costagliola

    2016-01-01

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

  7. Leishmania major infection in a dog with cutaneous manifestations.

    Science.gov (United States)

    Baneth, Gad; Nachum-Biala, Yaarit; Shabat Simon, Maytal; Brenner, Ori; Gaier, Sarit; Rojas, Alicia; Yasur-Landau, Daniel

    2016-05-10

    Leishmania major is a main cause of cutaneous leishmaniasis in humans in an area that stretches from India through Central Asia, the Middle East, to North and West Africa. In Israel, it is a common infection of humans with rodents as the reservoir hosts and Phlebotomus papatasi as its sand fly vector. A 6 months old spayed female mixed breed dog was referred to the Hebrew University Veterinary Teaching Hospital with a large ulcerative dermal lesion on the muzzle, and lesions in the foot pads and left hind leg. Histopathology of a skin biopsy found chronic lymphohistiocytic dermatitis with the presence of Leishmania spp. amastigotes in the muzzle. Physical examination indicated that the dog was overall in a good clinical condition and the main findings were the skin lesions and enlarged prescapular lymph nodes. Complete blood count and serum biochemistry profile were within reference ranges. Serology by ELISA was positive for Leishmania spp. and PCR of the prescapular lymph node was positive by an ITS1 region PCR-high resolution melt analysis. However, the melt curve and subsequent DNA sequencing indicated that infection was caused by L. major and not L. infantum, which is the main causative agent of canine leishmaniosis in the Mediterranean region. DNA was extracted from the paraffin embedded muzzle biopsy and PCR with sequencing also indicated L. major. The dog's young age and the absence of hyperglobulinemia and anemia were not typical of L. infantum infection. The dog was treated with allopurinol and the skin lesions improved and later disappeared when the dog was re-evaluated. This is the first molecularly-confirmed case of L. major infection in a dog. Two previous reports of L. major in dogs originated from Saudi-Arabia and Egypt in 1985 and 1987 were confirmed by enzymatic biochemical techniques. Serology for L. infantum was positive probably due to the well documented serological cross-reactivity between Leishmania spp. Although dogs and wild carnivores are

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

    Science.gov (United States)

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

    2017-05-01

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

  9. Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola

    NARCIS (Netherlands)

    Vilhena, Hugo; Granada, Sara; Oliveira, Ana Cristina; Schallig, Henk D. F. H.; Nachum-Biala, Yaarit; Cardoso, Luís; Baneth, Gad

    2014-01-01

    Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania

  10. Antigen Requirements for Efficient Priming of CD8+ T Cells by Leishmania major-Infected Dendritic Cells

    Science.gov (United States)

    Bertholet, Sylvie; Debrabant, Alain; Afrin, Farhat; Caler, Elisabeth; Mendez, Susana; Tabbara, Khaled S.; Belkaid, Yasmine; Sacks, David L.

    2005-01-01

    CD4+ and CD8+ T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4+ or OT-I CD8+ T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3′ nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8+ T cells. The ability of L. major transgenic parasites to activate OT-I CD8+ T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level. PMID:16177338

  11. Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

    Science.gov (United States)

    Moreno, Elizabeth Castro; Gonçalves, Andréa Vieira; Chaves, Anderson Vieira; Melo, Maria Norma; Lambertucci, José Roberto; Andrade, Antero Silva Ribeiro; Negrão-Corrêa, Deborah; Antunes, Carlos Mauricio de Figueiredo; Carneiro, Mariângela

    2009-01-01

    Background One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. Methodology Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. Findings The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). Conclusions Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias

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

    Science.gov (United States)

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

    2008-09-01

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

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

    Science.gov (United States)

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

    2017-07-01

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

  14. Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

    Science.gov (United States)

    Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

    2013-01-11

    Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

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

    Directory of Open Access Journals (Sweden)

    Luiza de Oliveira Ramos Pereira

    2013-08-01

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

  16. Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

    Science.gov (United States)

    Carvalho, A K; Carvalho, K; Passero, L F D; Sousa, M G T; da Matta, V L R; Gomes, C M C; Corbett, C E P; Kallas, G E; Silveira, F T; Laurenti, M D

    2016-01-01

    Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

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

    Science.gov (United States)

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

    2005-04-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

    International Nuclear Information System (INIS)

    Hashim, Amna Osman Yousif

    1997-06-01

    Leishmaniasis, manifesting on it's different clinical forms is endemic in different regions of the Sudan. diagnosis of the disease in the Sudan is usually done using simple methods such as microscopical examination of slit smirs, Histological sections and cultures. Serological diagnosis using enzymes linked immunosorbent assay (ELISA)- and direct agglutination test (DAT) are sometimes used as more sensitive tool has thrown light on the epidemiology of the disease in the Sudan. This study was conducted on 126 subjects to identify the parasites-causing the different clinical manifestations, to determine the genetic diversity of different isolates of Lieshmania- and to detect parasites in the peripheral blood of subjects from highly endemic foci. The study population consisted of 7 with suspected VL, 12 with suspected ML, 14 with suspected PKDL, 2 with sporotrichoid CL and 89 healthy games wardens and army soldiers from highly endemic foci. Parasites were cultured in biphasic medium and subcultured in liquid medium until mass production was stabilized. Extraction of DNA was done using three methods which were phenol/ chloroform/ isoamylalcohol, K buffer and proteinase K as well as lysing of the parasite with distilled water. The KDNA was amplified using species namely AJSI and DeB8. The products were analysed on 1.5% agarose gel-and were visualized and photographed with U.V. transilluminator and camera. Characteristic bands of 700 and 800 b.p corresponding to the full length of mini circle of L.major and L.donovani respectively were obtained on amplification of KDNA from patients with VL and CL. In some cases lower bands of 400 and 500 b.p PKDL and multiple bands for sporotrichoid CL.Leishmania DNA was detected from the conjucativa of the eye of a patient with PKDL. The genetic diversity of Leishmania parasite was determined by digesting PCR products from PKDL, sporotrichoids CL and VL patients. Different patterns were produced for each digesting product. This result

  20. The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

    Energy Technology Data Exchange (ETDEWEB)

    Hashim, Amna Osman Yousif [Department of Zoology, Faculty of Science, University of Khartoum, Khartoum (Sudan)

    1997-06-01

    Leishmaniasis, manifesting on it`s different clinical forms is endemic in different regions of the Sudan. diagnosis of the disease in the Sudan is usually done using simple methods such as microscopical examination of slit smirs, Histological sections and cultures. Serological diagnosis using enzymes linked immunosorbent assay (ELISA)- and direct agglutination test (DAT) are sometimes used as more sensitive tool has thrown light on the epidemiology of the disease in the Sudan. This study was conducted on 126 subjects to identify the parasites-causing the different clinical manifestations, to determine the genetic diversity of different isolates of Lieshmania- and to detect parasites in the peripheral blood of subjects from highly endemic foci. The study population consisted of 7 with suspected VL, 12 with suspected ML, 14 with suspected PKDL, 2 with sporotrichoid CL and 89 healthy games wardens and army soldiers from highly endemic foci. Parasites were cultured in biphasic medium and subcultured in liquid medium until mass production was stabilized. Extraction of DNA was done using three methods which were phenol/ chloroform/ isoamylalcohol, K buffer and proteinase K as well as lysing of the parasite with distilled water. The KDNA was amplified using species namely AJSI and DeB8. The products were analysed on 1.5% agarose gel-and were visualized and photographed with U.V. transilluminator and camera. Characteristic bands of 700 and 800 b.p corresponding to the full length of mini circle of L.major and L.donovani respectively were obtained on amplification of KDNA from patients with VL and CL. In some cases lower bands of 400 and 500 b.p PKDL and multiple bands for sporotrichoid CL.Leishmania DNA was detected from the conjucativa of the eye of a patient with PKDL. The genetic diversity of Leishmania parasite was determined by digesting PCR products from PKDL, sporotrichoids CL and VL patients. Different patterns were produced for each digesting product. This result

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

    Directory of Open Access Journals (Sweden)

    Rita C Bittar

    2007-08-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Changes in Macrophage Gene Expression Associated with Leishmania (Viannia braziliensis Infection.

    Directory of Open Access Journals (Sweden)

    Clemencia Ovalle-Bracho

    Full Text Available Different Leishmania species cause distinct clinical manifestations of the infectious disease leishmaniasis. It is fundamentally important to understand the mechanisms governing the interaction between Leishmania and its host cell. Little is known about this interaction between Leishmania (Viannia braziliensis and human macrophages. In this study, we aimed to identify differential gene expression between non-infected and L. (V braziliensis-infected U937-derived macrophages. We deployed a whole human transcriptome microarray analysis using 72 hours post-infection samples and compared those samples with their non-infected counterparts. We found that 218 genes were differentially expressed between infected and non-infected macrophages. A total of 71.6% of these genes were down-regulated in the infected macrophages. Functional enrichment analyses identified the steroid and sterol/cholesterol biosynthetic processes between regulatory networks down-regulated in infected macrophages. RT-qPCR further confirmed this down-regulation in genes belonging to these pathways. These findings contrast with those from studies involving other Leishmania species at earlier infection stages, where gene up-regulation for this metabolic pathway has been reported. Sterol biosynthesis could be an important biological process associated with the expression profile of macrophages infected by L. (V. braziliensis. Differential transcriptional results suggest a negative regulation of the genetic regulatory network involved in cholesterol biosynthesis.

  5. A radioattenuated Leishmania major vaccine markedly increases the resistance of CBA mice to subsequent infection with Leishmania mexicana mexicana

    International Nuclear Information System (INIS)

    Alexander, J.

    1982-01-01

    Vaccinating CBA mice with radioattenuated Leishmania major amastigotes but not with radioattenuated L. mexicana amastigotes rendered them highly resistant to subsequent infection with L. m. mexicana. Unvaccinated CBA mice were highly susceptible to infection with L. m. mexicana producing rapidly growing non-ulcerating cutaneous lesions. Two manifestations of resistance were induced in vaccinated animals depending on the timing of the challenge infection: no lesions appeared at the site of subcutaneous challenge in animals vaccinated four or more weeks previously, while lesions grew rapidly but ulcerated and healed in animals vaccinated less than 3 weeks beforehand. L. major amastigotes were found to be markedly more resistant to γ irradiation than L. m.mexicana amastigotes both as measured by their ability to infect susceptible strains of mice and to transform and multiply as promastigotes in NNN medium. (author)

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

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

    Science.gov (United States)

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

    2016-08-19

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

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

    Science.gov (United States)

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

    2014-12-20

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

  9. Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

    Science.gov (United States)

    Saraiva, Lara; Carvalho, Gustavo M L; Gontijo, Célia M F; Quaresma, Patrícia F; Lima, Ana C V M R; Falcão, Alda L; Andrade Filho, José D

    2009-09-01

    Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (= Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (= Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

  10. NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway.

    Science.gov (United States)

    Zamora-Chimal, Jaime; Fernández-Figueroa, Edith A; Ruiz-Remigio, Adriana; Wilkins-Rodríguez, Arturo A; Delgado-Domínguez, José; Salaiza-Suazo, Norma; Gutiérrez-Kobeh, Laila; Becker, Ingeborg

    2017-02-01

    NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes. We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

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

    Science.gov (United States)

    2013-01-01

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

  12. Expression of hsa Let-7a MicroRNA of Macrophages Infected by Leishmania Major

    Directory of Open Access Journals (Sweden)

    Nooshin Hashemi

    2016-10-01

    Full Text Available Leishmaniasis is a vector-born disease caused by species of the genus Leishmania and is transmitted from host to host through the bite of an infected sandfly. MicroRNAs (miRNAs are non-coding small RNAs with 22-nucleotide length. They are involved in some biological and cellular processes. We aimed to evaluate the expression of let-7a in human macrophages miRNA when are infected by Leishmania major. We also evaluated the impact of Leishmania major infection on the expression of let-7a at two different times, 24 and 48 hours, after infection. Blood samples were collected from ten healthy volunteers with no history of leishmaniasis. Development of macrophages from peripheral monocytes and infection with stationary phase of Leishmania major promastigotes were done through serial cultures under 5% CO2 environment and 37C. To measure the expression levels of let-7a real-time PCR was performed with specific related primers using the SYBR® Green master mix Kit™. The real-time PCR showed let-7a was expressed in cells infected with parasites after 24 and 48h post-infection. Comparison of let-7a miRNA expression after 24 and 48 h revealed that let-7a miRNAs were down-regulated at 48 h post-infection more than 24h after infection. The results of this study suggest that according to the main function of miRNA in repression of mRNA translation it could be possible to manipulate host cells in order to alter miRNA levels and regulate macrophage functions after establishment of intracellular parasites such as Leishmania.

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

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    Kelly Ben L

    2010-02-01

    -specific manner, however it augments the production of other proinflammatory cytokines. Our findings highlight the complexity of inflammatory cytokine signalling regulation in the context of the macrophage and Leishmania interaction and confirm the utility of the Leishmania/macrophage infection model as an experimental system for further studies of inflammatory regulation. Such studies may advance the development of therapies against inflammatory disease.

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

    OpenAIRE

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

    2010-01-01

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

  15. Evaluation of four molecular methods to detect Leishmania infection in dogs.

    Science.gov (United States)

    Albuquerque, Andreia; Campino, Lenea; Cardoso, Luís; Cortes, Sofia

    2017-03-13

    Canine leishmaniasis, a zoonotic disease caused by Leishmania infantum vectored by phlebotomine sand flies, is considered a relevant veterinary and public health problem in various countries, namely in the Mediterranean basin and Brazil, where dogs are considered the main reservoir hosts. Not only diseased dogs but also those subclinically infected play a relevant role in the transmission of L. infantum to vectors; therefore, early diagnosis is essential, under both a clinical and an epidemiological perspective. Molecular tools can be a more accurate and sensitive approach for diagnosis, with a wide range of protocols currently in use. The aim of the present report was to compare four PCR based protocols for the diagnosis of canine Leishmania infection in a cohort of dogs from the Douro region, Portugal. A total of 229 bone marrow samples were collected from dogs living in the Douro region, an endemic region for leishmaniasis. Four PCR protocols were evaluated for Leishmania DNA detection in canine samples, three single (ITS1-PCR, MC-PCR and Uni21/Lmj4-PCR) and one nested (nested SSU rRNA-PCR). Two of the protocols were based on nuclear targets and the other two on kinetoplastid targets. The higher overall percentage of infected dogs was detected with the nested SSU rRNA-PCR (37.6%), which also was able to detect Leishmania DNA in a higher number of samples from apparently healthy dogs (25.3%). The ITS1-PCR presented the lowest level of Leishmania detection. Nested SSU rRNA-PCR is an appropriate method to detect Leishmania infection in dogs. Accurate and early diagnosis in clinically suspect as well as apparently healthy dogs is essential, in order to treat and protect animals and public health and contribute to the control and awareness of the disease.

  16. Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola.

    Science.gov (United States)

    Vilhena, Hugo; Granada, Sara; Oliveira, Ana Cristina; Schallig, Henk D F H; Nachum-Biala, Yaarit; Cardoso, Luís; Baneth, Gad

    2014-03-24

    Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection. One hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection. Although other cases of CanL have previously been described in the country, this is the first reported study of canine Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.

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

    Science.gov (United States)

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

    2008-06-01

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

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

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    Fabiana de Oliveira Lara-Silva

    2014-01-01

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

  19. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

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    Marie Samanovic

    2009-01-01

    Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

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

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    André Luiz Rodrigues Roque

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

  1. 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.

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

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    Sameh Rabhi

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

  3. Molecular characterization of leishmania infection from naturally infected sand flies caught in a focus of cutaneous leishmaniasis (eastern iran.

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    Mohammad Akhoundi

    2013-12-01

    Full Text Available Cutaneous leishmaniasis due to Leishmania major is a serious and increasing problem affecting many rural areas of 17 out of 31 provinces in Iran. Little is known about sand fly fauna and leishmaniases in Eastern Iran and no study has been carried out in Sarbisheh County. The aim of this study was to determine sand flies composition and probable Leishmania infection to find the probable vectors of leishmaniasis in Sarbisheh district.Sand flies were caught using both sticky papers and CDC light traps in August 2010. They were identified morphologically and analyzed for Leishmania infection by amplification of ITS-rDNA.Totally, 842 specimens were caught and 8 species recorded. They belonged to the genera Phlebotomus and Sergentomyia: P. (Phlebotomus papatasi, P. (Paraphlebotomus sergenti, P. (Pa. caucasicus, P. (Pa. mongolensis, P. (Pa. jacusieli, S. (Sergentomyia dentata, S. (Se. sintoni and S. (Sintonius clydei. All collected females were processed for Leishmania DNA detection by PCR amplifying of Internal Transcribed Spacer1 (partial sequence, 5.8S (complete sequence and ITS2 (partial sequence fragments. Thirteen females were positive for Leishmania DNA. The sequencing of the 430 bp amplicons indicated that 9 P. papatasi and 3 females belonging to the Caucasicus group carried L. major DNA whereas one P. sergenti carried L. tropica DNA.Phlebotomus papatasi and P. sergenti are, like in several places, the probable vectors of cutaneous leishmaniases in this emerging or unknown focus of cutaneous leishmaniases.

  4. Histopathological and parasitological investigations of ear healthy skin of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

    Science.gov (United States)

    Figueiredo, Maria Marta; Moura, Eliane Perlatto; Costa, Miriam Maria; Ribeiro, Vitor Marcio; Michalick, Marilene Suzan; Tafuri, Washington Luiz; Tafuri, Wagner Luiz

    2010-07-01

    Although 90% of clinical cases of American visceral leishmaniasis (AVL) occur in the northeastern region of Brazil, the incidence of cases in recent years has increased in southeastern states such as Minas Gerais (MG), where the disease has been reported in several cities, including Belo Horizonte, the state capital. Some studies have shown a strong correlation between the incidence of AVL and canine visceral leishmaniasis (CVL) in Belo Horizonte. A study of 108 dogs with parasite Leishmania chagasi detected by immuno-histochemistry in healthy ear skin was obtained from two distinct geographical areas: 55 from a metropolitan area of the municipality (Santa Luzia, MG) and 53 dogs from a central area of Belo Horizonte. In parallel, a group of 10 beagles were experimentally infected with L. chagasi. Considering the clinical aspects of all naturally infected dogs, symptomatic dogs were more frequent than asymptomatic ones, especially animals from the metropolitan area compared with the central area (79.6% and 20.3%, respectively). A chronic exudate was observed in the ear of 51 out of 55 dogs naturally infected from the metropolitan area (92.7%) and 45 out of 53 dogs naturally infected from the central area (84.9%). Importantly, asymptomatic dogs from the central area harbor more parasites in the skin than the asymptomatic ones from the metropolitan area. In addition, a profound difference was noted in the intensity of the inflammatory reaction and parasite load in the skin of experimental infected dogs.

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

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    Carreira João Carlos

    2012-06-01

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

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

    Science.gov (United States)

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

    2012-06-07

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

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

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    MICHALSKY Érika M.

    2002-01-01

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

  8. 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....

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

    OpenAIRE

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

    2007-01-01

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

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

    OpenAIRE

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

    2007-01-01

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

  11. Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

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    Nestor Añez

    2003-06-01

    Full Text Available A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV and the thoracic mid-gut (TMG of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG. Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads, paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.

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

    Science.gov (United States)

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

    2015-08-24

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

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

    Science.gov (United States)

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

    2016-05-10

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

  14. Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

    Science.gov (United States)

    Farias, L H S; Rodrigues, A P D; Coêlho, E C; Santos, M F; Sampaio, S C; Silva, E O

    2017-09-01

    American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

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

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    Pinto Aldair JW

    2011-12-01

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

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

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    Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-11-01

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

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

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    C. Vasconcellos

    1999-07-01

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

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

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    Rezvan, H; Rees, R; Ali, SA

    2011-01-01

    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. PMID:22347315

  19. Presence of sandflies infected with Leishmania infantum and Massilia virus in the Marseille urban area.

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    Faucher, B; Bichaud, L; Charrel, R; Mary, C; Izri, A; de Lamballerie, X; Piarroux, R

    2014-05-01

    Leishmaniasis is considered a rural disease in Europe. However, circumstantial evidence has indicated urban transmission of leishmaniasis and phleboviruses in the urban area of Marseille, France. To investigate this urban transmission, sandflies were trapped in 33 locations in the urban area (horse farms, public gardens and a residential area). Sandflies were always captured: 87.8% were Phlebotomus perniciosus, a vector of Leishmania infantum and Toscana and Massilia viruses. RT-PCR and cell culture inoculation identified the Massilia virus in 2/99 pools of sandflies, and PCR identified Leishmania in 5/99. No dual infection was observed, but both pathogens were detected in samples from the same trapping site. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  20. Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

    Czech Academy of Sciences Publication Activity Database

    Grekov, Igor; Svobodová, M.; Nohýnková, E.; Lipoldová, Marie

    2011-01-01

    Roč. 87, č. 3 (2011), s. 273-277 ISSN 0167-7012 R&D Projects: GA MŠk(CZ) LC06009; GA ČR GA310/08/1697 Institutional research plan: CEZ:AV0Z50520514 Keywords : Leishmania * SNB-9 * biphasic culture medium * parasite infectivity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.086, year: 2011

  1. Characterization of Leishmania isolates from Nepalese patients with visceral leishmaniasis.

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    Pandey, Kishor; Yanagi, Testuo; Pandey, Basu Dev; Mallik, Arun Kumar; Sherchand, Jeevan Bahadur; Kanbara, Hiroji

    2007-05-01

    In Nepal, visceral leishmaniasis (VL) is endemic in 13 districts of the central and eastern regions. A total of 166 bone-marrow aspirates were obtained from patients with suspected VL. Ninety-seven were identified as positive by microscopy, and 29 of those were successfully isolated and cultured. We characterized these isolates by molecular analysis and by their ability to infect mice. PCR-restriction fragment length polymorphism analysis of the mini-exon and the cysteine proteinase b gene showed that all isolates were Leishmania donovani, and the restriction pattern of the Nepalese isolates corresponded to the standard Indian strain of L. donovani but differed from that of the Kenyan strain. The single-strand conformation polymorphism analysis of ribosomal internal transcribed spacer showed no genetic heterogeneity within Nepalese isolates. Intraperitoneal inoculation with the promastigotes of all isolates resulted in amastigote proliferation in the spleen of 20 nude mice, of which ten isolates were highly infective, and ten were moderately infective, including one BALB/c mouse. Of the 20 amastigotes isolated from the spleen of nude mice, only the ten highly infective isolates infected BALB/c mice, of which, two isolates were considered to have low infectivity, three isolates were considered to be moderately infective, and five isolates were considered to be highly infective.

  2. Leishmania Infection: Laboratory Diagnosing in the Absence of a “Gold Standard”

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    Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

    2010-01-01

    There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2. PMID:20134001

  3. Risk of Infection with Leishmania spp. in the Canine Population in the Netherlands

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    Slappendel RJ

    2002-12-01

    Full Text Available The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania-endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey. The questionnaire was sent to 1478 at random chosen families in the Netherlands. Of the 59.0% responders 28.0% had one or more dogs and 4.8% of these dogs had visited Southern Europe during the summer period of that year. On a total population of 1,200,000 dogs in the Netherlands, this means that each year some 58,000 dogs are at risk of being exposed to a Leishmania infection in Southern Europe. During the period 1990–1992 blood was collected for serology in 1911 dogs presented to the Utrecht University Clinic because of clinical problems not related to leishmaniasis, of which 434 had been in Southern Europe in the foregoing years. None was serologically positive. From these data it can be deduced that the highest chance to obtain leishmaniasis during a vacation in Southern Europe is mathematically less than 1/434 or less than 0.23%. Serology was also performed during the period 1989–1993 in 597 dogs that had been in Southern Europe and were suspected of leishmaniasis. Titers were positive in 145 of these samples. Sixty-four of these dogs were born in the Mediterranean and had been imported into the Netherlands. Excluding these imported dogs, it was calculated that at least 0.027% of the 58,000 dogs yearly taken to Southern Europe during holidays become infected with Leishmania. In order to establish the risk of disease transmission for people in close contact with an infected dog, serum samples of owners and house mates of 37 dogs with leishmaniasis were tested. All 112 sera tested

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

    Science.gov (United States)

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

    2009-05-01

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

  5. Infection parameters in the sand fly vector that predict transmission of Leishmania major.

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    Stamper, Lisa W; Patrick, Rachel L; Fay, Michael P; Lawyer, Phillip G; Elnaiem, Dia-Eldin A; Secundino, Nagila; Debrabant, Alain; Sacks, David L; Peters, Nathan C

    2011-08-01

    To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.

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

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

    2015-01-01

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

  7. Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon.

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    Zorrilla, Victor; De Los Santos, Maxy B; Espada, Liz; Santos, Rocío Del Pilar; Fernandez, Roberto; Urquia, Albino; Stoops, Craig A; Ballard, Sarah-Blythe; Lescano, Andres G; Vásquez, Gissella M; Valdivia, Hugo O

    2017-11-01

    Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission. Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification. A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the

  8. Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon.

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    Victor Zorrilla

    2017-11-01

    Full Text Available Cutaneous leishmaniasis (CL is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission.Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR and nested Real time PCR for species identification.A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species and Brumptomyia (2 species were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia spp. (24%, Lu. whitmani (19.4% and Lu. yucumensis (15.8% in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools, Mavila (10 pools and Arca Pacahuara (1 pool and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the

  9. Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon

    Science.gov (United States)

    Zorrilla, Victor; De Los Santos, Maxy B.; Espada, Liz; Santos, Rocío del Pilar; Fernandez, Roberto; Urquia, Albino; Stoops, Craig A.; Ballard, Sarah-Blythe; Lescano, Andres G.; Vásquez, Gissella M.; Valdivia, Hugo O.

    2017-01-01

    Background Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission. Methods Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification. Results A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu

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

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

    2018-01-01

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

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

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    Helena Messlinger

    2018-01-01

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

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

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    Sreenivas eGannavaram

    2012-07-01

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

  13. Transcriptomic signature of Leishmania infected mice macrophages: a metabolic point of view.

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    Imen Rabhi

    Full Text Available We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL, inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection.

  14. Production of interferon-¿ and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein

    DEFF Research Database (Denmark)

    Kemp, M; Kurtzhals, J A; Christensen, C B

    1993-01-01

    The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani in....... The results show that both IFN-gamma producing (Th1-like) and IL-4 producing (Th2-like) T cells recognizing LPGAP are expanded after infection with L. donovani in humans....... infections. LPGAP induced vigorous proliferation and production of interferon-gamma (IFN-gamma) by the cells. In addition PBMC incubated with LPGAP released interleukin-4 (IL-4) after pulsing with ionomycin and phorbol myristate acetate. Single cells were isolated from LPGAP-stimulated cell lines...

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

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    Everton Falcão de Oliveira

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  18. The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection

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    Ana M. Montalvo-Álvarez

    2008-01-01

    Full Text Available Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP in Leishmania, belonging to the small HSP (sHSP family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.

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

    Science.gov (United States)

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

    2015-12-01

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

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

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    Mariana Rosales-Chilama

    2015-12-01

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

  1. Cell-mediated immune response to Leishmania chagasi experimental infection of BALB/c immunosuppressed mice

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    JG Machado

    2010-01-01

    Full Text Available Leishmaniasis, a zoonosis of worldwide distribution, presents a significant impact on immunosupressed patients. This study aimed to evaluate Leishmania chagasi infection in BALB/c mice immunosuppressed with dexamethasone. Spleen cells stimulated or not with L. chagasi were cultured for cytokine quantification (IFN-γ, IL-2, IL-4 and IL-10 by sandwich ELISA. Parasite loads in the spleen and liver were determined by means of culture microtitration. Immunosuppressed groups showed statistically lower spleen weight and CD4-cell percentage in blood on the day of infection and produced Th1 and Th2 cytokines on other days of the study. The other infected groups, weather immunosupressed or not, also produced Th1 and Th2 cytokines. Parasite loads in the spleen and liver were not statistically different among the groups. It was concluded that L. chagasi infection was not affected by dexamethasone-induced immunosuppression, probably due the reversible effect of the treatment.

  2. The Role of Vitamin D and Vitamin D Receptor in Immunity to Leishmania major Infection

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    James P. Whitcomb

    2012-01-01

    Full Text Available Vitamin D signaling modulates a variety of immune responses. Here, we assessed the role of vitamin D in immunity to experimental leishmaniasis infection in vitamin D receptor-deficient mice (VDRKO. We observed that VDRKO mice on a genetically resistant background have decreased Leishmania major-induced lesion development compared to wild-type (WT mice; additionally, parasite loads in infected dermis were significantly lower at the height of infection. Enzymatic depletion of the active form of vitamin D mimics the ablation of VDR resulting in an increased resistance to L. major. Conversely, VDRKO or vitamin D-deficient mice on the susceptible Th2-biased background had no change in susceptibility. These studies indicate vitamin D deficiency, either through the ablation of VDR or elimination of its ligand, 1,25D3, leads to an increase resistance to L. major infection but only in a host that is predisposed for Th-1 immune responses.

  3. Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi.

    Science.gov (United States)

    Corrêa, Ana Paula Ferreira Lopes; Dossi, Ana Cláudia Silva; de Oliveira Vasconcelos, Rosemeri; Munari, Danísio Prado; de Lima, Valéria Marçal Felix

    2007-02-28

    The aims of this study were to evaluate the immunomodulatory role of TGF-beta1, IL-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta1, IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta1 was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta1 levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta1 and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.

  4. Molecular detection and identification of Leishmania infection in naturally infected sand flies in a focus of cutaneous leishmaniasis in northern Morocco

    OpenAIRE

    Es-Sette, Nargys; Ajaoud, Malika; Laamrani-Idrissi, Abderrahman; Mellouki, Fouad; Lemrani, Meryem

    2014-01-01

    Background Cutaneous leishmaniasis is an infectious disease caused by various species of the flagellate protozoan Leishmania. During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. The main objective of this study was to study the occurrence of Leishmania infection in vectors and to identify sand fly blood meal sources in an endemic locality of cutaneous leishmaniasis within Sefrou province, where the vectors of leishmaniasis were still unkno...

  5. IL-17 mediates immunopathology in the absence of IL-10 following Leishmania major infection.

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    Claudia Gonzalez-Lombana

    2013-03-01

    Full Text Available Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.

  6. Protection mediated by chemokine CXCL10 in BALB/c mice infected by Leishmania infantum

    Science.gov (United States)

    Figueiredo, Webertty Mayk Eufrásio; Viana, Sayonara de Melo; Alves, Dorotheia Teixeira; Guerra, Priscila Valera; Coêlho, Zirlane Castelo Branco; Barbosa, Helene Santos; Teixeira, Maria Jania

    2017-01-01

    BACKGROUND Visceral leishmaniasis (VL) caused by Leishmania infantum is characterised by the loss of the ability of the host to generate an effective immune response. Chemokines have a direct involvement in the pathogenesis of leishmaniasis, causing a rapid change in the expression of these molecules during infection by Leishmania. OBJECTIVES Herein, it was investigated the role of CXCL10 in controlling infection by L. infantum. METHODS RAW 264.7 macrophages were infected with L. infantum in vitro and treated or not with CXCL10 (25, 50 and 100 ng/mL). Parasite load, as well as nitric oxide (NO), IL-4 and IL-10 production were assessed at 24 and 48 h after infection. In vivo, BALB/c mice were infected and treated or not with CXCL10 (5 μg/kg) at one, three and seven days of infection. Parasite load, IFN-g, IL-4, TGF-β and IL-10 were evaluated one, seven and 23 days post treatment. FINDINGS In vitro, CXCL10 reduced parasitic load, not dependent on NO, and inhibited IL-10 and IL-4 secretion. In vivo, CXCL10 was able to reduce the parasite load in both liver and spleen, four weeks after infection, representing a higher decrease in the number of parasites in these organs, also induced IFN-γ at day 23 after treatment, correlating with the decrease in parasite load, and reduced IL-10 and TGF-β. MAIN CONCLUSIONS This study suggests a partial protective role of CXCL10 against L. infantum, mediated by IFN-g, not dependent on NO, and with suppression of IL-10 and TGF-β. These data may provide information for the development of new approaches for future therapeutic interventions for VL. PMID:28767981

  7. Leishmania infantum, Dirofilaria spp. and other endoparasite infections in kennel dogs in central Italy

    Science.gov (United States)

    Sauda, Federica; Malandrucco, Livia; Macrì, Gladia; Scarpulla, Manuela; De Liberato, Claudio; Terracciano, Giuliana; Fichi, Gianluca; Berrilli, Federica; Perrucci, Stefania

    2018-01-01

    Prevalence and risk factors of Leishmania infantum, Dirofilaria spp. and other potentially zoonotic or canine-specific endoparasite infections were assessed in 639 kennel dogs from central Italy. To this end, individual blood and fecal samples were examined using parasitological, immunological and molecular techniques. The presence of compatible clinical pictures, as well as age and gender were considered as putative risks factors. To evaluate risk factors, multivariable analysis with logistic regression and univariable analysis with a Chi square test and a Fischer’s exact test were performed. Overall, 52.6% of dogs (95% CI 48.6-56.5) were found positive, while 39.6% of dogs (95% CI 35.8-43.5) were infected by potentially zoonotic species. Leishmania infantum and Dirofilaria repens showed prevalences of 2.5% (95% CI 1.5-4.1) and 2.8% (95% CI 1.7-4.5), respectively. The prevalence of cardiorespiratory parasites was 7.8% (95% CI 5.9-10.3) and included the species Angiostrongylus vasorum, Eucoleus aerophilus, Eucoleus boehmi and D. immitis; the latter showed a prevalence of 0.2% (95% CI 0.001-1). Intestinal parasites were significantly prevalent (38.8%, 95% CI 35-42.7) and they consisted mainly of species of major zoonotic concern, including ancylostomatids, Toxocara canis, Giardia duodenalis, Dipylidium caninum, Taeniidae, Strongyloides stercoralis and Cryptosporidium parvum. Endoparasites were significantly prevalent in clinically suspected dogs. Leishmania infantum and cardiorespiratory nematodes were prevalent in older dogs, while intestinal parasites were prevalent in younger dogs. Results show high dog and public health risks in kennels in central Italy, and suggest the need for more effective control measures. PMID:29388550

  8. Leishmania infantum, Dirofilaria spp. and other endoparasite infections in kennel dogs in central Italy

    Directory of Open Access Journals (Sweden)

    Sauda Federica

    2018-01-01

    Full Text Available Prevalence and risk factors of Leishmania infantum, Dirofilaria spp. and other potentially zoonotic or canine-specific endoparasite infections were assessed in 639 kennel dogs from central Italy. To this end, individual blood and fecal samples were examined using parasitological, immunological and molecular techniques. The presence of compatible clinical pictures, as well as age and gender were considered as putative risks factors. To evaluate risk factors, multivariable analysis with logistic regression and univariable analysis with a Chi square test and a Fischer’s exact test were performed. Overall, 52.6% of dogs (95% CI 48.6-56.5 were found positive, while 39.6% of dogs (95% CI 35.8-43.5 were infected by potentially zoonotic species. Leishmania infantum and Dirofilaria repens showed prevalences of 2.5% (95% CI 1.5-4.1 and 2.8% (95% CI 1.7-4.5, respectively. The prevalence of cardiorespiratory parasites was 7.8% (95% CI 5.9-10.3 and included the species Angiostrongylus vasorum, Eucoleus aerophilus, Eucoleus boehmi and D. immitis; the latter showed a prevalence of 0.2% (95% CI 0.001-1. Intestinal parasites were significantly prevalent (38.8%, 95% CI 35-42.7 and they consisted mainly of species of major zoonotic concern, including ancylostomatids, Toxocara canis, Giardia duodenalis, Dipylidium caninum, Taeniidae, Strongyloides stercoralis and Cryptosporidium parvum. Endoparasites were significantly prevalent in clinically suspected dogs. Leishmania infantum and cardiorespiratory nematodes were prevalent in older dogs, while intestinal parasites were prevalent in younger dogs. Results show high dog and public health risks in kennels in central Italy, and suggest the need for more effective control measures.

  9. Detection of Leishmania infantum DNA in hamsters infested with ticks collected from naturally infected dogs

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    Valter dos Anjos Almeida

    2016-12-01

    Full Text Available ABSTRACT. Almeida V. dos A., da Hora T.N., Leça Júnior N.F., Carvalho F.S., da Silva A.L., Wenceslau A.A., Albuquerque G.R. & Silva F.L. Detection of Leishmania infantum DNA in hamsters infested with ticks collected from naturally infected dogs. [Detecção do DNA de Leishmania infantum em hamsters infestados com carrapatos coletados de cães naturalmente infectados.] Revista Brasileira de Medicina Veterinária, 38(4:329-333, 2016. Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Hospital Veterinário, Km 16, Rodovia Jorge Amado, Ilhéus, BA 45662-900, Brasil. E-mail: fabiana.lessa@gmail.com The aim of this study was to investigate the role of Rhipicephalus sanguineus, the brown dog tick, in the transmission of Leishmania infantum. To accomplish this, we used 24 adult golden hamsters of both genders, and divided them into two groups: a control group (n = 4 and an experimental group (n = 20. The animals from the experimental group were infested with ticks obtained from dogs naturally infected with L. infantum. Hamsters of the control group were not infested and were maintained at the same conditions, as the infested animals. After three months of observation, animals were euthanized and they were posted to obtain samples of their blood, spleen, liver, lymph nodes, and skin. These samples were then processed by histopathology, immunohistochemistry, and polymerase chain reaction (PCR. Fourteen hamsters (70% of the experimental group tested PCR-positive for L. infantum DNA in samples of buffy coat. The results of this study indicated that R. sanguineus ticks can transmit some forms or parts of L. infantum to parasitized hamsters.

  10. [Infecting glial cells with antimony resistant Leishmania tropica: A new ex-vivo model].

    Science.gov (United States)

    Zorbozan, Orçun; Harman, Mehmet; Evren, Vedat; Erdoğan, Mümin Alper; Kılavuz, Aslı; Tunalı, Varol; Çavuş, İbrahim; Yılmaz, Özlem; Özbilgin, Ahmet; Turgay, Nevin

    2018-01-01

    Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote

  11. Leishmania infection and blood food sources of phlebotomines in an area of Brazil endemic for visceral and tegumentary leishmaniasis

    Science.gov (United States)

    Guimarães-e-Silva, Antônia Suely; Silva, Soraia de Oliveira; Ribeiro da Silva, Rosa Cristina; Pinheiro, Valéria Cristina Soares; Rebêlo, José Manuel Macário; Melo, Maria Norma

    2017-01-01

    The aims of the study were to determine the blood feeding preferences of sandflies and to identify species of Leishmania that infected phlebotomines in Caxias, Maranhão, Brazil, an area that is highly endemic for leishmaniasis. Sandflies were captured in light traps located in the peridomiciliary environments of randomly selected houses in urban and rural settings between 1800 and 0600 hours on new moon days between March 2013 and February 2015. DNA extracts from 982 engorged female sandflies were submitted to fragment length polymorphism analysis to identify infecting species of Leishmania, and blood sources were identified for 778 of these specimens. Infection by Leishmania infantum was detected in Lutzomyia longipalpis, Lu. whitmani and Lu. termitophila; L. infantum/L. braziliensis in Lu. longipalpis, Lu. whitmani and Lu. trinidadensis; L. shawi in Lu. longipalpis; L. mexicana in Lu. longipalpis; L. braziliensis in Lu. longipalpis and Lu. whitmani; L. guyanensis in Lu. longipalpis and Lu. termitophila; L. amazonensis in Lu. longipalpis and L. lainsoni or L. naiffi in Lu. longipalpis, while Lu. longipalpis and Lu. trinidadensis were infected with unidentified Leishmania sp. Blood sources were identified in 573 individual phlebotomines and the preferred hosts were, in decreasing order, chicken, dog, rodent and human with lower preferences for pig, horse, opossum and cattle. Lu. longipalpis and Lu. whitmani performed mixed feeding on man, dog and rodent, while Lu. longipalpis was the most opportunistic species, feeding on the blood of all hosts surveyed, but preferably on dog/chicken, dog/rodent and rodent/chicken. Our findings reveal the concomitant circulation of Leishmania species that cause visceral leishmaniasis and tegumentary leishmaniasis in the study area, and explain the occurrence of autochthonous human cases of both clinical forms of leishmaniasis in Caxias, Maranhão. The results support our hypothesis that, in the municipality of Caxias, transmission

  12. Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain.

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    Fernández-Bellon, H; Solano-Gallego, L; Rodríguez-Cortés, A; Ferrer, L; Gallego, M; Alberola, J; Ramis, A

    2008-08-01

    Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.

  13. Diagnosis of Leishmania (Leishmania chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil

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    Julio Gomes Bigeli

    2012-02-01

    Full Text Available INTRODUCTION: The aim of the present study was to identify the presence of Leishmania (Leishmania chagasi infection in dogs in the City of Palmas, Tocantins, Brazil, using the PCR technique to list the hot spots of infected dogs in the city and associate their occurrence to significant environmental changes at capture sites. METHODS: DNA was extracted from blood of dogs, and the PCR were performed with primers RV1/RV2. After screening the population studied, the regions of the city that had the highest occurrence of canine infection were detected. These sites were visited, and ecological parameters denoting anthropogenic disturbance were evaluated. RESULTS: Some important features were listed in the regions visited, such as low urbanization, lack of public collection of sewage, limited garbage collection, vacant lots with tall vegetation, decaying organic matter, and, most importantly, the occurrence of stray dogs and poultry in homes. CONCLUSIONS: The methodology for screening the population was very efficient, especially in evaluating a large number of individuals in a short time, with a high degree of automation. The results indicate an association between the observed parameters and the occurrence of infection in dogs. The model presented in the city is ideal for studies of disease progression and expansion and for the evaluation of control measures adopted for canine VL.

  14. Experimental Infection of Lutzomyia (Nyssomyia) whitmani (Diptera: Psychodidae: Phlebotominae) With Leishmania (Viannia) braziliensis and Leishmania (L.) amazonensis, Etiological Agents of American Tugumentary Leishmaniasis.

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    Fonteles, Raquel S; Pereira Filho, Adalberto A; Moraes, Jorge L P; Kuppinger, Oliver; Rebêlo, José M M

    2016-01-01

    Leishmania (L.) amazonensis (Lainson & Shaw, 1972) and Leishmania (Viannia) braziliensis (Vianna, 1911) are the principal causative agents of American tegumentary leishmaniasis (ATL) in Brazil. L. amazonensis also causes diffuse cutaneous leishmaniasis (DCL) vectored principally by Lutzomyia flaviscutellata and secondarily by Lutzomyia whitmani (Antunes & Coutinho, 1939). The latter is the most common phlebotomine in the state of Maranhão, and it is the focal species for potential ATL transmission. For this reason, we tested the ability of L. whitmani to become infected with Lutzomyia parasites. Phlebotomines were derived from a colony maintained in the laboratorial conditions. The first generation, uninfected females were offered a bloodmeal with mice infected with the strains of both parasites. We found that L. whitmani can become infected with both parasite species, with infection rates of 65.2% (L. braziliensis) and 47.4% (L. amazonensis). We conclude that in Maranhão, L. whitmani is likely an important vector in the transmission of ATL and may function as a vector of DCL. This possibility should be further investigated. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections.

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    Omar A Saldarriaga

    2016-04-01

    Full Text Available Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V. braziliensis, L. (V. panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V. braziliensis (n = 33, L. (V. guyanensis (n = 17, L. (V. panamensis (n = 9. The less common L. (V. lainsoni, L. (V. shawi, and L. (V. naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania subgenus. In a small number of clinical samples (n = 13 we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.

  16. 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.

  17. Leishmania infantum and Epstein-Barr virus co-infection in a patient with hemophagocytosis

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    Zied Gaifer

    2017-01-01

    Full Text Available The authors describe a rare case of a 27-year old previously healthy male presenting with high grade fever, pancytopenia, hepatosplenomegaly, high levels of ferritin and triglyceride, suggesting a diagnosis of hemophagocytic lymphohistiocytosis (HLH syndrome. Other investigations showed a positive Leishmania infantum serology and high Epstein-Barr virus (EBV viremia. The diagnosis of a visceral leishmaniasis was confirmed by bone morrow biopsy, which showed Leishman-Donovan bodies and evidence of HLH. The patient received liposomal amphotericin B and he had a complete resolution of his symptoms and clearance of EBV viremia. This case of HLH associated with visceral leishmaniasis and EBV co-infection raises the question about the significance of EBV in patients with HLH. The treatment of actual etiological agent can lead to complete cure while using current recommend chemotherapy for HLH-related EBV in a patient with hidden infection may have deleterious effects.

  18. Cytokine and Phenotypic Cell Profiles of Leishmania infantum Infection in the Dog

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    Carla Maia

    2012-01-01

    Full Text Available Leishmaniasis has reemerged in recent years showing a wider geographic distribution and increased global incidence of human and canine disease than previously known. Dogs are the main domestic/peridomestic reservoir hosts of zoonotic visceral leishmaniasis caused by Leishmania infantum. Since the evolution of leishmaniasis and clinical appearance is a consequence of complex interactions between the parasite and host immune response, a profound knowledge about the immune profile developed in dog's infection is crucial for vaccine and immunomodulatory therapy design. The main goal of this paper is to compile the recent advances made on cytokine and phenotypic cell profiles in different tissues and organs of dogs infected with L. infantum. This paper also stressed that the knowledge of the immune responses developed, namely, in liver, lymph node, and spleen is very limited. All data emphasizes that more research on canine leishmaniasis is necessary for the development of new and efficacious tools to control zoonotic leishmaniasis.

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

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

    2013-01-01

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

  20. Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

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    Bichaud, Laurence; Souris, Marc; Mary, Charles; Ninove, Laëtitia; Thirion, Laurence; Piarroux, Raphaël P; Piarroux, Renaud; De Lamballerie, Xavier; Charrel, Rémi N

    2011-09-01

    Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between

  1. Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

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    Laurence Bichaud

    2011-09-01

    Full Text Available Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus, an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i in individuals and (ii at a spatial level in the city of Marseille (south-eastern France. Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of

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

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

    2015-11-21

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

  3. Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

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    Stella Maria Barrouin-Melo

    2004-03-01

    Full Text Available The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test in symptomatic (28 out of 33 animals than in asymptomatic animals (19 out of 31 animals. These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

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

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    Lenilton Silva da Silveira-Júnior

    2017-01-01

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

  5. Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

    Science.gov (United States)

    Costa, Rúbia S; Carvalho, Lucas P; Campos, Taís M; Magalhães, Andréa S; Passos, Sara T; Schriefer, Albert; Silva, Juliana A; Lago, Ednaldo; Paixão, Camilla S; Machado, Paulo; Scott, Phillip; Carvalho, Edgar M

    2018-02-14

    Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  6. Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp.

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    Yuan, Dongmei; Qin, Hanxiao; Zhang, Jianguo; Liao, Lin; Chen, Qiwei; Chen, Dali; Chen, Jianping

    2017-02-01

    Leishmaniasis is a worldwide epidemic disease caused by the genus Leishmania, which is still endemic in the west and northwest areas of China. Some viewpoints of the traditional taxonomy of Chinese Leishmania have been challenged by recent phylogenetic researches based on different molecular markers. However, the taxonomic positions and phylogenetic relationships of Chinese Leishmania isolates remain controversial, which need for more data and further analysis. In this study, the heat shock protein 70 (HSP70) gene and cytochrome b (cyt b) gene were used for phylogenetic analysis of Chinese Leishmania isolates from patients, dogs, gerbils, and sand flies in different geographic origins. Besides, for the interesting Leishmania sp. in China, the ultrastructure of three Chinese Leishmania sp. strains (MHOM/CN/90/SC10H2, SD, GL) were observed by transmission electron microscopy. Bayesian trees from HSP70 and cyt b congruently indicated that the 14 Chinese Leishmania isolates belong to three Leishmania species including L. donovani complex, L. gerbilli, and L. (Sauroleishmania) sp. Their identity further confirmed that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China is closely related to L. tarentolae. The phylogenetic results from HSP70 also suggested the classification of subspecies within L. donovani complex: KXG-918, KXG-927, KXG-Liu, KXG-Xu, 9044, SC6, and KXG-65 belong to L. donovani; Cy, WenChuan, and 801 were proposed to be L. infantum. Through transmission electron microscopy, unexpectedly, the Golgi apparatus were not observed in SC10H2, SD, and GL, which was similar to previous reports of reptilian Leishmania. The statistical analysis of microtubule counts separated SC10H2, SD, and GL as one group from any other reference strain (L. donovani MHOM/IN/80/DD8; L. tropica MHOM/SU/74/K27; L. gerbilli MRHO/CN/60/GERBILLI). The ultrastructural characteristics of Leishmania sp. partly lend support to the phylogenetic inference that

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

    Science.gov (United States)

    Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

    2016-02-01

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

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

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    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.

  9. Pathology of dogs in Campo Grande, MS, Brazil naturally co-infected with Leishmania infantum and Ehrlichia canis

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    Gisele Braziliano Andrade

    2014-12-01

    Full Text Available Different parasites that commonly occur concomitantly can influence one another, sometimes with unpredictable effects. We evaluated pathological aspects of dogs naturally co-infected with Leishmania infantum and Ehrlichia canis. The health status of the dogs was investigated based on histopathological, hematological and biochemical analyses of 21 animals infected solely with L. infantum and 22 dogs co- infected with L. infantum and E. canis. The skin of both groups showed chronic, predominantly lymphohistioplasmacytic inflammatory reaction. The plasmacytosis in the lymphoid tissues was likely related with the hypergammaglobulinemia detected in all the dogs. The disorganization of extracellular matrix found in the reticular dermis of the inguinal region and ear, characterized by the substitution of thick collagen fibers for thin fibers, was attributed to the degree of inflammatory reaction, irrespective of the presence of parasites. In addition, the histopathological analysis revealed that twice as many dogs in the co-infected group presented Leishmania amastigotes in the ear skin than those infected solely with Leishmania, increasing the possibility of becoming infected through sand fly vectors. Our findings highlight the fact that the health of dogs infected concomitantly with L. infantum and E. canis is severely compromised due to their high levels of total plasma protein, globulins, alkaline phosphatase and creatine kinase, and severe anemia.

  10. Temporal and spatial distribution of Leishmania mexicana infections in a population of Neotoma micropus

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    Raymond Russell W

    2003-01-01

    Full Text Available A 19-month mark-release-recapture study of Neotoma micropus with sequential screening for Leishmania mexicana was conducted in Bexar County, Texas, USA. The overall prevalence rate was 14.7% and the seasonal prevalence rates ranged from 3.8 to 26.7%. Nine incident cases were detected, giving an incidence rate of 15.5/100 rats/year. Follow-up of 101 individuals captured two or more times ranged from 14 to 462 days. Persistence of L. mexicana infections averaged 190 days and ranged from 104 to 379 days. Data on dispersal, density, dispersion, and weight are presented, and the role of N. micropus as a reservoir host for L. mexicana is discussed.

  11. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Moreno, Elizabeth Castro; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela; Fernandes, Octavio

    2002-01-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with 32 P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with 32 P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  12. Effects of trans-stilbene and terphenyl compounds on different strains of Leishmania and on cytokines production from infected macrophages.

    Science.gov (United States)

    Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Giacomini, Elisa; Roberti, Marinella; Colomba, Claudia; Cascio, Antonio; Tolomeo, Manlio

    2018-01-01

    Most of the antileishmanial modern therapies are not satisfactory due to high toxicity or emergence of resistance and high cost of treatment. Previously, we observed that two compounds of a small library of trans-stilbene and terphenyl derivatives, ST18 and TR4, presented the best activity and safety profiles against Leishmania infantum promastigotes and amastigotes. In the present study we evaluated the effects of ST18 and the TR4 in 6 different species of Leishmania and the modifications induced by these two compounds in the production of 8 different cytokines from infected macrophages. We observed that TR4 was potently active in all Leishmania species tested in the study showing a leishmanicidal activity higher than that of ST18 and meglumine antimoniate in the most of the species. Moreover, TR4 was able to decrease the levels of IL-10, a cytokine able to render the host macrophage inactive allowing the persistence of parasites inside its phagolysosome, and increase the levels of IL-1β, a cytokine important for host resistance to Leishmania infection by inducible iNOS-mediated production of NO, and IL-18, a cytokine implicated in the development of Th1-type immune response. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Serum chemistry reference values for the common genet (Genetta genetta): variations associated with Leishmania infantum infection.

    Science.gov (United States)

    Millán, Javier; Chirife, Andrea D; Altet, Laura

    2015-03-01

    The role of wildlife in the epidemiology of leishmaniosis in under debate, and determining whether infection with Leishmania infantum causes illness in wild carnivores is important to determine its potential role as a reservoir. To provide for the first time serum biochemistry reference values for the common genet (Genetta genetta), and to determine variations associated with L. infantum infection. Twenty-five serum biochemistry parameters were determined in 22 wild-caught genets. Blood samples were analyzed for L. infantum DNA by means of real-time polymerase chain reaction (PCR). Two female genets were positive for L. infantum DNA but did not show any external clinical sign upon physical examination. Among other variations in the biochemistry values of these genets, one presented a higher concentration of gamma-globulins and cholesterol, whereas the other genet presented increased creatinine, bilirubin, and chloride levels when compared to uninfected females. Sex-related differences in some parameters were also reported. Infection with L. infantum may sometimes be accompanied by abnormal serum biochemistry in wild carnivores. Clinical disease may occur in L. infantum-infected wild carnivores. This has implications in the epidemiology of leishmaniosis. In addition, the data provided here would also be useful as reference values for researchers or rehabilitators working with the common genet.

  14. First Human Cases of Leishmania (Viannia) lainsoni Infection and a Search for the Vector Sand Flies in Ecuador.

    Science.gov (United States)

    Kato, Hirotomo; Bone, Abdon E; Mimori, Tatsuyuki; Hashiguchi, Kazue; Shiguango, Gonzalo F; Gonzales, Silvio V; Velez, Lenin N; Guevara, Angel G; Gomez, Eduardo A; Hashiguchi, Yoshihisa

    2016-05-01

    An epidemiological study of leishmaniasis was performed in Amazonian areas of Ecuador since little information on the prevalent Leishmania and sand fly species responsible for the transmission is available. Of 33 clinical specimens from patients with cutaneous leishmaniasis (CL), causative parasites were identified in 25 samples based on cytochrome b gene analysis. As reported previously, Leishmania (Viannia) guyanensis and L. (V.) braziliensis were among the causative agents identified. In addition, L. (V.) lainsoni, for which infection is reported in Brazil, Bolivia, Peru, Suriname, and French Guiana, was identified in patients with CL from geographically separate areas in the Ecuadorian Amazon, corroborating the notion that L. (V.) lainsoni is widely distributed in South America. Sand flies were surveyed around the area where a patient with L. (V.) lainsoni was suspected to have been infected. However, natural infection of sand flies by L. (V.) lainsoni was not detected. Further extensive vector searches are necessary to define the transmission cycle of L. (V.) lainsoni in Ecuador.

  15. Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

    Science.gov (United States)

    Barrera, Maria Claudia; Rojas, Laura Jimena; Weiss, Austin; Fernandez, Olga; McMahon-Pratt, Diane; Saravia, Nancy G; Gomez, Maria Adelaida

    2017-12-01

    The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results

  16. Digital holographic microscopy as a technique to monitor macrophages infected by leishmania

    Science.gov (United States)

    Mendoza-Rodríguez, E.; Organista-Castelblanco, C.; Camacho, M.; Monroy-Ramírez, F.

    2017-06-01

    The Digital Holographic Microscopy in Transmission technique (DHM) is considered a useful tool in the noninvasive quantifying of transparent biological objects like living cells. In this work, we propose this technique to study and to monitor control macrophages infected by Leishmania (mouse lineJ774.A1). When the promastigotes enter in contact with healthy macrophages, they got phagocytosed and latterly confined in the formed parasitophorous vacuole. These processes change the morphology and density of the host macrophage. Both parameters can be measured in a label-free analysis of cells with the aid of the DHM technique. Our technique begins with the optical record of the holograms using a modified Mach-Zehnder interferometer and the reconstruction of the complex optical field transmitted by macrophages. In the latter point, we employ the angular spectrum algorithm. With the complex optical field reconstruction, we compute the field amplitude and the phase difference maps, which leads to describe one morphological characterization for the samples. Using phase difference maps is possible to measure internal variations for the integral refractive index, estimating the infection level of macrophages. Through the changes in the integral refractive index, it is also possible to describe and quantify in two different states the evolution of the infection. With these results some parameters of cells have been quantified, making the DHM technique a viable tool for diagnosis of biological samples under the presence of some pathogen.

  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

    Science.gov (United States)

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

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  19. Immunization with LJM11 salivary protein protects against infection with Leishmania braziliensis in the presence of Lutzomyia longipalpis saliva.

    Science.gov (United States)

    Cunha, Jurema M; Abbehusen, Melissa; Suarez, Martha; Valenzuela, Jesus; Teixeira, Clarissa R; Brodskyn, Cláudia I

    2018-01-01

    Leishmania is transmitted in the presence of sand fly saliva. Protective immunity generated by saliva has encouraged identification of a vector salivary-based vaccine. Previous studies have shown that immunization with LJM11, a salivary protein from Lutzomyia longipalpis, is able to induce a Th1 immune response and protect mice against bites of Leishmania major-infected Lutzomyia longipalpis. Here, we further investigate if immunization with LJM11 recombinant protein is able to confer cross-protection against infection with Leishmania braziliensis associated with salivary gland sonicate (SGS) from Lutzomyia intermedia or Lu. longipalpis. Mice immunized with LJM11 protein exhibited an increased production of anti-LJM11 IgG, IgG1 and IgG2a and a DTH response characterized by an inflammatory infiltrate with the presence of CD4 + IFN-γ + T cells. LJM11-immunized mice were intradermally infected in the ear with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia SGS. A significant reduction of parasite numbers in the ear and lymph node in the group challenged with L. braziliensis plus Lu. longipalpis SGS was observed, but not when the challenge was performed with L. braziliensis plus Lu. intermedia SGS. A higher specific production of IFN-γ and absence of IL-10 by lymph node cells were only observed in LJM11 immunized mice after infection. After two weeks, a similar frequency of CD4 + IFN-γ + T cells was detected in LJM11 and BSA groups challenged with L. braziliensis plus Lu. longipalpis SGS, suggesting that early events possibly triggered by immunization are essential for protection against Leishmania infection. Our findings support the specificity of saliva-mediated immune responses and reinforce the importance of identifying cross-protective salivary antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

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    José Cláudio Carneiro de Freitas

    2012-02-01

    Full Text Available INTRODUCTION: Canine visceral leishmaniasis (CVL is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ were used, selected by immunofluorescence assay (IFA and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85 were grouped according to clinical signs: negative (CN = 7, subclinical (CS = 10, and clinical (CC = 68. Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%, keratitis (61.8%, and lymphadenopathy (55.9%, and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%, hematocrit (72%, and hemoglobin (62%, as well as leukocytosis (33%, neutropenia (28%, thrombocytopenia (50%, uremia (45%, hyperproteinemia (53%, p<0.05, hypergammaglobulinemia (62%, p<0.01, and hypoalbuminemia (58%. CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported.

  1. Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

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    Raphael De Souza Vasconcellos

    2014-11-01

    Full Text Available Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of

  2. Leishmania infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase-2 is an Apyrase Involved in Macrophage Infection and Expressed in Infected Dogs

    Science.gov (United States)

    Vasconcellos, Raphael De Souza; Mariotini-Moura, Christiane; Gomes, Rodrigo Saar; Serafim, Tiago Donatelli; Firmino, Rafaela de Cássia; Silva e Bastos, Matheus; de Castro, Felipe Freitas; de Oliveira, Claudia Miranda; Borges-Pereira, Lucas; de Souza, Anna Cláudia Alves; de Souza, Ronny Francisco; Gómez, Gabriel Andres Tafur; Pinheiro, Aimara da Costa; Maciel, Talles Eduardo Ferreira; Silva-Júnior, Abelardo; Bressan, Gustavo Costa; Almeida, Márcia Rogéria; Baqui, Munira Muhammad Abdel; Afonso, Luís Carlos Crocco; Fietto, Juliana Lopes Rangel

    2014-01-01

    Background Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. Methodology/Principal Findings We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. Conclusions/Significance In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in

  3. Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi.

    Science.gov (United States)

    Lage, R S; Oliveira, G C; Busek, S U; Guerra, L L; Giunchetti, R C; Corrêa-Oliveira, R; Reis, A B

    2007-01-15

    Recent studies suggest that asymptomatic dogs infected with canine visceral leishmaniasis (CVL) develop a Th1 immunological profile whilst oligosymptomatic and symptomatic CVL-infected animals present a Th2 profile. In the present study, an RT-PCR method has been standardised and employed to evaluate the frequency and the semi-quantitative level of expression of the cytokines IL-4, IL-10, IL-12, INF-gamma and TNF-alpha in splenocytes of 30 dogs naturally infected with Leishmania chagasi and of 7 non-infected dogs (NID). An increase in the level of expression of IL-12 (p=0.059) was detected in all CVL-infected dogs compared with NID. In dogs exhibiting high parasitism, the frequency of expression of IL-10 was higher (p=0.011) than in animals presenting low parasitism or medium parasitism (MP) and in NID animals, whilst the level of expression of IL-10 was higher (p=0.0094) than in animals exhibiting MP and in the NID group. Positive correlations between the levels of expression of IL-10 with respect to the progression of the disease (IL-10: r=0.3510; p=0.0337) and the levels of expression of IL-10 and INF-gamma increase in parasitism (IL-10: r=0.3428; p=0.0438 and INF-gamma: r=0.4690; p=0.0045) were observed. Such data suggest that CVL is marked by a balanced production of Th1 and Th2 cytokines, with a predominant accumulation of IL-10 as a consequence of an increase in parasitic load and progression of the disease, and INF-gamma was related with the increase in parasitic load.

  4. Leishmania mexicana amazonensis: heterogeneity in 5-nucleotidase and peroxidase activities of mononuclear phagocytes during in vivo and in vitro infection

    OpenAIRE

    Côrte-Real, Suzana; Grimaldi Junior, Gabriel; Meirelles, Maria de Nazareth Leal de

    1988-01-01

    The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonenesis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastrctural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells, and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. onl...

  5. Survey of Wild and Domestic Mammals for Infection with Leishmania infantum following an Outbreak of Desert Zoonotic Visceral Leishmaniasis in Jiashi, People's Republic of China.

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    Chun-Hua Gao

    Full Text Available In 2008 and 2009, an outbreak of desert-subtype zoonotic visceral leishmaniasis occurred in Jiashi county, Xinjiang, China. So far, no animal reservoir has been identified for this type of visceral leishmaniasis. Therefore, we surveyed the most common mammals (wild and domestic for Leishmania infections during the outbreak in 2008 and 2009 in order to identify the source of the visceral leishmaniasis in this region. Spleen, liver, bone marrow and blood samples collected from 86 wood mice (Apodemus sylvaticus, 61midday jirds (Meriones meridianus and 27 Yarkand hares (Lepus yarkandensis were tested for the presence of Leishmania by microscopy, culture and PCR. All of the animals were found to be negative for Leishmania infections; On the other hand, Leishmania DNA was detected in blood samples collected from livestock reared in the outbreak area: 30.36% (17/56 of sheep, 21.57% (11/51 of goats, 17.78% (8/45 of cattle, and 21.62 (8/37 of donkeys were positive for Leishmania DNA by PCR. The amplified kDNA sequences from the livestock samples matched Leishmania DNA sequences isolated from patients with visceral leishmaniasis in the study area. We suggest that these domestic mammals are a possible reservoir host for Leishmania infantum in the outbreak area.

  6. First molecular detection of Leishmania major within naturally infected Phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern Iran.

    Science.gov (United States)

    Azizi, K; Fakoorziba, M R; Jalali, M; Moemenbellah-Fard, M D

    2012-03-01

    Human cutaneous leishmaniasis (CL) is a major notifiable public health problem in many parts of Iran. It is often caused by the zooflagellate parasite Leishmania major which is mainly transmitted by the bites of female phlebotomine sandflies belonging to the genus Phlebotomus (Diptera: Psychodidae). The annual incidence of CL in Fars province, southern Iran, was about 108-144 in 2007. The leishmanial infections of wild sandflies that may act as vectors were thus investigated at an endemic focus in this province. In all 330 female Phlebotomus sandflies were screened for the detection of Leishmania-specific kinetoplast DNA (kDNA) by polymerase chain reaction (PCR) methods. A two stage nested PCR protocol was used to establish the identity of Leishmania major species in naturally infected sandflies. The L. major kDNA was detected in 18 (5.5%) individual sandflies which belonged to four different Phlebotomus species (Phlebotomus papatasi, Phlebotomus salehi, Phlebotomus sergenti and P. major group). For the first time, one naturally infected P. salehi specimen contained the kDNA of the protozoan parasite, L. major, with a main band of 560 base pairs identified using the nested PCR method. It seems most likely therefore that P. salehi is potentially a rare sylvatic vector of L. major parasites in parts of this province. This is the first combined morphological and molecular studies of P. salehi in Iran.

  7. Case report of canine co-infection with Leishmania infantum and Ehrlichia canis

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    Stefanovska Jovana

    2011-05-01

    Full Text Available Canine leishmaniasis (CanL due to Leishmania infantum and canine monocytic ehrilichiosis (CME due to Ehrlichia canis are common diseases with zoonotic potential in the Mediterranean area. Their prevalence in R. Macedonia as a neighboring Mediterranean county is expected. In both diseases similar clinical symptoms can be manifested in dogs such as: lethargy, anorexia, weight loss, epistaxis, fever, pale mucous membranes, enlarged lymph nodes, splenomegaly, ocular signs. This case report present an atypical case of 11 year old female Samoyed with starting single clinical symptom epistaxys. Initial diagnostic procedures revealed the presence only of CanL, which was diagnosed using indirect immunofluorescence method and ELISA. First laboratory findings showed normal hematological and renal profiles. Dog was put on a treatment with Allopurinol (20mg/kg, p/o for at least 9 months. Termination of the therapy after 6 months brought a numerous clinical symptoms involving weakness, dehydration, pale mucous membranes lost pupilar reflex, uremic breath and biochemical parameters revealed a renal failure. Using a commercial ELISA kit Ehrlichia canis as a co infection was diagnosed. Most probably the second infectious agent was induced in the past 6 months, causing more severe pathological effects than CanL infection alone.

  8. Evidence of Leishmania infantum Infection in Rabbits (Oryctolagus cuniculus in a Natural Area in Madrid, Spain

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    Nerea García

    2014-01-01

    Full Text Available Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT and direct (PCR, culture techniques. Fifty-seven (82.6% of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

  9. Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

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    Mauro Maciel de Arruda

    2016-01-01

    Full Text Available In Brazil, human and canine visceral leishmaniasis (CVL caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA and indirect immunofluorescence assays (IFA as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA, decreased sensitivity (83.3% and increased specificity (92.5% were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

  10. Lutzomyia sand fly diversity and rates of infection by Wolbachia and an exotic Leishmania species on Barro Colorado Island, Panama.

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    Jorge Azpurua

    2010-03-01

    Full Text Available Sand flies (Diptera, Psychodidae, Phlebotominae in the genus Lutzomyia are the predominant vectors of the protozoan disease leishmaniasis in the New World. Within the watershed of the Panama Canal, the cutaneous form of leishmaniasis is a continuous health threat for residents, tourists and members of an international research community. Here we report the results of screening a tropical forest assemblage of sand fly species for infection by both Leishmania and a microbe that can potentially serve in vector population control, the cytoplasmically transmitted rickettsia, Wolbachia pipientis. Knowing accurately which Lutzomyia species are present, what their evolutionary relationships are, and how they are infected by strains of both Leishmania and Wolbachia is of critical value for building strategies to mitigate the impact of this disease in humans.We collected, sorted and then used DNA sequences to determine the diversity and probable phylogenetic relationships of the Phlebotominae occurring in the understory of Barro Colorado Island in the Republic of Panama. Sequence from CO1, the DNA barcoding gene, supported 18 morphology-based species determinations while revealing the presence of two possible "cryptic" species, one (Lu. sp. nr vespertilionis within the Vespertilionis group, the other (Lu. gomezi within the Lutzomyia-cruciata series. Using ITS-1 and "minicircle" primers we detected Leishmania DNA in 43.3% of Lu. trapidoi, 26.3% of Lu. gomezi individuals and in 0% of the other 18 sand fly species. Identical ITS-1 sequence was obtained from the Leishmania infecting Lu. trapidoi and Lu. gomezi, sequence which was 93% similar to Leishmania (viannia naiffi in GenBank, a species previously unknown in Panama, but recognized as a type of cutaneous leishmaniasis vectored broadly across northern and central South America. Distinct strains of the intracellular bacterium Wolbachia were detected in three of 20 sand fly species, including Lu. trapidoi

  11. Leishmania (Viannia braziliensis infection in two Colombian dogs: a note on infectivity for sand flies and response to treatment

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    Bruno L. Travi

    2006-10-01

    Full Text Available Introduction. Although canine cutaneous leishmaniasis has been reported in several foci of South America, no published information from Colombia is available. Objective. We report on two cases found in the Pacific coast region of this country, which presented as a single scrotal ulcer in one dog, and two ulcers on the external surface of the ear in a second dog. Materials and methods. Parasites were isolated by culture in Senekjie’s culture medium and identified using monoclonal antibodies. The capacity of these dogs to transmit the parasites to sand fly vectors (Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia longipalpis, Lutzomyia youngi was tested by allowing the flies to feed on the lesion borders. Results. Both isolates were identified as Leishmania (Viannia braziliensis. No infections were detected upon dissection of engorged flies. A single peri-and sub-lesional injection of 1-2 ml of pentavalent antimony in the dog with ear lesions resulted in clinical cure 6 weeks post-treatment. Conclusions. These observations suggest that although dogs are susceptible to L. braziliensis, their reservoir competence could be low. However, if further studies indicate that canines are capable reservoir hosts of L. Viannia spp., the local treatment of lesions could become a feasible approach to diminish the risk of human infection in the peridomestic setting, without sacrificing infected dogs.

  12. Leishmania (Viannia) braziliensis infection in two Colombian dogs: a note on infectivity for sand flies and response to treatment.

    Science.gov (United States)

    Travi, Bruno L; Tabares, Carlos Javier; Cadena, Horacio

    2006-10-01

    Although canine cutaneous leishmaniasis has been reported in several foci of South America, no published information from Colombia is available. We report on two cases found in the Pacific coast region of this country, which presented as a single scrotal ulcer in one dog, and two ulcers on the external surface of the ear in a second dog. Parasites were isolated by culture in Senekjie's culture medium and identified using monoclonal antibodies. The capacity of these dogs to transmit the parasites to sand fly vectors (Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia longipalpis, Lutzomyia youngi) was tested by allowing the flies to feed on the lesion borders. Both isolates were identified as Leishmania (Viannia) braziliensis. No infections were detected upon dissection of engorged flies. A single peri-and sub-lesional injection of 1-2 ml of pentavalent antimony in the dog with ear lesions resulted in clinical cure 6 weeks post-treatment. These observations suggest that although dogs are susceptible to L. braziliensis, their reservoir competence could be low. However, if further studies indicate that canines are capable reservoir hosts of L. Viannia spp., the local treatment of lesions could become a feasible approach to diminish the risk of human infection in the peridomestic setting, without sacrificing infected dogs.

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

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    Ralph Lainson

    1987-09-01

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

  14. Molecular detection and identification of Leishmania infection in naturally infected sand flies in a focus of cutaneous leishmaniasis in northern Morocco.

    Science.gov (United States)

    Es-Sette, Nargys; Ajaoud, Malika; Laamrani-Idrissi, Abderrahman; Mellouki, Fouad; Lemrani, Meryem

    2014-07-02

    Cutaneous leishmaniasis is an infectious disease caused by various species of the flagellate protozoan Leishmania. During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. The main objective of this study was to study the occurrence of Leishmania infection in vectors and to identify sand fly blood meal sources in an endemic locality of cutaneous leishmaniasis within Sefrou province, where the vectors of leishmaniasis were still unknown. 2650 sand flies were collected using CDC miniature light traps and identified morphologically. The identified sand flies were tested for Leishmania infection by nested PCR. The source of blood meal of 10 freshly engorged females: 6 Phlebotomus longicuspis and 4 Phlebotomus sergenti, was determined using the Cyt b sequence. The collected sand flies consisted of 10 species, seven of which belonged to the genus Phlebotomus and three to the genus Sergentomyia. The most abundant species was P. longicuspis, accounting for 72% of the total sand flies collected. In females of three P. longicuspis and four P. sergenti, Leishmania infantum and Leishmania tropica DNA was detected, respectively.The source of blood meal of engorged females showed that all sand flies tested fed on humans. We report for the first time the natural infection of P. longicuspis with L. infantum in Morocco. The high frequency of this species in this region, in addition to its anthropophilic character make P. longicuspis the putative vector of L. infantum in this cutaneous leishmaniasis focus where L. tropica is confirmed as the causative agent of the disease and P. sergenti as its vector. The presence of L. infantum, and its presumed vector in this area, makes this a site of high risk of visceral leishmaniasis, mostly because of the proximity of a focus of human and canine visceral leishmaniasis.

  15. Man-biting sand fly species and natural infection with the Leishmania promastigote in leishmaniasis-endemic areas of Ecuador.

    Science.gov (United States)

    Gomez, Eduardo A; Kato, Hirotomo; Hashiguchi, Yoshihisa

    2014-12-01

    A countrywide surveillance of sand flies was performed to obtain information on their geographical distribution and natural infection by Leishmania protozoa in Ecuador. A total of 18,119 sand flies were collected by human landing collections during 32 years from 1982 to 2014, and 29 species were recognized. The most prevalent 10 species were Lutzomyia gomezi, Lu. robusta, Lu. hartmanni, Lu. shannoni, Lu. trapidoi, Lu. panamensis, Lu. maranonensis, Lu. ayacuchensis, Lu. tortura and Lu. yuilli yuilli, and their topographical and vertical distributions were identified. Among all the sand flies, only 197 (1.09%) flies of four Lutzomyia species, Lu. gomezi, Lu. trapidoi, Lu. tortura and Lu. ayacuchensis, were positive for Leishmania. Endotrypanum, a flagellate parasite not pathogenic to humans, were detected in five Lutzomyia species, Lu. robusta, Lu. hartmanni, Lu. trapidoi, Lu. panamensis and Lu. yuilli yuilli, suggesting wide vector-ranges of Endotrypanum species. These data on the genus Lutzomyia and their natural infections with Leishmania and Endotrypanum will be useful for transmission studies and surveillance of leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. B-1 cells modulate the murine macrophage response to Leishmania major infection.

    Science.gov (United States)

    Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

    2017-05-26

    To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major ( L. major ) in vitro . Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE 2 ) were determined using a PGE 2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE 2 -neutralizing drugs inhibited PGE 2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major . We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major -infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE 2 in supernatants of L. major -infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major -infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. Our results show that elevated levels of PGE 2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell

  17. First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species.

    Science.gov (United States)

    Kato, Hirotomo; Calvopiña, Manuel; Criollo, Hipatia; Hashiguchi, Yoshihisa

    2013-12-01

    Epidemiological surveillance of leishmaniasis was conducted in a northern Amazonian region of Ecuador, in which cutaneous leishmaniasis cases were recently reported. Sand flies were captured in the military training camp, and the natural infection of sand flies by Leishmania species was examined. Out of 334 female sand flies dissected, the natural infection by flagellates was microscopically detected in 3.9% of Lutzomyia yuilli yuilli and 3.7% of Lutzomyia tortura, and the parasite species were identified as Endotrypanum and Leishmania (Viannia) naiffi, respectively. After the sand fly surveillance, specimens from cutaneous leishmaniasis (CL) patients considered to have acquired the infection in the training camp area were obtained, and the infected parasite species were identified as L. (V.) naiffi. The present study reported first cases of CL caused by L. (V.) naiffi infection in Ecuador. In addition, a high ratio of infection of Lu. tortura by L. (V.) naiffi in the same area strongly suggested that Lu. tortura is responsible for the transmission of L. (V.) naiffi in this area. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Extracellular vesicles from Leishmania-infected macrophages confer an anti-infection cytokine-production profile to naïve macrophages.

    Directory of Open Access Journals (Sweden)

    André Cronemberger-Andrade

    2014-09-01

    Full Text Available Extracellular vesicles (EVs are structures with phospholipid bilayer membranes and 100-1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles.In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50-300 nm-diameter extracellular vesicles (EVs. The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1β, and TNF-α.EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1β and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.

  19. Interleukin-27 Early Impacts Leishmania infantum Infection in Mice and Correlates with Active Visceral Disease in Humans

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    Begoña Pérez-Cabezas

    2016-11-01

    Full Text Available The complexity of Leishmania-host interactions, one of the main leishmaniasis issues, is yet to be fully understood. We detected elevated IL-27 plasma levels in European patients with active visceral disease caused by Leishmania infantum, which returned to basal levels after successful treatment, suggesting this cytokine as a probable infection mediator. We further addressed this hypothesis recurring to two classical susceptible visceral leishmaniasis mouse models. BALB/c, but not C57BL/6 mice, showed increased IL-27 systemic levels after infection, which was associated with an upregulation of IL-27p28 expression by dendritic cells and higher parasite burdens. Neutralization of IL-27 in acutely infected BALB/c led to decreased parasite burdens and a transient increase in IFN-γ+ splenic T cells, while administration of IL-27 to C57BL/6 promoted a local anti-inflammatory cytokine response at the site of infection and increased parasite loads. Overall we show that, as in humans, BALB/c IL-27 systemic levels are infection-dependently upregulated and may favor parasite installation by controlling inflammation.

  20. Qualitative and quantitative immunohistochemical evaluation of iNOS expression in the spleen of dogs naturally infected with Leishmania chagasi.

    Science.gov (United States)

    dos Santos, Fernando Rocha; Vieira, Paula Melo Abreu; Correa-Oliveira, Rodrigo; Giunchetti, Rodolfo Cordeiro; Carneiro, Claudia Martins; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme Cotta

    2011-06-01

    Nitric oxide (NO), the product of the nitric oxide synthase enzymes has been detected in Leishmania-infected animals. Besides its role on the immunity to infection, the role of NO and the inducible nitric oxide synthase (iNOS) in the pathogenesis of canine visceral leishmaniasis (CVL) is not well understood. This study aimed at evaluating immunohistochemically the iNOS expression in the spleen of dogs naturally infected (ID) with Leishmania (L.) chagasi compared with non-infected dogs (NID). The ID was grouped according to the clinical form and the parasite load. Symptomatic dogs (SD) presented higher parasite load in relation to oligosymptomatic (OD) and asymptomatic (AD). The qualitative expression of iNOS was observed only in ID. SD presented strong and prominent labeling of iNOS, followed by OD and AD. Quantitatively, the results showed that the median expression of iNOS was higher in SD and OD compared to NID. Also, dog spleens with high parasitism load showed marked iNOS expression. Taken together, the results suggest that the expression of iNOS in the spleen of infected dogs with CVL was associated with clinical worsening of the disease and with high parasitism.

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

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    Descalzo Miguel A

    2011-04-01

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

  2. Asymptomatic Leishmania Infected Children: A Seroprevalence and Molecular Survey in a Rural Area of Fars Province, Southern Iran

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    Akram Layegh Gigloo

    2018-01-01

    Full Text Available The current study aimed to evaluate the seroprevalence of visceral leishmaniasis in asymptomatic healthy children in a rural area of Fars province, Southern Iran. Blood samples were taken from 617 asymptomatic healthy children and serum samples along with buffy coat were separated from the blood. The serum samples were assessed for antibodies against Leishmania infantum by an indirect ELISA and the buffy coats were tested for the presence of L. infantum DNA by molecular method. Of the 617 recruited children, 297 (48.1% were female and 317 (51.4% were male. Anti-Leishmania antibodies were detected in 17 (2.8% of the children. From those 17 seropositive cases, 5 (29.4% were male and 12 (70.6% cases were female. Children aged 5–8 years had the highest seroprevalence rate; however, no associations were found between seropositivity to Leishmania and gender or age of the children. Moreover, L. infantum DNA was detected in buffy coat of 8 (1.3% of 617 children. Three of the PCR-positive cases were seropositive whereas 14 of seropositive subjects (82.3% were PCR-negative. Findings of the current study revealed a considerable subclinical leishmanial infection in children in the studied rural area in the south of Iran. Results of the current study could be used for surveillance, prevention, and control of VL in the area.

  3. 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.

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

    Science.gov (United States)

    de Moura, Tatiana R; Oliveira, Fabiano; Carneiro, Marcia W; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M C; Valenzuela, Jesus G; de Oliveira, Camila I

    2013-01-01

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

  5. 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

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune...... to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes....... 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...

  6. Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (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

    2017-10-01

    Human leishmaniases are widespread 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 mucocutaneous 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 the identification of microorganisms, 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 the performance of MSI using 268 samples, 231 of which were Leishmania strains. All Leishmania strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the Leishmania donovani , L. guyanensis , and L. braziliensis complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being 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 users' computers. Copyright © 2017 American Society for Microbiology.

  7. Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937 Experimental infection of Equus asinus with Leishmania chagasi Cunha & Chagas, 1937

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    Elúzio José Lima Cerqueira

    2003-12-01

    Full Text Available Quatro Equus asinus foram inoculados com promastigotas de Leishmania chagasi Cunha & Chagas, 1937 e acompanhados durante 12 meses através de: pesquisa de amastigotas em esfregaços e culturas de sangue periférico em fragmentos de tecido do lábio inferior, medula óssea, baço e fígado e de testes de ELISA e TRALd. Estes foram positivos nos 8º, 10º e 12º meses após a inoculação. O exame histopatológico pós necropsia, demonstrou discreto número de amastigotas no fígado de dois dos eqüídeos inoculados. Apesar de desafiados com elevado número de promastigotas, os animais não desenvolveram infecções patentes e não infectaram experimentalmente a vetora Lutzomya longipalpis. Os resultados induzem a acreditar que os eqüídeos são desprovidos de importância como reservatórios na cadeia de transmissão da leishmaniose visceral, embora sirvam como boa fonte de alimentação sangüínea e proliferação da vetora Lutzomyia longipalpis.Four Equus asinus were challenged with promastigotes of Leishmania chagasi Cunha & Chagas, 1937, and followed up for 12 months. They were observed by means of direct testing for promastigotes in smears and culture of peripheral blood, fragments from inferior lip, bone marrow, spleen and liver and the immunological assays ELISA and TRALd. The post-necropsy histological examination demonstrated a small number of amastigotes in the liver of two animals. ELISA and TRALd tests were positive at the 8th, 10th and 12th month after inoculation. The results suggest that the donkeys were able to overcome the experimental leishmanial infection and did not infect the vector Lutzomyia longipalpis in the laboratory. Consequently they can not be considered an important reservoir in the epidemiological chain of transmission of visceral leishmaniasis, although they represent an important blood source for the vector and its proliferation.

  8. Leptomonas seymouri: Adaptations to the dixenous life cycle analyzed by genome sequencing, transcriptome profiling and co-infection with Leishmania donovani

    Czech Academy of Sciences Publication Activity Database

    Kraeva, N.; Butenko, A.; Hlaváčová, J.; Kostygov, A.; Myšková, J.; Grybchuk, D.; Leštinová, T.; Votýpka, Jan; Volf, P.; Opperdoes, F.; Flegontov, P.; Lukeš, Julius; Yurchenko, V.

    2015-01-01

    Roč. 11, č. 8 (2015), e1005127 E-ISSN 1553-7374 R&D Projects: GA ČR(CZ) GA14-23986S EU Projects: European Commission(XE) 316304 Institutional support: RVO:60077344 Keywords : parasites * Costa Rica * RNA virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.003, year: 2015

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

    Directory of Open Access Journals (Sweden)

    Lívia O Santos

    Full Text Available BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania

  10. Conditions Influencing the Efficacy of Vaccination with Live Organisms against Leishmania major Infection

    Science.gov (United States)

    Tabbara, Khaled S.; Peters, Nathan C.; Afrin, Farhat; Mendez, Susana; Bertholet, Sylvie; Belkaid, Yasmine; Sacks, David L.

    2005-01-01

    Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-γ)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-γ+ CD4+ cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4+ T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens. PMID:16040984

  11. 17-AAG kills intracellular Leishmania amazonensis while reducing inflammatory responses in infected macrophages.

    Science.gov (United States)

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

    2012-01-01

    Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory. We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25-500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O(2) (-)) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment. The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor's potential in the development of new generations of anti-leishmanials.

  12. High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

    Directory of Open Access Journals (Sweden)

    Grinsztejn Beatriz

    2010-12-01

    Full Text Available Abstract Background Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL and tegumentary leishmaniasis (ATL have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods To address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3. Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

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

    Science.gov (United States)

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

    2017-01-01

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

  14. First Human Cases of Leishmania (Viannia) lainsoni Infection and a Search for the Vector Sand Flies in Ecuador

    Science.gov (United States)

    Kato, Hirotomo; Bone, Abdon E.; Mimori, Tatsuyuki; Hashiguchi, Kazue; Shiguango, Gonzalo F.; Gonzales, Silvio V.; Velez, Lenin N.; Guevara, Angel G.; Gomez, Eduardo A.; Hashiguchi, Yoshihisa

    2016-01-01

    An epidemiological study of leishmaniasis was performed in Amazonian areas of Ecuador since little information on the prevalent Leishmania and sand fly species responsible for the transmission is available. Of 33 clinical specimens from patients with cutaneous leishmaniasis (CL), causative parasites were identified in 25 samples based on cytochrome b gene analysis. As reported previously, Leishmania (Viannia) guyanensis and L. (V.) braziliensis were among the causative agents identified. In addition, L. (V.) lainsoni, for which infection is reported in Brazil, Bolivia, Peru, Suriname, and French Guiana, was identified in patients with CL from geographically separate areas in the Ecuadorian Amazon, corroborating the notion that L. (V.) lainsoni is widely distributed in South America. Sand flies were surveyed around the area where a patient with L. (V.) lainsoni was suspected to have been infected. However, natural infection of sand flies by L. (V.) lainsoni was not detected. Further extensive vector searches are necessary to define the transmission cycle of L. (V.) lainsoni in Ecuador. PMID:27191391

  15. First Human Cases of Leishmania (Viannia lainsoni Infection and a Search for the Vector Sand Flies in Ecuador.

    Directory of Open Access Journals (Sweden)

    Hirotomo Kato

    2016-05-01

    Full Text Available An epidemiological study of leishmaniasis was performed in Amazonian areas of Ecuador since little information on the prevalent Leishmania and sand fly species responsible for the transmission is available. Of 33 clinical specimens from patients with cutaneous leishmaniasis (CL, causative parasites were identified in 25 samples based on cytochrome b gene analysis. As reported previously, Leishmania (Viannia guyanensis and L. (V. braziliensis were among the causative agents identified. In addition, L. (V. lainsoni, for which infection is reported in Brazil, Bolivia, Peru, Suriname, and French Guiana, was identified in patients with CL from geographically separate areas in the Ecuadorian Amazon, corroborating the notion that L. (V. lainsoni is widely distributed in South America. Sand flies were surveyed around the area where a patient with L. (V. lainsoni was suspected to have been infected. However, natural infection of sand flies by L. (V. lainsoni was not detected. Further extensive vector searches are necessary to define the transmission cycle of L. (V. lainsoni in Ecuador.

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

    OpenAIRE

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

    2007-01-01

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

  17. After infection with Leishmania infantum, Golden Hamsters (Mesocricetus auratus) become more attractive to female sand flies (Lutzomyia longipalpis).

    Science.gov (United States)

    Nevatte, T M; Ward, R D; Sedda, L; Hamilton, J G C

    2017-07-21

    In Brazil, human and canine visceral leishmaniasis is caused by infection with Leishmania infantum, a Protist parasite transmitted by blood-feeding female Lutzomyia longipalpis sand flies. The objective of this study was to determine if the odour of hamsters, infected with Le. infantum, was more attractive than the odour of the same hamsters, before they were infected. The attractiveness of odour collected from individual hamsters (n = 13), before they were infected, was compared in a longitudinal study, with the attractiveness of the odour of the same hamster in a Y-tube olfactometer bioassay, at a late stage of infection. The odour of six of the golden hamsters was significantly more attractive to 50% of the female sand flies at the end of infection compared to before infection and the odour of four of the golden hamsters was significantly more attractive to 75% of the female sand flies at the end of infection. These results strongly indicate that hamsters infected with Le. infantum become significantly more attractive to a greater proportion of female sand flies as the infection progresses.

  18. 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. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Low seroprevalence of Leishmania infantum infection in cats from northern Portugal based on DAT and ELISA

    NARCIS (Netherlands)

    Cardoso, Luís; Lopes, Ana Patrícia; Sherry, Kate; Schallig, Henk; Solano-Gallego, Laia

    2010-01-01

    Cats have been considered playing a role in the epidemiology of leishmaniasis caused by Leishmania infantum, an endemic zoonosis in countries of the Mediterranean basin. The present study assessed the prevalence of antibodies to L. infantum in 316 domestic cats from northern Portugal, by means of

  20. DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice.

    Science.gov (United States)

    Maspi, N; Ghaffarifar, F; Sharifi, Z; Dalimi, A; Khademi, S Z

    2017-12-01

    Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (pTSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (pTSA and TSA groups than LACK group after challenge (pTSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

  1. Natural infections of man-biting sand flies by Leishmania and Trypanosoma species in the northern Peruvian Andes.

    Science.gov (United States)

    Kato, Hirotomo; Gomez, Eduardo A; Cáceres, Abraham G; Vargas, Franklin; Mimori, Tatsuyuki; Yamamoto, Kento; Iwata, Hiroyuki; Korenaga, Masataka; Velez, Lenin; Hashiguchi, Yoshihisa

    2011-05-01

    The natural infection of sand flies by Leishmania species was studied in the Andean areas of Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured by human bait and Center for Disease Control (CDC) light trap catches at Nambuque and Padregual, Department of La Libertad, Peru, and morphologically identified. Among 377 female sand flies dissected, the two dominant man-biting species were Lutzomyia (Helcocyrtomyia) peruensis (211 flies) and Lutzomyia (Helcocyrtomyia) caballeroi (151 flies). Another sand fly species captured by light trap was Warileya phlebotomanica (15 flies). The natural infection of sand flies by flagellates was detected in 1.4% of Lu. (H.) peruensis and 2.6% of Lu. (H.) caballeroi, and the parasite species were identified as Le. (V.) peruviana and Trypanosoma avium, respectively, by molecular biological methods. The results indicated that the vector species responsible for the transmission of leishmaniasis in the study areas is Lu. (H.) peruensis. In addition, the presence of Trypanosoma in man-biting sand fly species means that more careful consideration is necessary for vector research in areas of Andean Peru where leishmaniasis is endemic.

  2. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

    Science.gov (United States)

    Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2014-04-01

    Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

    Science.gov (United States)

    Lyda, Todd A.; Joshi, Manju B.; Andersen, John F.; Kelada, Andrew Y.; Owings, Joshua P.; Bates, Paul A.; Dwyer, Dennis M.

    2015-01-01

    Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts. PMID:25763714

  4. Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST)

    NARCIS (Netherlands)

    Cardoso, Luís; Schallig, Henk D. F. H.; Neto, Francisco; Kroon, Nel; Rodrigues, Manuela

    2004-01-01

    Leishmaniasis caused by Leishmania infantum is a prevalent disease in dogs and humans. A serological survey of Leishmania infection in dogs was carried out in the endemic region of Alto Douro (north Portugal). Two hundred and ninety-four dogs from the municipality of Peso da Régua were examined for

  5. Serum Cytokine Responses over the Entire Clinical-Immunological Spectrum of Human Leishmania (L. infantum chagasi Infection

    Directory of Open Access Journals (Sweden)

    Patrícia Karla Ramos

    2016-01-01

    Full Text Available The clinical-immunological spectrum of human Leishmania (L. infantum chagasi infection in Amazonian Brazil was recently reviewed based on clinical, DTH, and IFAT (IgG evaluations that identified five profiles: three asymptomatic (asymptomatic infection, AI; subclinical resistant infection, SRI; and indeterminate initial infection, III and two symptomatic (symptomatic infection, SI; American visceral leishmaniasis, AVL; and subclinical oligosymptomatic infection, SOI. TNF-α, IL-4, IL-6, and IL-10 serum cytokines were analyzed using multiplexed Cytometric Bead Array in 161 samples from endemic areas in the Brazilian Amazon: SI [AVL] (21 cases, III (49, SRI (19, SOI (12, AI (36, and a control group [CG] (24. The highest IL-6 serum levels were observed in the SI profile (AVL; higher IL-10 serum levels were observed in SI than in SOI or CG and in AI and III than in SOI; higher TNF-α serum levels were seen in SI than in CG. Positive correlations were found between IL-6 and IL-10 serum levels in the SI and III profiles and between IL-6 and TNF-α and between IL-4 and TNF-α in the III profile. These results provide strong evidence for associating IL-6 and IL-10 with the immunopathogenesis of AVL and help clarify the role of these cytokines in the infection spectrum.

  6. Experimental infection of Leishmania (L. chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae

    Directory of Open Access Journals (Sweden)

    Felio J Bello

    2005-10-01

    Full Text Available The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5 cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6% and on day 4 in the J774 cells (51%. This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

  7. 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.

  8. Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs.

    Science.gov (United States)

    Solcà, Manuela da Silva; Guedes, Carlos Eduardo Sampaio; Nascimento, Eliane Gomes; Oliveira, Geraldo Gileno de Sá; dos Santos, Washington Luis Conrado; Fraga, Deborah Bittencourt Mothé; Veras, Patrícia Sampaio Tavares

    2012-03-23

    Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic

  9. Diagnosis of Leishmania infantum infection by Polymerase Chain Reaction in wild mammals

    Directory of Open Access Journals (Sweden)

    Mayara C. Lombardi

    2014-12-01

    Full Text Available Visceral leishmaniasis is a chronic infectious disease caused by Leishmania infantum (synonym: Leishmania chagasi and transmitted by the sandfly Lutzomyia longipalpis in Brazil. It is an endemic zoonosis in several regions of the country, including Belo Horizonte (State of Minas Gerais. In urban areas, the domestic dog is susceptible and considered the most important animal reservoir. However, L. infantum has been previously diagnosed in other species, including captive primates and canids. This study aimed to evaluate the presence of the agent DNA in captive animals as well as some free ranging animals from the Zoo-Botanical Foundation of Belo Horizonte by Polymerase Chain Reaction. Eighty one blood samples from primates, carnivores, ruminants, edentates, marsupial, and a monogastric herbivore were analyzed. Three primates Alouatta guariba (brown howler monkey, and two canids Speothos venaticus (bush dog were positive, demonstrating the importance of leishmaniasis control in endemic areas for preservation of wildlife species in captivity.

  10. An investigation on vertical transmission of Leishmania infantum in experimentally infected dogs and assessment of offspring's infectiousness potential by xenodiagnosis.

    Science.gov (United States)

    Ben Slimane, T; Chouihi, E; Ben Hadj Ahmed, S; Chelbi, I; Barhoumi, W; Cherni, S; Zoghlami, Z; Gharbi, M; Zhioua, E

    2014-12-15

    Dogs are the main reservoir host of Leishmania infantum, etiologic agent of human visceral leishmaniasis (HVL) and canine visceral leishmaniasis (CVL). Transmission of L. infantum to humans and dogs is mainly through the bite of infected sand flies. In the Western Mediterranean basin, Phlebotomus perniciosus is the main vector of L. infantum. However, occasional vertical transmission of L. infantum has been reported. This study investigated L. infantum vertical transmission in offspring of experimentally infected dogs. Among 14 surviving puppies from three female beagle dogs that developed CVL following an experimental infection with L. infantum, one was tested positive by indirect immunofluorescence antibody test, by PCR and by xenodiagnosis with a high parasite burden in the spleen at 14 months old. None of the remaining puppies were tested positive for L. infantum. These findings strongly suggest that infected puppies following vertical transmission can sustain infection and contribute in infecting sand flies with L. infantum. Any strategy for controlling CVL should take into consideration the vertical transmission of L. infantum. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Czech Academy of Sciences Publication Activity Database

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Imidazole-containing phthalazine derivatives inhibit Fe-SOD performance in Leishmania species and are active in vitro against visceral and mucosal leishmaniasis.

    Science.gov (United States)

    Sánchez-Moreno, M; Gómez-Contreras, F; Navarro, P; Marín, C; Ramírez-Macías, I; Rosales, M J; Campayo, L; Cano, C; Sanz, A M; Yunta, M J R

    2015-07-01

    The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1-4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

  15. The paratransgenic sand fly: a platform for control of Leishmania transmission.

    Science.gov (United States)

    Hurwitz, Ivy; Hillesland, Heidi; Fieck, Annabeth; Das, Pradeep; Durvasula, Ravi

    2011-05-19

    Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes. This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Pesticide resistance in sand fly populations, environmental toxicity, and limited resources confound this approach. A novel paratransgenic strategy aimed at control of vectorial transmission of L. donovani is presented using Bacillus subtilis, a commensal bacterium isolated from the sand fly gut. In this work, B. subtilis expressing Green Fluorescent Protein (GFP) was added to sterilized larval chow. Control pots contained larval chow spiked either with untransformed B. subtilis or phosphate-buffered saline. Fourth-instar P. argentipes larvae were transferred into the media and allowed to mature. The number of bacterial colony forming units, relative abundance and the mean microbial load were determined per developmental stage. Addition of B. subtilis to larval chow did not affect sand fly emergence rates. B. cereus and Lys fusiformis were identified at each developmental stage, revealing transstadial passage of endogenous microbes. Larvae exposed to an exogenous bolus of B. subtilis harbored significantly larger numbers of bacteria. Bacterial load decreased to a range comparable to sand flies from control pots, suggesting an upper limit to the number of bacteria harbored. Emerging flies reared in larval chow containing transformed B. subtilis carried large numbers of these bacteria in their gut lumens. Strong GFP expression was detected in these paratransgenic flies with no spread of transformed bacteria to other compartments of the insects. This is the first demonstration of paratransgenic manipulation of P. argentipes. Paratransgenic manipulation of P. argentipes appears feasible. Expression of leishmanicidal molecules via commensal bacteria commonly found at breeding sites of P. argentipes could render adult sand flies refractory

  16. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi; PCR associado a hibridizacao com sondas radioativas de DNA para a identificacao de infeccao subclinica causada por Leishmania Chagasi

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Moreno, Elizabeth Castro [Fundacao Nacional de Saude, Belo Horizonte, MG (Brazil). Coordenacao Regional de Minas Gerais; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical

    2002-07-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with {sup 32} P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with {sup 32} P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  17. First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen.

    Science.gov (United States)

    Mahdy, Mohammed A K; Al-Mekhlafi, Abdulsalam M; Abdul-Ghani, Rashad; Saif-Ali, Reyadh; Al-Mekhlafi, Hesham M; Al-Eryani, Samira M; Lim, Yvonne A L; Mahmud, Rohela

    2016-01-01

    Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved.

  18. 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. © The American Society of Tropical Medicine and Hygiene.

  19. The Effect of Removing Potentially Infectious Dogs on the Numbers of Canine Leishmania infantum Infections in an Endemic Area with High Transmission Rates

    OpenAIRE

    Grimaldi, Gabriel; Teva, Antonio; Santos, Claudiney B.; Ferreira, Adelson L.; Falqueto, Aloísio

    2012-01-01

    To assess the effect of the rapid removal of potentially infectious dogs on the prevalence and incidence of canine infections, a prospective study was undertaken in an area endemic for Leishmania infantum. We used serological testing based on the rapid DPP rK28 fusion protein chromatographic immunoassay for this dog screening-and-culling intervention trial. The outcome was evaluated by measuring seropositivity and sero-conversion/-reversion rates for canine infection. Our estimates indicated ...

  20. Molecular detection and identification of Leishmania spp. in naturally infected Phlebotomus tobbi and Sergentomyia dentata in a focus of human and canine leishmaniasis in western Turkey.

    Science.gov (United States)

    Özbel, Yusuf; Karakuş, Mehmet; Arserim, Suha K; Kalkan, Şaban Orçun; Töz, Seray

    2016-03-01

    Human visceral leishmaniasis (VL) is reported from 38 provinces of Turkey and dogs are accepted as main reservoir hosts. Kuşadası town, belonging to Aydın province and located in western part of Turkey, is endemic for human and canine visceral leishmaniasis caused by Leishmania infantum MON1 and MON98. In this study, phlebotomine survey was conducted to determine the vector sand fly species and to identify sand fly blood meal sources. In August and September 2012, 1027 sand fly specimens were caught using CDC light traps. Eight Phlebotomus and two Sergentomyia species with the dominancy of Phlebotomus tobbi (61.34%) were detected. A total of 622 female sand flies (571 Phlebotomus; 51 Sergentomyia) were checked for Leishmania infection by direct dissection of the midgut. The half of the midgut content was inoculated into NNN culture for isolation of the parasite. Leishmania species-specific ITS1 real time PCR, conventional PCR assays of ITS1 and hsp70 genes and subsequent sequencing were performed from extracted DNAs. A region of cytochrome b (cyt-b) gene of vertebrates based PCR was used to determine the source of blood meal of sand flies. In microscopical examinations, two female specimens (0.32%) were found naturally infected with high number and different stages of promastigotes. No growth was observed in NNN culture but Leishmania DNA was obtained from both specimens. First positive specimen was identified as P. tobbi and L. infantum DNA was detected. Second specimen was Sergentomyia dentata, but Leishmania DNA could not be identified on species level. A total of 16 blood-fed female P. tobbi specimens were used for blood meal analysis and eight, three and one specimens were positive for human, dog and mouse, respectively. This is the first detection of Leishmania promastigotes using microscopical examination in P. tobbi and S. dentata in human and canine visceral leishmaniasis endemic area in western part of Turkey. Our results indicate that, (i) P. tobbi is

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

    Directory of Open Access Journals (Sweden)

    É. Labat

    2010-06-01

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

  2. Phlebotomine Sand Fly Fauna and Leishmania Infection in the Vicinity of the Serra do Cipó National Park, a Natural Brazilian Heritage Site

    Directory of Open Access Journals (Sweden)

    Rosana Silva Lana

    2015-01-01

    Full Text Available In the New World, the leishmaniases are primarily transmitted to humans through the bites of Leishmania-infected Lutzomyia (Diptera: Psychodidae phlebotomine sand flies. Any or both of two basic clinical forms of these diseases are endemic to several cities in Brazil—the American cutaneous leishmaniasis (ACL and the American visceral leishmaniasis (AVL. The present study was conducted in the urban area of a small-sized Brazilian municipality (Jaboticatubas, in which three cases of AVL and nine of ACL have been reported in the last five years. Jaboticatubas is an important tourism hub, as it includes a major part of the Serra do Cipó National Park. Currently, no local data is available on the entomological fauna or circulating Leishmania. During the one-year period of this study, we captured 3,104 phlebotomine sand flies belonging to sixteen Lutzomyia species. In addition to identifying incriminated or suspected vectors of ACL with DNA of the etiological agent of AVL and vice versa, we also detected Leishmania DNA in unexpected Lutzomyia species. The expressive presence of vectors and natural Leishmania infection indicates favorable conditions for the spreading of leishmaniases in the vicinity of the Serra do Cipó National Park.

  3. Phlebotomine Sand Fly Fauna and Leishmania Infection in the Vicinity of the Serra do Cipó National Park, a Natural Brazilian Heritage Site

    Science.gov (United States)

    Lana, Rosana Silva; Michalsky, Érika Monteiro; Fortes-Dias, Consuelo Latorre; França-Silva, João Carlos; Lara-Silva, Fabiana de Oliveira; Lima, Ana Cristina Vianna Mariano da Rocha; Moreira de Avelar, Daniel; Martins, Juliana Cristina Dias; Dias, Edelberto Santos

    2015-01-01

    In the New World, the leishmaniases are primarily transmitted to humans through the bites of Leishmania-infected Lutzomyia (Diptera: Psychodidae) phlebotomine sand flies. Any or both of two basic clinical forms of these diseases are endemic to several cities in Brazil—the American cutaneous leishmaniasis (ACL) and the American visceral leishmaniasis (AVL). The present study was conducted in the urban area of a small-sized Brazilian municipality (Jaboticatubas), in which three cases of AVL and nine of ACL have been reported in the last five years. Jaboticatubas is an important tourism hub, as it includes a major part of the Serra do Cipó National Park. Currently, no local data is available on the entomological fauna or circulating Leishmania. During the one-year period of this study, we captured 3,104 phlebotomine sand flies belonging to sixteen Lutzomyia species. In addition to identifying incriminated or suspected vectors of ACL with DNA of the etiological agent of AVL and vice versa, we also detected Leishmania DNA in unexpected Lutzomyia species. The expressive presence of vectors and natural Leishmania infection indicates favorable conditions for the spreading of leishmaniases in the vicinity of the Serra do Cipó National Park. PMID:25793193

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

    Directory of Open Access Journals (Sweden)

    L. Ryan

    1987-09-01

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

  5. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Uveitis associated to the infection by Leishmania chagasi in dog from the Olinda city, Pernambuco, Brazil

    Directory of Open Access Journals (Sweden)

    Brito Fábio Luiz da Cunha

    2004-01-01

    Full Text Available Among the parasitic diseases, Canine Visceral Leishmaniasis (CVL is included in the systemic illnesses of chronic evolution that attack men and dogs, presenting varied clinical manifestations as cachexia, dermatologic lesions, peripheral lymphadenopathies, besides the ocular lesions. This work report the case of a dog clinically suspected of having CVL, presenting skin lesions, cachexia, gryphosis, and ocular signs of uveitis. The parasitological diagnosis was accomplished for Canine Leishmaniasis through the visualization of amastigote forms of Leishmania chagasi in smears of bone marrow fluid aspirate, of non-lesioned, and lesioned skin. Alterations in the ocular structures are characterized mainly by mononuclear-plasmocitic infiltrate.

  7. Different genetic control of cutaneous and visceral disease after Leishmania major infection in mice

    Czech Academy of Sciences Publication Activity Database

    Vladimirov, Vladimir; Badalová, Jana; Svobodová, M.; Havelková, Helena; Hart, A. A. M.; Blažková, Hana; Demant, P.; Lipoldová, Marie

    2003-01-01

    Roč. 71, č. 4 (2003), s. 2041-2046 ISSN 0019-9567 R&D Projects: GA ČR GA310/00/0760; GA ČR GA310/03/1381; GA MŠk OK 394 Grant - others:Howard Hughes Medical Institute(US) HHMI55000323; EC(XE) ERBI-C15-CT98-0317; EC(XE) BIO-4-CT98-0445 Institutional research plan: CEZ:AV0Z5052915 Keywords : genetic control * Leishmania major Subject RIV: EC - Immunology Impact factor: 3.875, year: 2003

  8. A minimally invasive approach to spleen histopathology in dogs: A new method for follow-up studies of spleen changes in the course of Leishmania infantum infection.

    Science.gov (United States)

    Santos, Silvana Ornelas; Fontes, Jonathan L M; Laranjeira, Daniela F; Vassallo, José; Barrouin-Melo, Stella Maria; Dos-Santos, Washington L C

    2016-10-01

    Severe forms of zoonotic visceral leishmaniosis (ZVL) are associated with disruption of the spleen structure. However, the study of spleen histology requires splenectomy or necropsy. In this work, we present a minimally invasive cell-block technique for studying spleen tissue histology in dogs with ZVL. We examined 13 dogs with and seven dogs without Leishmania infantum infection. The dogs with Leishmania infection had a lower frequency of lymphoid follicles (2/13, Fisher's test, Pdogs (5/7 exhibiting lymphoid follicles and a plasma cell score of 1). The dogs with Leishmania infection also presented with granulomas (8/13) and infected macrophages (5/13). These differences in the histological presentations of spleen tissue from infected and uninfected dogs corresponded to changes observed in conventional histology. Hence, the cell-block technique described here may be used in the follow-up care and study of dogs with ZVL and other diseases in both clinical practice and research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. 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

    of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-gamma production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating...... call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.......Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-gamma was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated...

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

    Science.gov (United States)

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

    2017-01-01

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

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

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    Valéria da Costa Oliveira

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

  12. Factors associated with Leishmania chagasi infection in domestic dogs from Teresina, State of Piauí, Brazil

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    João Pereira da Silva

    2012-08-01

    Full Text Available INTRODUCTION: Many studies have evaluated risk factors for human visceral leishmaniasis, but few have focused on the infection among dogs. The objective of this study was to assess the association between peridomestic socioeconomic and environmental factors and the presence of dogs seropositive for Leishmania chagasi in the City of Teresina, Brazil. METHODS: This case-control study was based on the results of a routine seroepidemiological survey among domestic dogs carried out in 2007. Serological tests were performed by means of indirect immunofluorescence antibody test. All dwellings in which at least one seropositive dog was detected were considered cases, and controls were a random sample of dwellings in which only seronegative dogs were identified. Associations between variables were expressed as odds ratios (OR and their respective 95% confidence intervals (95%CI estimated using multivariate logistic regression. RESULTS: Dwellings with a history of dogs removed by the visceral leishmaniasis control program in the last 12 months had five-fold higher odds of having at least one seropositive dog as compared with dwellings having no history of dog removal (OR = 5.19; 95%CI = 3.20-8.42. Dwellings with cats had 58% increased odds of dog infection as compared with those having no cats (OR = 1.58; 95%CI = 1.01-2.47. CONCLUSIONS: Identification of factors associated with canine visceral leishmaniasis might be used for the delimitation of areas of higher risk for human visceral leishmaniasis, since infection in dogs generally precedes the appearance of human cases.

  13. Engagement of NKG2D on bystander memory CD8 T cells promotes increased immunopathology following Leishmania major infection.

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    Erika J Crosby

    2014-02-01

    Full Text Available One of the hallmarks of adaptive immunity is the development of a long-term pathogen specific memory response. While persistent memory T cells certainly impact the immune response during a secondary challenge, their role in unrelated infections is less clear. To address this issue, we utilized lymphocytic choriomeningitis virus (LCMV and Listeria monocytogenes immune mice to investigate whether bystander memory T cells influence Leishmania major infection. Despite similar parasite burdens, LCMV and Listeria immune mice exhibited a significant increase in leishmanial lesion size compared to mice infected with L. major alone. This increased lesion size was due to a severe inflammatory response, consisting not only of monocytes and neutrophils, but also significantly more CD8 T cells. Many of the CD8 T cells were LCMV specific and expressed gzmB and NKG2D, but unexpectedly expressed very little IFN-γ. Moreover, if CD8 T cells were depleted in LCMV immune mice prior to challenge with L. major, the increase in lesion size was lost. Strikingly, treating with NKG2D blocking antibodies abrogated the increased immunopathology observed in LCMV immune mice, showing that NKG2D engagement on LCMV specific memory CD8 T cells was required for the observed phenotype. These results indicate that bystander memory CD8 T cells can participate in an unrelated immune response and induce immunopathology through an NKG2D dependent mechanism without providing increased protection.

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

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

    2018-01-01

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

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

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

    2017-10-10

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

  16. Cryopreservation of Leishmania Species in Manisa Province.

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    Çavuş, İbrahim; Ocak, Fulya; Kaya, Tuğba; Özbilgin, Ahmet

    2017-09-01

    It was aimed to assess the success of the cryopreservation process which is carried out in order to preserve the genetic material and the virulence of the Leishmania species that are an important health problem in our region. Leishmania tropica, L. infantum, L. major, and L. donovani strains in Novy-MacNeal-Nicolle (NNN) medium in MCBU were used. Promastigotes cultured in the NNN medium were transferred to RPMI 1640 medium; promastigotes in the logarithmic phase were washed three times with PBS, and 15% dimethylsulfoxide (DMSO) was added. Leishmania species were transferred to 12 separate tubes. The tubes were stored at -86°C for one night by placing them in Coolcell boxes. The tubes were transferred into a liquid nitrogen tank. One cryotube per Leishmania strain is thawed monthly and cultured in NNN medium. For the duration of study it was observed that each Leishmania isolate preserved 60-65% of their viability and entered the logarithmic phase on the 7th day following the inoculation in the NNN medium. Abnormalities in the structures and movements of the promastigotes were not observed in microscopic examinations. The following conclusions were made: cryopreservation is important for studies planned related to leishmaniasis and cryopreservation with DMSO is successful.

  17. Factors associated with Leishmania asymptomatic infection: results from a cross-sectional survey in highland northern Ethiopia.

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    Estefanía Custodio

    Full Text Available BACKGROUND: In northern Ethiopia the prevalence of visceral leishmaniasis is steadily rising posing an increasing public health concern. In order to develop effective control strategies on the transmission of the disease it is important to generate knowledge on the epidemiological determinants of the infection. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional survey on children 4-15 years of age using a multi staged stratified cluster sampling on high incidence sub-districts of Amhara regional state, Ethiopia. The survey included a socio-demographic, health and dietary questionnaire, and anthropometric measurements. We performed rK39-ICT and DAT serological tests in order to detect anti-Leishmania antibodies and carried out Leishmanin Skin Test (LST using L.major antigen. Logistic regression models were used. Of the 565 children surveyed 56 children were positive to infection (9.9%. The individual variables that showed a positive association with infection were increasing age, being male and sleeping outside [adjusted odds ratios (95% CI: 1.15 (1.03, 1.29, 2.56 (1.19, 5.48 and 2.21 (1.03, 4.71 respectively] and in relation to the household: past history of VL in the family, living in a straw roofed house and if the family owned sheep [adjusted OR (95% CI: 2.92 (1.25, 6.81, 2.71 (1.21, 6.07 and 4.16 (1.41, 12.31 respectively]. CONCLUSIONS/SIGNIFICANCE: A behavioural pattern like sleeping outside is determinant in the transmission of the infection in this area. Protective measures should be implemented against this identified risk activity. Results also suggest a geographical clustering and a household focalization of the infection. The behaviour of the vector in the area needs to be clarified in order to establish the role of domestic animals and house materials in the transmission of the infection.

  18. Sand flies naturally infected by Leishmania (L.) mexicana in the peri-urban area of Chetumal city, Quintana Roo, México.

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    Sánchez-García, Laura; Berzunza-Cruz, Miriam; Becker-Fauser, Ingeborg; Rebollar-Téllez, Eduardo A

    2010-06-01

    The surveillance of prevalent Leishmania sand fly vectors is an important issue for epidemiological studies in populated areas where leishmaniasis is endemic. In this study, we collected sand flies from a peri-urban area in the southeast of Mexico. Natural infection with Leishmania (L.) mexicana was studied by PCR using a Leishmania internal transcribed spacer of the ribosomal RNA gene for amplification. Infected Lutzomyia olmeca olmeca, Lu. shannoni and Lu. cruciata sand flies were collected mainly during the high transmission season (November to March), coinciding with the highest sand fly densities. Additionally, positive specimens of Lu. olmeca olmeca were also captured during July and August. The infected sand flies were from primary forest (subperennial forest) and secondary forest (18-25 years old and 10-15 years old respectively). Sand flies collected with Disney and Shannon traps were the ones found to be infected with L. (L.) mexicana. We conclude that the high-risk period in which L. (L.) mexicana is transmitted in the peri-urban area of Chetumal City is from July to March and that transmission is associated with both the subperennial forest and the secondary forest. 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

  19. 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

  20. Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease

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    Eduardo Ontoria

    2018-06-01

    Full Text Available Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4 or remain activated throughout the experiment (Il18bp. The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi, suggesting the generation of a classical “protective response” against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  2. [Natural infection with Leishmania infantum chagasi in Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) sandflies captured in the municipality of Janaúba, State of Minas Gerais, Brazil].

    Science.gov (United States)

    Michalsky, Erika Monteiro; Guedes, Karla de Sena; Lara e Silva, Fabiana de Oliveira; França-Silva, João Carlos; Dias, Consuelo Latorre Fortes; Barata, Ricardo Andrade; Dias, Edelberto Santos

    2011-01-01

    Visceral leishmaniasis has been notified in nearly all states of Brazil, and particularly in the north of Minas Gerais, where the disease is endemic. The aim of this study was to detect natural infection of Lutzomyia longipalpis and, through the PCR/RFLP technique, identify Leishmania species found in sandflies in the municipality of Janaúba. Using light traps, 1,550 females of L. longipalpis were caught and grouped into pools of 10 specimens to be subjected to DNA extraction and amplification, by means of generic PCR and cacophony. Out of the 155 pools, six were positive for Leishmania sp., and thus the infection rate in the municipality was 3.9%. Through PCR/RFLP, the digestion pattern among the positive samples was found to be similar to that of the reference strain of Leishmania chagasi (MHOM/BR/74/PP75). The detection of natural infection associated with studies on the epidemiology of visceral leishmaniasis suggests that L. longipalpis is involved in transmission of L. infantum chagasi in Janaúba, particularly in areas of intense transmission of visceral leishmaniasis.

  3. Factores dermicos que condicionan la infeccion de Lutzomyia townsendi (Ortiz, 1959 por Leishmania spp. de Venezuela Dermal factors which condition infection of Lutzomyia townsendi (Ortiz, 1959 by Leishmania spp. of Venezuela

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    Maritza Carnevali

    1982-12-01

    Full Text Available Se estudia la susceptibilidad de Lutzomyia townsendi a la infección con Leishmania spp. sobre lesiones experimentales de hamsteres. Se estudia la frecuencia y distribución de los amastigotos en la dermis, relacionándola con la profundidad que alcanza el estilete bucal del insecto. Una correlación positiva, con significante coeficiente de correlación, se establece entre la frecuencia de los parásitos a una profundidad de 100-150 nm en la dermis y el éxito de la infección de los flebótomos.The susceptibility of Lutzomyia townsendi to infection with amastigotes of Leishmania spp. from experimental lesions in hamsters is studied. A good correlation is found when the number of infected sandflies are compared with the distribution and density of parasites in the dermis, taking into account the size of the fascicle, particularly the length of labrun and dental depth in maxillae. Considerations are made on the susceptibilities of some anthropophilic sandflies from America that are considered as good vectors.

  4. Clinical forms of canine visceral Leishmaniasis in naturally Leishmania infantum-infected dogs and related myelogram and hemogram changes.

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    Roney de Carvalho Nicolato

    Full Text Available Hematological analysis has limited applications for disease diagnosis in Leishmania infantum-infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum-infected dogs: asymptomatic I (AD-I: serum negative/PCR+, asymptomatic II (AD-II: serum positive, oligosymptomatic (OD, and symptomatic (SD. Results were compared with those from noninfected dogs (NID. The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia, that contribute to CVL prognosis.

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

    Science.gov (United States)

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

    2016-10-15

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

  6. Infection by Leishmania spp. in Free-Ranging Opossums (Didelphis albiventris) in an Environmentally Protected Area Inhabited by Humans in Southeastern Brazil.

    Science.gov (United States)

    Paiz, Laís Moraes; Donalisio, Maria Rita; Richini-Pereira, Virgínia Bodelão; Motoie, Gabriela; Castagna, Claudio Luiz; Tolezano, José Eduardo

    2016-11-01

    There is a growing concern about the participation of wild hosts and reservoirs in the epidemiology of leishmaniasis, particularly within the context of increasingly frequent environmental changes and the expansion of the One Health concept. This work is a molecular research of infection by Leishmania spp. among the wildlife of an environmentally protected area located in the municipality of Campinas, São Paulo, Brazil. The studied area has a history of intense environmental changes, with notifications of human cases of cutaneous leishmaniasis in the 1990s, and a focus of canine visceral leishmaniasis since 2009. Eighty-two wild mammals were sampled by monthly captures in this region over a 1-year period. Blood samples were collected from each animal and subjected to DNA extraction and PCR using primers for the region of the internal transcribed spacer-1. The results of gene sequencing for the first time revealed the infection of opossums (Didelphis albiventris) by Leishmania spp., subgenera Leishmania and Viannia, in Campinas. These findings, in addition to environmental and historical characteristics of the studied area, indicate a possible role of wildlife in the introduction and/or maintenance of natural foci of leishmaniasis transmission.

  7. Estimation of canine Leishmania infection prevalence in six cities of the Algerian littoral zone using a Bayesian approach.

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    Amel Adel

    Full Text Available A large-scale study on canine Leishmania infection (CanL was conducted in six localities along a west-east transect in the Algerian littoral zone (Tlemcen, Mostaganem, Tipaza, Boumerdes, Bejaia, Jijel and covering two sampling periods. In total 2,184 dogs were tested with an indirect fluorescent antibody test (IFAT and a direct agglutination test (DAT. Combined multiple-testing and several statistical methods were compared to estimate the CanL true prevalence and tests characteristics (sensitivity and specificity. The Bayesian full model showed the best fit and yielded prevalence estimates between 11% (Mostaganem, first period and 38% (Bejaia, second period. Sensitivity of IFAT varied (in function of locality between 86% and 88% while its specificity varied between 65% and 87%. DAT was less sensitive than IFAT but showed a higher specificity (between 80% and 95% in function of locality or/and season. A general increasing trend of the CanL prevalence was noted from west to east. A concordance between the present results and the incidence of human cases of visceral leishmaniasis was observed, where also a maximum was recorded for Bejaia. The results of the present study highlight the dangers when using IFAT as a gold standard.

  8. First report of Warileya rotundipennis (Psychodidae: Phlebotominae) naturally infected with Leishmania (Viannia) in a focus of cutaneous leishmaniasis in Colombia.

    Science.gov (United States)

    Moreno, Mabel; Ferro, Cristina; Rosales-Chilama, Mariana; Rubiano, Luisa; Delgado, Marcela; Cossio, Alexandra; Gómez, Maria Adelaida; Ocampo, Clara; Saravia, Nancy Gore

    2015-08-01

    The expansion of transmission of cutaneous leishmaniasis from sylvatic ecosystems into peri-urban and domestic settings has occurred as sand flies have adapted to anthropogenic environmental modifications. Assessment of the intradomiciliary presence of sand flies in households of the settlement "La Cabaña", in the Department of Risaralda, Colombia, revealed an abundance of Warileya rotundipennis. This unexpected observation motivated further analyses to evaluate the participation of this species in the transmission of cutaneous leishmaniasis. Collections using CDC light traps were conducted during two consecutive nights in May and August 2011.The total of 667 sand flies collected were classified into five species: W. rotundipennis (n=654; 98.05%), Nyssomyia trapidoi (n=7; 1.04%); Lutzomyia (Helcocyrtomyia) hartmanni (n=3; 0.44%); Lutzomyia lichyi (n=2; 0.29%) and Psychodopygus panamensis (n=1; 0.14%). The striking predominance of W. rotundipennis within households during both wet (May) and dry (August) seasons, anthropophilic behavior demonstrated by human blood in 95.23% (60/63) evaluable blood-engorged specimens, and natural infection (5/168-3%) with genetically similar parasites of the Leishmania (Viannia) subgenus observed in a patient in this community, support the involvement of W. rotundipennis in the domestic transmission of cutaneous leishmaniasis in "La Cabaña". Copyright © 2015 Elsevier B.V. All rights reserved.

  9. [Presence of infected vectors of Leishmania (V.) panamensis within dwellings in two endemic foci in the foothill of the middle Magdalena valley, western Boyacá, Colombia].

    Science.gov (United States)

    Santamaría, Erika; Ponce, Nubia; Zipa, Yaneth; Ferro, Cristina

    2006-10-01

    Case records of leishmaniasis of the years 1997 to 2003 of the department of Boyaca showed that since the year 2000 the department experienced an unusual rise in the incidence of cutaneous leishmaniasis that might correspond to an epidemic outbreak in the western region of the department. Age and gender distribution of cases supported a domestic transmission. This research was designed to identify the vectors of cutaneous leishmaniasis in the municipalities of Otanche and Pauna through their presence in dwellings and their natural infection with the same species of Leishmania isolated from patients. Sampling of sand flies was done with CDC traps in and around dwellings. Samples from patients and pooled females of the most abundant species of Lutzomyia were used to identify the parasite by PCR. Monoclonal antibody typing was also used to confirm the identification of the parasite in samples from patients. In both municipalities L. trapidoi was the most abundant anthropophilic species of Lutzomyia indoors and around dwellings. L. hartmanni and L. yuilli were also abundant in Otanche, and L. gomezi and L. panamensis in Pauna. Leishmania (V) panamensis was identified both in patients and in the sand flies: L. yuilli, L. gomezi and L. panamensis.. Our findings prove the presence of infected vectors of Leishmania panamensis within dwellings in the towns of Otanche and Pauna in Western Boyacá. Since L. trapidoi was the most abundant species, it may be considered as the principal vector of Leishmania (V.) panamensis. The evidence of transmission within human dwellings warrants vector control at least in this environment.

  10. Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection.

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    Magdalena Radwanska

    2007-05-01

    Full Text Available Effector responses induced by polarized CD4+ T helper 2 (Th2 cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha. IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(creIL-4Ralpha(-/lox deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(creIL-4Ralpha(-/lox mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(creIL-4Ralpha(-/lox mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L

  11. Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey.

    Science.gov (United States)

    Karaku Ş, M; Pekağ Irba Ş, M; Demir, S; Eren, H; Töz, S; Özbel, Y

    2017-06-01

    Leishmaniasis is an arthropod-borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Aydın province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS-1 real-time PCR. The detection of Leishmania DNA in wild-caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Aydın province. © 2016 The Royal Entomological Society.

  12. Serological survey for Leishmania sp. infection in wild animals from the municipality of Maringá, Paraná state, Brazil

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    EM Voltarelli

    2009-01-01

    Full Text Available Leishmania sp. infection was investigated in wild animals from the Ingá Park, in the municipality of Maringá, Paraná state, Brazil, where American cutaneous leishmaniasis (ACL is an endemic disease. Sixty-five mammals, comprising Didelphis albiventris, Cerdocyon thous, Lycalopex vetulus, Cebus apella, Dasyprocta azarae, Dasypus novemcinctus, Procyon cancrivorus and Nasua nasua, were captured. Blood samples were collected for parasite cultivation. Antibodies were investigated by direct agglutination test (DAT using Leishmania (Viannia braziliensis as antigen. Flagellates were observed in blood cultures of 14 (35.9% Didelphis albiventris. Anti-Leishmania antibodies were detected in 31 (51.6% specimens of Cerdocyon thous, Lycalopex vetulus, Cebus apella, Dasyprocta azarae, Procyon cancrivorus and Nasua nasua. These results suggest that Cerdocyon thous and Lycalopex vetulus (crab-eating fox, Cebus apella (capuchin monkey, Dasyprocta azarae (agouti, Procyon cancrivorus (crab-eating raccoon and Nasua nasua (coati play an important role in the ACL transmission cycle in the northwestern region of Paraná, Brazil.

  13. Natural Leishmania (Viannia) infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil.

    Science.gov (United States)

    de Souza, Adelson Alcimar Almeida; da Rocha Barata, Iorlando; das Graças Soares Silva, Maria; Lima, José Aprígio Nunes; Jennings, Yara Lúcia Lins; Ishikawa, Edna Aoba Yassui; Prévot, Ghislaine; Ginouves, Marine; Silveira, Fernando Tobias; Shaw, Jeffrey; Dos Santos, Thiago Vasconcelos

    2017-01-01

    From 1996 to 1999 multi-trapping methods (Center of Diseases Control, CDC) light traps, light-baited Shannon traps, and aspiration on tree bases) were used to study the phlebotomine fauna of the "Serra do Navio" region of the Brazilian State of Amapá, which is part of the Guiana Shield. Fifty-three species were identified among 8,685 captured individuals. The following species, associated with the transmission of American cutaneous leishmaniasis in Amazonian Brazil, were captured: Nyssomyia umbratilis (3,388), Psychodopygus squamiventris maripaensis (995), Ny. anduzei (550), Trichophoromyia ubiquitalis (400), Ny. whitmani (291), Ps. paraensis (116), and Bichromomyia flaviscutellata (50). Flagellate infections were detected in 45 flies. Of the 19 parasites isolated in vitro, 15 were Leishmania (Viannia) guyanensis (13 in Ny. umbratilis, 1 in Ny. whitmani, 1 in Ny. anduzei) and three were L. (V.) naiffi (2 in Ps. s. maripaensis, 1 in Ny. anduzei). The results indicate the participation of three phlebotomine species in the transmission of L. (V.) guyanensis and two species in that of L. (V.) naiffi, and show that the same phlebotomine species is involved in the transmission of different Leishmania (Viannia) species in the Guianan/Amazon region. A review of the literature together with the results of the present study, and other published and unpublished results, indicate that eight phlebotomine species potentially participate in the transmission of Leishmania (Viannia) naiffi in Amazonia. © A.A.A. de Souza et al., published by EDP Sciences, 2017.

  14. Native rodent species are unlikely sources of infection for Leishmania (Viannia braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

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    Lisa A Shender

    Full Text Available An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V. braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%, Hylaeamys perenensis (15.7%, and Proechimys spp. (12%. All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V. braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V. braziliensis transmission to human inhabitants in this highly prevalent region.

  15. Native rodent species are unlikely sources of infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

    Science.gov (United States)

    Shender, Lisa A; De Los Santos, Maxy; Montgomery, Joel M; Conrad, Patricia A; Ghersi, Bruno M; Razuri, Hugo; Lescano, Andres G; Mazet, Jonna A K

    2014-01-01

    An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region.

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

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    Celia Maria Vieira Vendrame

    2014-01-01

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

  17. Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

    Science.gov (United States)

    Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

    2005-12-01

    The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

  18. Presence of amastigotes in the central nervous system of hamsters infected with Leishmania sp. Presença de amastigotas em sistema nervoso central de hamster infectado com Leishmania sp.

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    Elisangela de Oliveira

    2011-06-01

    Full Text Available Visceral leishmaniasis (VL is a severe chronic disease caused by Leishmania (Leishmania infantum chagasi. Better knowledge on the effects caused by this disease can help develop adequate clinical management and treatment. Parasitological and immunohistochemical studies were performed golden hamsters Mesocricetus auratus infected with bone marrow from individuals with VL in the State of Mato Grosso do Sul, central-west Brazil. The effects of parasitism in the spleen, liver, kidneys, lungs, heart and brain of the animals were examined. Eighteen hamsters were inoculated intraperitoneally, and six healthy animals were used as negative controls. The animals were kept in the animal house and checked for clinical signs. Specimens of each organ were examined for the presence of amastigotes. Immunohistochemical technique was performed in all brain specimens and organs negative on the direct examination of parasites. Direct examination of amastigotes was positive in the spleen and liver of all infected animals; 33.3% showed the parasite in the kidneys and lungs, and 16.7% in the heart. Parasitic forms were seen in 83.3% (15/18 of the brain examined. Immunohistochemistry confirmed the results of the direct examination, except in two specimens of lung tissue and in the brain specimens. Other studies are needed to further clarify the effect of the parasite in the central nervous system.A leishmaniose visceral (LV é uma doença crônica grave, causada pelo parasito Leishmania (Leishmania infantum chagasi. Esclarecer as alterações provocadas pela doença é fundamental para que se adotem condutas clínicas e de tratamento adequadas. Com o objetivo de analisar a infecção experimental em hamsters da linhagem golden, Mesocricetus auratus, infectados com tecido de medula óssea de pacientes com LV no Estado de Mato Grosso do Sul, foram realizados estudos parasitológicos e de imunomarcação. Foi verificada a distribuição do parasitismo no baço, f

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

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    Edézio Ferreira Cunha-Júnior

    2017-01-01

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

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

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    Somayeh GHOTLOO

    2015-12-01

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

  1. Leishmania infantum infection in dogs from maroon communities in the Eastern Amazon

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    Andréia Ferreira da Silva

    Full Text Available ABSTRACT: This study was designed to detect L. infantum infection in dogs and to evaluate the factors associated with canine visceral leishmaniasis in the maroon communities of Menino Jesus de Petimandeua and Itaboca in the municipality of Inhangapi, Pará, Brazil. Whole blood and intact skin samples were collected from 143 dogs, and a questionnaire was applied. L. infantum DNA was detected by polymerase chain reaction (PCR using primers RV1 and RV2. Collection sites were georeferenced to obtain a spatial distribution of the residences visited and infected dogs. L. infantum DNA was detected in 8.4% (12/143 of the skin samples and in 1.4% (2/143 of the blood samples. On the risk map, three clusters were observed in Itaboca and one was observed in Menino Jesus de Petimandeua. We observed that most of the inhabitants in these maroon communities live close to forested areas and do not use protection against insect vectors. The presence of canine reservoirs of L. infantum associated to environment characteristics (preserved forests and deforested areas and habits of dog owners (living near forested areas and not using any protection against insects may favor the transmission of L. infantum in the studied areas.

  2. Salivary Gland Extract Modulates the Infection of Two Leishmania enriettii Strains by Interfering With Macrophage Differentiation in the Model of Cavia porcellus

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    Lucélia J. Pinheiro

    2018-05-01

    Full Text Available The subgenus Mundinia includes several Leishmania species that have human and veterinary importance. One of those members, Leishmania Mundinia enriettii was isolated from the guinea pig Cavia porcellus in the 1940s. Several histopathological studies have already been performed in this species in the absence of salivary gland extract (SGE, which are determinant and the early and future events of the infection. Our main hypothesis is that SGE could differentially modulate the course of the lesion and macrophage differentiation caused by avirulent and virulent L. enriettii strains. Here, the C. porcellus nasal region was infected using needles with two strains of L. enriettii (L88 and Cobaia in the presence/absence of SGE and followed for 12 weeks. Those strains vary in terms of virulence, and their histopathological development was characterized. Some L88-infected animals could develop ulcerated/nodular lesions, whereas Cobaia strain developed non-ulcerated nodular lesions. Animals experimentally inoculated developed a protuberance and/or lesion after the 4th and 5th weeks of infection. Macroscopically, the size of lesion in L88-infected animals was smaller in the presence of SGE. Remarkable differences were detected microscopically in the presence of SGE for both strains. After the 6th and 7th weeks, L88-infected animals were heavily parasitized with an intense inflammatory profile bearing amastigotes and pro-inflammatory cells compared to those infected by Cobaia strain. Morphometry analysis revealed that L1+ macrophages were abundant in the L88 infection, but not in the Cobaia infection. In the presence of SGE, an increased CD163+ macrophage infiltrate by both strains was detected. Interestingly, this effect was more pronounced in Cobaia-infected animals. This study showed the role of SGE during the course of L. enriettii (strains L88 and Cobaia infection and its role in modulating macrophage attraction to the lesion site. SGE decreased L1

  3. Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice

    Czech Academy of Sciences Publication Activity Database

    Bádalová, J.; Svobodová, M.; Havelková, Helena; Vladimirov, Vladimir; Vojtíšková, Jarmila; Engová, J.; Pilčík, Tomáš; Volf, P.; Demant, P.; Lipoldová, Marie

    2002-01-01

    Roč. 2002, č. 3 (2002), s. 187-195 ISSN 1466-4879 Institutional research plan: CEZ:AV0Z5052915 Keywords : IgE * quantitative trait * Leishmania major, Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.060, year: 2002

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

    NARCIS (Netherlands)

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

    2015-01-01

    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

  5. The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

    Czech Academy of Sciences Publication Activity Database

    Rohoušová, I.; Hostomská, J.; Vlková, M.; Kobets, Tetyana; Lipoldová, Marie; Volf, P.

    2011-01-01

    Roč. 41, č. 5 (2011), s. 481-485 ISSN 0020-7519 R&D Projects: GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : Phlebotomus duboscqi * Leishmania major * anti-saliva IgG Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.393, year: 2011

  6. 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...

  7. Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

    Directory of Open Access Journals (Sweden)

    Pedro J Alcolea

    Full Text Available Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1, the glyoxylase I (GLO1, the hydrophilic surface protein B (HASPB, the methylmalonyl-CoA epimerase (MMCE and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

  8. The Seminested PCR Based Detection of Leishmania infantum Infection in Asymptomatic Dogs in a New Endemic Focus of Visceral Leishmaniasis in Iran

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    Y Rassi

    2007-06-01

    Full Text Available Visceral Leishmaniasis (Kala-azar is a serious health problem in some northern and south western parts of Iran. The incidence of kala-azar caused by Leishmania infantum has recently increased in Nourabad-Mamassani district of Fars Province, in the south of the country. This study was designed to determine the role of asymptomatic dogs as host reservoir of L. infantum in this new formed focus and detection of prevalence of infection near them. A total of 20 as¬ymptomatic stray and sheep dogs were randomly sampled. The Buffy coat layer of their peripheral blood was used for DNA extraction and PCR. A species specific seminested PCR was used for DNA amplification using LINR4, LIN17 and LIN19 primers. These primers amplified variable area of the minicircle kDNA of Leishmania parasites. Of the 20 sampled dogs checked for leishmanial kDNA, six (30% were found naturally infected. It is concluded that, dogs (Canis familiaris even if asympto¬matic, is considered as the domestic host reservoir of kala-azar in this endemic focus.

  9. Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

    Science.gov (United States)

    Alcolea, Pedro J; Alonso, Ana; Moreno-Izquierdo, Miguel A; Degayón, María A; Moreno, Inmaculada; Larraga, Vicente

    2016-01-01

    Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

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

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    Élide Aparecida Oliveira

    2011-01-01

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

  11. 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.

  12. Anti-Leishmania and cytotoxic activities of perillaldehyde epoxide synthetic positional isomers.

    Science.gov (United States)

    Keesen, Tatjana Souza Lima; da Silva, Larisse Virgolino; da Câmara Rocha, Juliana; Andrade, Luciana Nalone; Lima, Tamires Cardoso; de Sousa, Damião Pergentino

    2018-03-13

    Leishmaniasis belongs to a complex of zoonotic disease caused by protozoa of the genus Leishmania and is considered a major public health problem. Several essential oil chemical components have inhibitory effect against protozoa, including Leishmania donovani. Thus, the aim of this study was to evaluate for the first time the anti-Leishmania activity of two p-menthane monoterpene isomers (EPER-1: perillaldehyde 1,2-epoxide and EPER-2: perillaldehyde 8,9-epoxide) against L. donovani promastigotes as well as evaluating cytotoxic effect on mononuclear peripheral blood cells. Results of anti-Leishmania assay revealed that EPER-2 (IC 50  = 3.8 μg.mL -1 ) was 16-fold more potent than its isomer EPER-1 (IC 50  = 64.6 μg.mL -1 ). In contrast to PBMC cells, EPER-2 was not cytotoxic (IC 50  > 400 μg.mL -1 ) when compared to positive control. These data suggest that the disposition of epoxide group into the p-menthane skeleton affects the anti-Leishmania activity, being that the presence of the exocyclic epoxide group considerably increased potency. Thus, it was possible to observe that the location of the epoxide group into the p-menthane skeleton resulted in different potencies.

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

    Science.gov (United States)

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

    2017-04-01

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

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

    Science.gov (United States)

    Dias-Teixeira, Karina Luiza; Calegari-Silva, Teresa Cristina; dos Santos, Guilherme R R M; Vitorino Dos Santos, José; Lima, Carolina; Medina, Jorge Mansur; Aktas, Bertal Huseyin; Lopes, Ulisses G

    2016-04-01

    Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR duringLeishmania amazonensisinfection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 μM) increasedL. amazonensisinfectivity in an IFN1-α receptor (IFNAR)-dependent manner. In Western blot assays, we showed thatL. amazonensisactivates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for theIfnbgene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude thatL. amazonensisactivation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-β expression.-Dias-Teixeira, K. L., Calegari-Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response inLeishmania amazonensismacrophage infection: the role of X-box binding protein 1 transcription factor. © FASEB.

  18. Age structure, blood-feeding behavior, and Leishmania chagasi infection in Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia.

    Science.gov (United States)

    Ferro, C; Morrison, A C; Torres, M; Pardo, R; Wilson, M L; Tesh, R B

    1995-09-01

    Ecological studies on the sand fly Lutzomyia longipalpis (Lutz & Neiva) were conducted during 1990-1992 in a small rural community in Colombia where American visceral leishmaniasis (AVL) is endemic. Subsamples of sand flies collected weekly from pigpens, the interior of houses, and natural outdoor resting sites were dissected to determine physiological age and Leishmania chagasi Cunha & Chagas infection rates. Eleven female L. longipalpis had flagellates in their gut, 2 of which were successfully cultured and identified as Leishmania chagasi. The reproductive status, stage of ovarian development, and trophic history of female sand flies varied among sites, habitats, and time of collection. The percentage of parous females ranged from about one-third to two-thirds overall and varied seasonally. Of most relevance to AVL transmission was the finding that 8% of L. longipalpis females were multiparous. In addition, our data suggest that L. longipalpis rest inside houses after blood-feeding outdoors, and that this species can blood-feed more than once during a single gonotrophic cycle.

  19. Effect of Leishmania spp infection on the survival, life expectancy, fecundity and fertility of Lutzomyia longipalpiss.l. and Lutzomyia pseudolongipalpis

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    Irma Fatima Agrela

    2015-08-01

    Full Text Available We evaluated the effects of Leishmaniaspp infection on several population parameters of Lutzomyia longipalpis sensu lato andLutzomyia pseudolongipalpis, vectors of visceral leishmaniasis in Venezuela, under experimental conditions during the first post-feeding period. Females of both species were allowed to feed and engorge on a suspension of fresh washed human red blood cells in foetal calf serum. These blood cells were either non-infected or infected with one of the fourLeishmaniaspp strains and were offered through a chicken skin membrane. The longevity, life expectancy and the fecundity of uninfected flies were similar in both species, but the fertility was significantly lower in uninfected Lu. longipalpis females. In all cases, the infection of Lu. longipalpis and Lu. pseudolongipalpis by the Leishmaniastrains resulted in significant detrimental effects, which exerted a fitness cost expressed by reduced survival and life expectancy, as well as decreased fertility and fecundity compared with the control groups. Nevertheless, differences in these parameters were observed between these vector species depending on whether they were infected with the autochthonous Venezuelan Leishmania infantum strain (NESA or the Brazilian reference strain (PP75. The experimental data obtained agree with field data on the natural infection of these vector species and the significance of this scenario is discussed.

  20. A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

    Science.gov (United States)

    Rampazzo, Rita de Cássia Pontello; Solcà, Manuela da Silva; Santos, Liliane Celestino Sales; Pereira, Lais de Novaes; Guedes, José Carlos Oliveira; Veras, Patrícia Sampaio Tavares; Fraga, Deborah Bittencourt Mothé; Krieger, Marco Aurélio; Costa, Alexandre Dias Tavares

    2017-11-15

    Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format

  1. Leishmania in sand flies: comparison of quantitative polymerase chain reaction with other techniques to determine the intensity of infection

    Czech Academy of Sciences Publication Activity Database

    Myšková, J.; Votýpka, Jan; Volf, P.

    2008-01-01

    Roč. 45, č. 1 (2008), s. 133-138 ISSN 0022-2585 R&D Projects: GA MŠk(CZ) LC06009 Grant - others:Univerzita Karlova v Praze(CZ) 195/2005/B-BIO/PrF Institutional research plan: CEZ:AV0Z60220518 Keywords : sand fly * Leishmania * Phlebotomus * parasite-vector interaction * RT-PCR Subject RIV: EG - Zoology Impact factor: 1.967, year: 2008

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

    Directory of Open Access Journals (Sweden)

    Verçosa BLA

    2011-01-01

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

  3. Epidemiological survey on Leishmania infection in red foxes (Vulpes vulpes) and hunting dogs sharing the same rural area in Southern Italy.

    Science.gov (United States)

    Piantedosi, Diego; Veneziano, Vincenzo; Di Muccio, Trentina; Manzillo, Valentina Foglia; Fiorentino, Eleonora; Scalone, Aldo; Neola, Benedetto; Di Prisco, Francesca; D'Alessio, Nicola; Gradoni, Luigi; Oliva, Gaetano; Gramiccia, Marina

    2016-12-01

    Southern Italy, particularly Campania region, is an area where canine leishmaniasis (CanL) and zoonotic human visceral leishmaniasis (VL) are endemic. The red fox (Vulpes vulpes) has been hypothesized to play a role in occurrence of CanL in Italy but specific studies are poor. The aim of the present survey was to investigate the prevalence of Leishmania infection in dogs and foxes living in the same rural area (Picentini hills). 123 sera from autochthonous fox-hunting dogs were examined by immunofluorescent-antibody test (IFAT) using a cut-off of 1:160. The seroprevalence of dogs examined was 17.9%. Moreover, 48 foxes were examined after having been shooted by hunters or road accidents. Spleen, liver and lymph node samples were analyzed by specific Leishmania nested PCR (n-PCR). 10 foxes were found infected by L. infantum (20.8%) of which 4 animals in spleen, 2 in lymph nodes and 4 both in spleen and lymph nodes. The overall n-PCR positivity was 17.4% for spleen samples and 13.3% for lymph nodes; all liver samples resulted negative. In positive PCR foxes no signs clearly referable to leishmaniasis were recorded at necropsy. The results confirmed the presence of L. infantum infection in red foxes from Southern Italy, with a moderate level of exposure. Because large proportions of dogs with ascertained progressive leishmaniasis show a prolonged "subpatent condition" during which they are only positive to n-PCR before seroconversion, our results allow to assume that exposure risk in foxes is lower than hunting dogs living in the studied area.

  4. Cutaneous leishmaniosis in naturally infected dogs in Paraná, Brazil, and the epidemiological implications of Leishmania (Viannia) braziliensis detection in internal organs and intact skin.

    Science.gov (United States)

    Marquez, Ellen de Souza; de Castro, Edilene Alcântara; Nabut, Luciene Biazono; da Costa-Ribeiro, Magda Clara Vieira; Dela Coletta Troiano Araújo, Ludmilla; Poubel, Saloe Bispo; Gonçalves, André Luiz; Cruz, Mariza Fordellone Rosa; Dos Santos Trad, Ana Paula Millet Evangelista; Dias, Rafael Andre Ferreira; Navarro, Italmar Teodorico; Thomaz-Soccol, Vanete

    2017-08-30

    Environmental changes have occurred over the years, altering the eco-epidemiological pattern of leishmaniosis in the State of Paraná, Brazil, involving the pillars of the cycle (parasite, vectors, reservoir, and environment) and their interaction. Much has been discussed about the dog's role as a reservoir of the Leishmania (Viannia) braziliensis Vianna, 1911 transmission cycle. However, this question remains unanswered. The purpose of this study was to investigate, using parasitological and molecular methods, different samples in eight naturally infected dogs from an endemic rural locality where only L. (V.) braziliensis is present, and where human cases have been previously notified. Blood and biopsied organ samples from naturally infected dogs were analyzed by culture media, PCR, random amplified polymorphic DNA and sequencing methodologies. Only skin lesions from all dogs yielded positive cultures and when PCR was performed, L. (V.) braziliensis DNA was amplified from intact skin, peripheral blood, bone marrow, spleen, liver and lymph nodes. RAPD was also applied to isolates from the skin lesions, exhibiting the genetic variability of the parasite identified. To confirm which species of Leishmania was amplified in PCR, the sequencing method was performed, verifying 100% similarity with the Viannia subgenus. This study showed that L. (V.) braziliensis can spread to other sites besides the ulcerous lesions, such as intact skin, peripheral blood and internal organs, making it possibility for dogs to serve as active sources of parasite transmission. For definitive proof, xenodiagnostic test on intact skin of infected dogs, should be done. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. First description of Leishmania (Viannia) infection in Evandromyia saulensis, Pressatia sp. and Trichophoromyia auraensis (Psychodidae: Phlebotominae) in a transmission area of cutaneous leishmaniasis in Acre state, Amazon Basin, Brazil

    Science.gov (United States)

    de Araujo-Pereira, Thais; de Pita-Pereira, Daniela; Boité, Mariana Côrtes; Melo, Myllena; da Costa-Rego, Taiana Amancio; Fuzari, Andressa Alencastre; Brazil, Reginaldo Peçanha; Britto, Constança

    2017-01-01

    Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites. PMID:28076470

  6. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies

    Directory of Open Access Journals (Sweden)

    Elnaiem Dia-Eldin

    2008-01-01

    Full Text Available Abstract Background In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Results Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5, two peritrophin like proteins, a trypsin like protein (Lltryp1, two chymotrypsin like proteins (LuloChym1A and 2 and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3, a trypsin like protein (Lltryp2 and an astacin-like metalloprotease (LuloAstacin. Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5, a Chymotrypsin (LuloChym1A and a carboxypeptidase (LuloCpepA1, among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1, a trypsin-like protein (Lltryp2 and an unknown protein. Conclusion This transcriptome analysis represents the largest set

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

    Science.gov (United States)

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

    2015-11-01

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

  8. Leishmania mexicana amazonensis: heterogeneity in 5-nucleotidase and peroxidase activities of mononuclear phagocytes during in vivo and in vitro infection Leishmania mexicana amazonensis: heterogeneidade da 5’-Nucleotidase e da peroxidase em fagócitos mononucleares durante infecção in vivo e in vitro

    OpenAIRE

    Suzana Côrte-Real; Gabriel Grimaldi Junior; Maria de Nazareth Leal de Meirelles

    1988-01-01

    The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonenesis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastrctural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells, and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. onl...

  9. Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay.

    Science.gov (United States)

    Acardi, Soraya Alejandra; Liotta, Domingo Javier; Santini, María Soledad; Romagosa, Carlo Mariano; Salomón, Oscar Daniel

    2010-09-01

    In this study, a genotypification of Leishmania was performed using polimerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing techniques to identify species of Leishmania parasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpis were grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantum by PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpis among the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantum in the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

  10. Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum.

    Science.gov (United States)

    Cajueiro, Ana Paula Bacellar; Goma, Ester Puna; Dos Santos, Hilton Antônio Mata; Almeida Rodrigues, Igor; Toma, Helena Keiko; Araújo, Silvana Marques; Bonamin, Leoni Villano; Gomes, Nelson Brêtas de Noronha; Castelo-Branco, Morgana Teixeira Lima; de Souza Dias, Edilma Paraguai; Dos Santos Pyrrho, Alexandre; Holandino, Carla

    2017-07-01

    The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n=138) infected with Leishmania infantum, and treated with Leishmania infantum 30× (10 -30 ) biotherapy - BioLi30×. We performed controlled experiments using 8-to-12-week-old mice, infected with 5×10 7 L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30× pre-treated), G4 (BioLi30× pre/post-treated), G5 (BioLi30× post-treated), G6 (Water 30× post-treated), G7 (Antimonium crudum 30× post-treated) and G8 (Glucantime® post-treated). G3-G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received Glucantime® (0.6mg/100µl of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals' serum was measured after 49days of infection, and cytokines (IFN-γ, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-γ/IL-4 and IFN-γ/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3-G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2-G8), and intense disruption (G2-G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-γ/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Phlebotomine sand fly survey in the focus of leishmaniasis in Madrid, Spain (2012-2014): seasonal dynamics, Leishmania infantum infection rates and blood meal preferences.

    Science.gov (United States)

    González, Estela; Jiménez, Maribel; Hernández, Sonia; Martín-Martín, Inés; Molina, Ricardo

    2017-08-01

    An unusual increase of human leishmaniasis cases due to Leishmania infantum is occurring in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys have shown that Phlebotomus perniciosus is the only potential vector. Direct xenodiagnosis in hares (Lepus granatensis) and rabbits (Oryctolagus cuniculus) collected in the focus area proved that they can transmit parasites to colonized P. perniciosus. Isolates were characterized as L. infantum. The aim of the present work was to conduct a comprehensive study of sand flies in the outbreak area, with special emphasis on P. perniciosus. Entomological surveys were done from June to October 2012-2014 in 4 stations located close to the affected area. Twenty sticky traps (ST) and two CDC light traps (LT) were monthly placed during two consecutive days in every station. LT were replaced every morning. Sand fly infection rates were determined by dissecting females collected with LT. Molecular procedures applied to study blood meal preferences and to detect L. infantum were performed for a better understanding of the epidemiology of the outbreak. A total of 45,127 specimens belonging to 4 sand fly species were collected: P. perniciosus (75.34%), Sergentomyia minuta (24.65%), Phlebotomus sergenti (0.005%) and Phlebotomus papatasi (0.005%). No Phlebotomus ariasi were captured. From 3203 P. perniciosus female dissected, 117 were infected with flagellates (3.7%). Furthermore, 13.31% and 7.78% of blood-fed and unfed female sand flies, respectively, were found infected with L. infantum by PCR. The highest rates of infected P. perniciosus were detected at the end of the transmission periods. Regarding to blood meal preferences, hares and rabbits were preferred, although human, cat and dog blood were also found. This entomological study highlights the exceptional nature of the Leishmania outbreak occurring in southwestern Madrid, Spain. It is confirmed that P. perniciosus is the only vector in the affected area

  12. Immune modulating effects of NKT cells in a physiologically low dose Leishmania major infection model after αGalCer analog PBS57 stimulation.

    Science.gov (United States)

    Griewank, Klaus G; Lorenz, Beate; Fischer, Michael R; Boon, Louis; Lopez Kostka, Susanna; von Stebut, Esther

    2014-06-01

    Leishmaniasis is a parasitic infection affecting ∼12 million people worldwide, mostly in developing countries. Treatment options are limited and no effective vaccines exist to date. Natural Killer T (NKT) cells are a conserved innate-like lymphocyte population with immunomodulating effects in various settings. A number of reports state a role of NKT cells in different models of Leishmania infection. Here, we investigated the effect of NKT cells in a physiologically relevant, intradermal low dose infection model. After inoculation of 103 infectious-stage L. major, comparable numbers of skin-immigrating NKT cells in both susceptible BALB/c mice and resistant C57BL/6 mice were noted. Compared to their wild type counterparts, NKT cell-deficient mice on a C57BL/6 background were better able to contain infection with L. major and showed decreased IL-4 production in cytokine analysis performed 5 and 8 weeks after infection. Low doses of the NKT cell stimulating αGalCer analog PBS57 applied at the time of infection led to disease exacerbation in C57BL/6 wild-type, but not NKT-deficient mice. The effect was dependent both on the timing and amount of PBS57 administered. The effect of NKT cell stimulation by PBS57 proved to be IL-4 dependent, as it was neutralized in IL-4-deficient C57BL/6 or anti-IL-4 antibody-treated wild-type mice. In contrast to C57BL/6 mice, administration of PBS57 in susceptible BALB/c mice resulted in an improved course of disease. Our results reveal a strain- and cytokine-dependent regulatory role of NKT cells in the development of immunity to low dose L. major infections. These effects, probably masked in previous studies using higher parasite inocula, should be considered in future therapy and immunization approaches.

  13. Dynamics and Predictive Potential of Antibodies against Insect-Derived Recombinant Leishmania infantum Proteins during Chemotherapy of Naturally Infected Dogs

    Science.gov (United States)

    Todolí, Felicitat; Galindo, Inmaculada; Gómez-Sebastián, Silvia; Pérez-Filgueira, Mariano; Escribano, José M.; Alberola, Jordi; Rodríguez-Cortés, Alhelí

    2010-01-01

    A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis. PMID:20439957

  14. Phylogenomic reconstruction supports supercontinent origins for Leishmania.

    Science.gov (United States)

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

    2016-03-01

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

  15. Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

    Science.gov (United States)

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L

    2014-01-01

    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, subunit, 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 Leishmania 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 LdCen(-/-) 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 in normal

  16. Biomarkers of Safety and Immune Protection for Genetically Modified Live Attenuated Leishmania Vaccines Against Visceral Leishmaniasis – Discovery and Implications

    Science.gov (United States)

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L.

    2014-01-01

    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, subunit, 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 Leishmania 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 LdCen−/− 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 in normal

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

    2017-01-01

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

  19. Experimental Acquisition, Development, and Transmission of Leishmania tropica by Phlebotomus duboscqi

    Science.gov (United States)

    2013-01-01

    ac ta t ropica Experimental acquisition, development, and transmission of Leishmania tropica by Phlebotomus duboscqi Hanafi A. Hanafi, El...August 2012 Accepted 2 September 2012 Available online 10 September 2012 Keywords: Phlebotomus duboscqi Leishmania tropica Transmission Vector...competency a b s t r a c t We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand

  20. Serologic evidence of Leishmania infection in free-ranging wild and domestic canids around a Brazilian National Park

    Directory of Open Access Journals (Sweden)

    Nelson Henrique de Almeida Curi

    2006-02-01

    Full Text Available Transmission of disease between wildlife, domestic animals, and humans is of great concern to conservation issues and public health. Here we report on the prevalence of anti-Leishmania sp. antibodies in 21 wild canids (7 Chrysocyon brachyurus, 12 Cerdocyon thous, and 2 Lycalopex vetulus and 74 free domestic dogs (Canis familiaris sampled around the Serra do Cipó National Park. In dogs, the apparent prevalence was 8.1% and in wild canids it was 19% (2 crab-eating foxes, C. thous, and 2 maned wolves, C. brachyurus. Management of the domestic dog population with evaluation of incidence changes in humans and wildlife, and enlightenment on the role of wild reservoirs are essential issues for future action and research.

  1. Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

    Directory of Open Access Journals (Sweden)

    Floriane Auderset

    Full Text Available The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+ T helper (Th 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1 and Notch2 (N2 are expressed on activated CD4(+ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

  2. Natural Leishmania (Viannia) spp. infections in phlebotomine sand flies (Diptera: Psychodidae) from the Brazilian Amazon region reveal new putative transmission cycles of American cutaneous leishmaniasis

    Science.gov (United States)

    de Souza, Adelson Alcimar Almeida; dos Santos, Thiago Vasconcelos; Jennings, Yara Lúcia Lins; Ishikawa, Edna Aoba Yassui; Barata, Iorlando da Rocha; Silva, Maria das Graças Soares; Lima, José Aprígio Nunes; Shaw, Jeffrey; Lainson, Ralph; Silveira, Fernando Tobias

    2016-01-01

    In Amazonian Brazil the etiological agents of American cutaneous leishmaniasis (ACL) belong to at least seven Leishmania species but little is known about the putative phlebotomine sand fly vectors in different biomes. In 2002–2003 a survey of the phlebotomine fauna was undertaken in the “Floresta Nacional do Tapajós”, Belterra municipality, in the lower Amazon region, western Pará State, Brazil, where we recently confirmed the presence of a putative hybrid parasite, L. (V.) guyanensis × L. (V.) shawi shawi. Sand flies were collected from Centers for Disease Control (CDC) light traps, Shannon traps and by aspiration on tree bases. Females were dissected and attempts to isolate any flagellate infections were made by inoculating homogenized midguts into Difco B45 medium. Isolates were characterized by monoclonal antibodies and isoenzyme electrophoresis. A total of 9,704 sand flies, belonging to 68 species or subspecies, were collected. Infections were found in the following sand flies: L. (V.) naiffi with Psychodopygus hirsutus hirsutus (1) and Ps. davisi (2); and L. (V.) shawi shawi with Nyssomyia whitmani (3) and Lutzomyia gomezi (1). These results provide strong evidence of new putative transmission cycles for L. (V.) naiffi and L. (V.) s. shawi. PMID:27235194

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

    Science.gov (United States)

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

    2017-03-13

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

  4. Erythema exsudativum multiforme after a Leishmania skin test

    NARCIS (Netherlands)

    Wind, Bas S.; Guimarães, Luiz H.; Machado, Paulo R. L.

    2014-01-01

    A 45-year-old otherwise healthy male from an endemic region for Leishmania braziliensis infection in Bahia, Brazil, presented with three erosive hemorrhagic infiltrated plaques on the left shin accompanied with lymphadenopathy in the groin since one month. A Leishmania skin test performed on the

  5. Clonal variation within a mucosal isolate derived from a patient with Leishmania (Viannia braziliensis infection Variação clonal de um isolado derivado de um paciente com infecção mucosa pela Leishmania (Viannia braziliensis

    Directory of Open Access Journals (Sweden)

    César Augusto Cuba-Cuba

    1991-10-01

    Full Text Available Three isolates over 5 years from a patient with persistent relapsing mucosal leishmaniasis due to Leishmania (Viannia braziliensis and 7 clones from one of these isolates were studied by zymodemes and scrodemes analysis. Results showed evidences of clonal phenotypic variation. Eight isoenzymes markers demonstrated clear differences on Cellulose Acetate (CA and thin starch gel electrophoresis. Also a panel of specific monoclonal antibodies showed such differences. Our observations provide additional evidence that Leishmania (Viannia braziliensis is composed by subpopulations of parasites with peculiar biochemical and antigenic characteristics.No transcurso de um período de 5 anos foram estudados 3 isolados de um paciente com leishmaniose mucosa recidivante causada pela Leishmania (Viannia braziliensis e 7 clones de um desses isolados. Este estudo foi feito pela análise dos serodemas e zimodemas. Os resultados indicaram a ocorrência de variações fenotípicas clonais. Oito marcadores isoenzimáticos demonstraram diferenças nos padrões eletroforéticos em Acetato de Celulose (AC, bem como em camada fina de amido. Da mesma forma foram consultadas diferenças em um painel de anticorpos monoclonais específicos e subespecíficos. Nossas observações indicam ainda que a Leishmania (Viannia braziliensis está composta por subpopulações de parasitas com características bioquímicas e antigênicas peculiares.

  6. Detection of Leishmania RNA virus in Leishmania parasites.

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    Haroun Zangger

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  8. BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production.

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    Diego L Costa

    Full Text Available BACKGROUND: Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis-infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R and LTCP15171(S, which are resistant and susceptible to antimony and nitric oxide (NO, respectively. Mice inoculated with LTCP393(R presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S. Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R-inoculated animals and also arginase I (Arg I expression. Moreover, anti-IL-4 monoclonal antibody (mAb treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R, but not in those inoculated with LTCP15171(S. CONCLUSION/SIGNIFICANCE: We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect

  9. Anti-L. donovani activity in macrophage/amastigote model of palmarumycin CP18 and its large scale production.

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    Ortega, Humberto E; Teixeira, Eliane de Morais; Rabello, Ana; Higginbotham, Sarah; Cubilla-Ríos, Luis

    2014-01-01

    Palmarumycin CP18, isolated from an extract of the fermentation broth and mycelium of the Panamanian endophytic fungus Edenia sp., was previously reported with strong and specific activity against Leishmania donovani. Here we report that when the same strain was cultured on different solid media--Harrold Agar, Leonian Agar, Potato dextrose Agar (PDA), Corn Meal Agar, Honey Peptone Agar, and eight vegetables (V8) Agar--in order to determine the optimal conditions for isolation of palmarumycin CP18, no signal for this compound was observed in any of the 1H NMR spectra of fractions obtained from these extracts. However, one extract, prepared from the fungal culture in PDA contained significant amounts of CJ-12,372, a possible biosynthetic precursor of palmarumycin CP18. Edenia sp. was cultivated on a large scale on PDA and CJ-12,372 was converted to palmarumycin CP18 by oxidation of its p-hydroquinone moiety with DDQ in dioxane. Palmarumycin CP18 showed anti-leishmanial activity against L. donovani in a macrophage/amastigote model, with IC50 values of 23.5 microM.

  10. Identification of immunodominant Leishmania major antigenic markers of the early C57BL/6 and BALB/c mice infection stages.

    Science.gov (United States)

    Sassi, Atfa; Kaak, Olfa; Elgaaied, Amel Benammar

    2015-08-24

    The C57BL/6 mouse strain is resistant to Leishmania (L.) major infection and, unlike susceptible BALB/c, develops small self healing cutaneous lesions. The specific antibody responses of C57BL/6 and BALB/c mice were previously characterized by the predominance of IgG2a ("resistant" isotype associated with Th1) and Ig