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Sample records for chemotypes targeting leishmania

  1. Target Oriented Drugs against Leishmania.

    Science.gov (United States)

    1980-01-31

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

  2. Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51.

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    Shamila S Gunatilleke

    Full Text Available BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51 for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50 <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50 of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5, fatty acid ω-hydroxylases (CYP4, 17α-hydroxylase/17,20-lyase (CYP17 and aromatase (CYP19. Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP

  3. Herbal extract targets in Leishmania tropica.

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

    2015-12-01

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

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

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    Rakesh N. R.

    2013-06-01

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

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

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    Canton, Johnathan; Kima, Peter E

    2012-10-01

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

  6. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

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    Miguel Angel Moreno

    2014-12-01

    Full Text Available Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.

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

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    Robert Duncan

    2011-01-01

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

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

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    Requena Jose M

    2010-01-01

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

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

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    Laura-Isobel McCall

    2015-03-01

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

  10. 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.

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

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

    2016-07-01

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

  12. Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

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    Gazanion, Élodie; Fernández-Prada, Christopher; Papadopoulou, Barbara; Leprohon, Philippe; Ouellette, Marc

    2016-01-01

    Gain-of-function screens using overexpression genomic libraries are powerful tools for discovering drug target/resistance genes, but several limitations make this technique less amenable to high-throughput screening. Using cosmid-based functional screening coupled to next-generation sequencing, an approach that we term Cosmid Sequencing (or “Cos-Seq”), we followed the dynamics of cosmid enrichment during drug pressure in Leishmania, the parasite responsible for leishmaniasis, a neglected trop...

  13. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples.

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    Mohammadiha, A; Mohebali, M; Haghighi, A; Mahdian, R; Abadi, A R; Zarei, Z; Yeganeh, F; Kazemi, B; Taghipour, N; Akhoundi, B

    2013-01-01

    Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (Preal-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively.

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

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

    2015-11-01

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

  15. Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

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    Gazanion, Élodie; Fernández-Prada, Christopher; Papadopoulou, Barbara; Leprohon, Philippe; Ouellette, Marc

    2016-05-24

    Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

  16. Efficacy of Withania somnifera chemotypes NMITLI - 101R, 118R and Withaferin A against experimental visceral leishmaniasis.

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    Tripathi, C D P; Gupta, R; Kushawaha, P K; Mandal, C; Misra Bhattacharya, S; Dube, A

    2014-06-01

    The immunoprophylactic and therapeutic potentials of root extracts of Withania somnifera chemotypes (NMITLI-118, NMITLI-101) and pure withanolide-withaferin A was investigated against Leishmania donovani infection in hamsters. The naive animals, fed orally with immunostimulatory doses of chemotypes 101R, 118R (10 and 3 mg/kg) and withaferin A (9 and 3 mg/kg) for five consecutive days and challenged with Leishmania parasites on day 6, were euthanized on days 30 and 45 p.c. for the assessment of parasite clearance, real-time analysis of mRNAs of Th1/Th2 cytokines (IFN-γ, IL-12, TNF-α, iNOS/IL-4, IL-10 and TGF-β), NO production, reactive oxygen species (ROS) generation, lymphocyte transformation test and antibody responses. By day 45 p.c., there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α but decrease in IL-4, IL-10 and TGF-β, an enhanced Leishmania-specific LTT response as well as ROS, NO and antileishmanial IgG2 levels in 101R-treated hamsters followed by 118R- and withaferin A-treated ones, respectively. When these chemotypes were given to L. donovani-infected hamsters at different doses, there was moderate therapeutic efficacy of chemotype 101R (~50%) at 30 mg/kg × 5 followed by the other two. The results established that the 101R is the most potential chemotype and can be evaluated for combination therapy along with available antileishmanials.

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

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    Pramod K Kushawaha

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

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

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    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

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

  19. Chemotyping of yeast mutants using robotics.

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    Rieger, K J; El-Alama, M; Stein, G; Bradshaw, C; Slonimski, P P; Maundrell, K

    1999-07-01

    By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

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

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    Zhang, Wen-Wei; Lypaczewski, Patrick

    2017-01-01

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

  1. Polypharmacology directed compound data mining: identification of promiscuous chemotypes with different activity profiles and comparison to approved drugs.

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    Hu, Ye; Bajorath, Jürgen

    2010-12-27

    Increasing evidence that many pharmaceutically relevant compounds elicit their effects through binding to multiple targets, so-called polypharmacology, is beginning to change conventional drug discovery and design strategies. In light of this paradigm shift, we have mined publicly available compound and bioactivity data for promiscuous chemotypes. For this purpose, a hierarchy of active compounds, atomic property based scaffolds, and unique molecular topologies were generated, and activity annotations were analyzed using this framework. Starting from ∼35 000 compounds active against human targets with at least 1 μM potency, 33 chemotypes with distinct topology were identified that represented molecules active against at least 3 different target families. Network representations were utilized to study scaffold-target family relationships and activity profiles of scaffolds corresponding to promiscuous chemotypes. A subset of promiscuous chemotypes displayed a significant enrichment in drugs over bioactive compounds. A total of 190 drugs were identified that had on average only 2 known target annotations but belonged to the 7 most promiscuous chemotypes that were active against 8-15 target families. These drugs should be attractive candidates for polypharmacological profiling.

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

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    Kumar, Devanand; Kumar, Diwakar; Saha, Swati

    2012-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Zehavi, U.; El-On, J.

    1981-01-31

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

  4. Soft coral Sarcophyton (Cnidaria: Anthozoa: Octocorallia species diversity and chemotypes.

    Directory of Open Access Journals (Sweden)

    Satoe Aratake

    Full Text Available Research on the soft coral genus Sarcophyton extends over a wide range of fields, including marine natural products and the isolation of a number of cembranoid diterpenes. However, it is still unknown how soft corals produce this diverse array of metabolites, and the relationship between soft coral diversity and cembranoid diterpene production is not clear. In order to understand this relationship, we examined Sarcophyton specimens from Okinawa, Japan, by utilizing three methods: morphological examination of sclerites, chemotype identification, and phylogenetic examination of both Sarcophyton (utilizing mitochondrial protein-coding genes MutS homolog: msh1 and their endosymbiotic Symbiodinium spp. (utilizing nuclear internal transcribed spacer of ribosomal DNA: ITS- rDNA. Chemotypes, molecular phylogenetic clades, and sclerites of Sarcophyton trocheliophorum specimens formed a clear and distinct group, but the relationships between chemotypes, molecular phylogenetic clade types and sclerites of the most common species, Sarcophyton glaucum, was not clear. S. glaucum was divided into four clades. A characteristic chemotype was observed within one phylogenetic clade of S. glaucum. Identities of symbiotic algae Symbiodinium spp. had no apparent relation to chemotypes of Sarcophyton spp. This study demonstrates that the complex results observed for S. glaucum are due to the incomplete and complex taxonomy of this species group. Our novel method of identification should help contribute to classification and taxonomic reassessment of this diverse soft coral genus.

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

    Science.gov (United States)

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

    2017-02-06

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

  6. 3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies.

    Science.gov (United States)

    Couto, Marcos; Sánchez, Carina; Dávila, Belén; Machín, Valentina; Varela, Javier; Álvarez, Guzmán; Cabrera, Mauricio; Celano, Laura; Aguirre-López, Beatriz; Cabrera, Nallely; de Gómez-Puyou, Marieta Tuena; Gómez-Puyou, Armando; Pérez-Montfort, Ruy; Cerecetto, Hugo; González, Mercedes

    2015-08-12

    The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.

  7. 3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies

    Directory of Open Access Journals (Sweden)

    Marcos Couto

    2015-08-01

    Full Text Available The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM. Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.

  8. Discrimination of fennel chemotypes applying IR and Raman spectroscopy – discovery of a new -asarone chemotype

    Directory of Open Access Journals (Sweden)

    Krähmer, Andrea

    2016-07-01

    Full Text Available Various vibrational spectroscopy methods have been applied to classify different fennel chemotypes according to their individual profile of volatile substances. Intact fennel fruits of different chemotypes could be successfully discriminated by Attenuated Total Reflectance Fourier transform Infrared (ATR-FTIR and Near Infrared (NIR spectroscopy. Solvent extracts (CCl4 of the considered fennel fruits showed characteristic fingerprints with marker bands related to the individual volatile components (trans-anethole, fenchone, estragole, piperitenone oxide, -asarone, limonene for ATR-FTIR and FT-Raman spectroscopy. Especially C=C and C=O absorption bands contribute to the different spectral profiles. Based on hierarchical cluster analysis, the considered fennel accessions were classified according to gas chromatographic (GC and vibrational spectroscopic data. Furthermore, even a discrimination of “sweet” and “bitter” fennel fruits, both belonging to the trans-anethole chemotype, could be successfully performed. All vibrational spectroscopical techniques used in this study are rapid and easy to apply. Hence, they allow different fennel chemotypes to be reliably distinguished and can also be used for on-site measurement in free nature.

  9. Discrimination of fennel chemotypes applying IR and Raman spectroscopy: discovery of a new γ-asarone chemotype.

    Science.gov (United States)

    Gudi, Gennadi; Krähmer, Andrea; Krüger, Hans; Hennig, Lothar; Schulz, Hartwig

    2014-04-23

    Various vibrational spectroscopy methods have been applied to classify different fennel chemotypes according to their individual profile of volatile substances. Intact fennel fruits of different chemotypes could be successfully discriminated by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and near infrared (NIR) spectroscopy. Solvent extracts (CCl4) of the considered fennel fruits showed characteristic fingerprints with marker bands related to the individual volatile components (trans-anethole, fenchone, estragole, piperitenone oxide, γ-asarone, limonene) for ATR-FTIR and FT-Raman spectroscopy. Especially νC═C and νC═O absorption bands contribute to the different spectral profiles. On the basis of hierarchical cluster analysis, the considered fennel accessions were classified according to gas chromatographic (GC) and vibrational spectroscopic data. Furthermore, even a discrimination of "sweet" and "bitter" fennel fruits, both belonging to the trans-anethole chemotype, could be successfully performed. All vibrational spectroscopical techniques used in this study are rapid and easy to apply. Hence, they allow different fennel chemotypes to be reliably distinguished and can also be used for on-site measurement in free nature.

  10. Leishmania Skin Test

    Science.gov (United States)

    2010-03-01

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

  11. The discovery of novel human androgen receptor antagonist chemotypes using a combined pharmacophore screening procedure.

    Science.gov (United States)

    Voet, Arnout; Helsen, Christine; Zhang, Kam Y J; Claessens, Frank

    2013-04-01

    Unraveling the mechanisms involved in castration- and therapy-resistant prostate cancer has led to a renewed interest in androgen receptor (AR)-targeted therapeutics. Anti-androgens that block the activity of the AR therefore remain a valid therapeutic option. However, they must be more effective than, or display a distinct mechanism of action or binding mode from those of bicalutamide and hydroxyflutamide, which are currently in clinical use. For that reason, the second-generation anti-androgen MDV3100 was developed. MDV3100, however, shares its 4-cyano-3-(trifluoromethyl)phenyl group with bicalutamide and hydroxyflutamide required for binding to the AR. In this work, we used a combined strategy to find new antagonist structures distinct from the 4-cyano-3-(trifluoromethyl)phenyl group to avoid cross-resistance for these compounds and to find structures without agonist activity on mutant ARs (AR W741C and AR T877A). We found two novel chemotypes with AR-antagonistic activity (IC(50): 3-6 μM) by virtual screening and confirmed their biological activity in an androgen-responsive reporter assay. The design of our computational approach was validated by the observation of strongly decreased or absence of agonistic activity on the two mutant ARs. Further structural derivatization to optimize the potency of these compounds can render these chemotypes into very promising, alternative AR antagonists for prostate cancer therapy.

  12. Species Identification and Molecular Typing of Leishmania Spp. Using Targeting HSP70 Gene in Suspected Patients of Cutaneous Leishmaniasis from Sistan and Baluchestan Province, Southeast Iran

    Science.gov (United States)

    MIRAHMADI, Hadi; SALIMI KHORASHAD, Alireza; SOHRABNAHAD, Alireza; HEYDARIAN, Peyman; BIZHANI, Negar

    2016-01-01

    Background: Leishmaniasis is a sand fly-borne disease caused by the protozoan parasites belonging to the genus Leishmania. Because of the preventing and controlling methods, clinical course, prognosis and choice of treatment are differing from species; differentiation of species is critical. The present study was aimed to detect the parasite species using the PCR-RFLP method. Methods: A total of 130 Giemsa-Stained slides from suspected Cutaneous leishmaniasis (CL) patients were examined under a light microscope at ×1000. DNA from each slide was extracted PCR method was undertaken with HSP70 genes and the PCR products were digested with a restriction enzyme HaeIII (BsuR1). The study was conducted in the laboratory of Zahedan University of Medical Sciences in the Sistan and Baluchestan Province, southeastern Iran in 2015. Results: From 130 suspected samples, 59 (45.3%) were positive by the microscopic examination, meanwhile 64 (49.2%) were positive by PCR-RFLP, Leishmania species were recognized, and L. tropica was introduced as predominant species in current study. Conclusion: PCR-RFLP is a valuable technique for distinguish of Leishmania species. Furthermore, anthroponotic CL is the dominant cause of CL in Sistan and Baluchestan Province. PMID:28127360

  13. Identification and characterization of a new chemotype of noncovalent SENP inhibitors.

    Science.gov (United States)

    Madu, Ikenna G; Namanja, Andrew T; Su, Yang; Wong, Steven; Li, Yi-Jia; Chen, Yuan

    2013-07-19

    Enzymes called SENPs catalyze both the maturation of small ubiquitin-like modifier (SUMO) precursors and removal of SUMO modifications, which regulate essential cellular functions such as cell cycle progression, DNA damage response, and intracellular trafficking. Some members, such as SENP1, are potential targets for developing cancer therapeutics. We searched for small molecule inhibitors of SENPs using in silico screening in conjunction with biochemical assays and identified a new chemotype of small molecule inhibitors that noncovalently inhibit SENPs. The inhibitors confer the noncompetitive inhibitory mechanism, as shown by nuclear magnetic resonance (NMR) and quantitative enzyme kinetic analysis. The NMR data also provided evidence for substrate-assisted inhibitor binding, which indicates the need for caution in using artificial substrates for compound screening, as the inhibitory effects could be significantly different from using the physiological substrates. This finding also suggests the possibility of designing inhibitors for this class of enzymes that are tuned for substrate-specificity.

  14. Trichothecene chemotype composition of Fusarium graminearum and related species in Finland and Russia

    Science.gov (United States)

    Fusarium graminearum and type B trichothecene producers can be divided into three chemotypes. Analysis of 290 single-spore isolates of F. graminearum and related Fusarium species revealed that all F. graminearum isolates from Finland (15) and western Russian (26) possessed the 3ADON chemotype, whil...

  15. Activity cliffs and activity cliff generators based on chemotype-related activity landscapes.

    Science.gov (United States)

    Pérez-Villanueva, Jaime; Méndez-Lucio, Oscar; Soria-Arteche, Olivia; Medina-Franco, José L

    2015-11-01

    Activity cliffs have large impact in drug discovery; therefore, their detection and quantification are of major importance. This work introduces the metric activity cliff enrichment factor and expands the previously reported activity cliff generator concept by adding chemotype information to representations of the activity landscape. To exemplify these concepts, three molecular databases with multiple biological activities were characterized. Compounds in each database were grouped into chemotype classes. Then, pairwise comparisons of structure similarities and activity differences were calculated for each compound and used to construct chemotype-based structure-activity similarity (SAS) maps. Different landscape distributions among four major regions of the SAS maps were observed for different subsets of molecules grouped in chemotypes. Based on this observation, the activity cliff enrichment factor was calculated to numerically detect chemotypes enriched in activity cliffs. Several chemotype classes were detected having major proportion of activity cliffs than the entire database. In addition, some chemotype classes comprising compounds with smooth structure activity relationships (SAR) were detected. Finally, the activity cliff generator concept was applied to compounds grouped in chemotypes to extract valuable SAR information.

  16. Greenhouse studies reveal increased aggressiveness of emergent Canadian Fusarium graminearum chemotypes in wheat

    Science.gov (United States)

    The role of Fusarium graminearum trichothecene-chemotypes in disease outcomes was evaluated in a series of wheat lines with different levels of resistance to Fusarium Head Blight (FHB). Four inocula, each consisting of a composite of four strains with either 15-acetyldeoxynivalenol (ADON) chemotypes...

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

    Directory of Open Access Journals (Sweden)

    Daniel Paape

    2014-12-01

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

  18. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    OpenAIRE

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

    2015-01-01

    Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host’s cells. In this work, we have Investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promasti...

  19. Chemotyping the distribution of vitamin D metabolites in human serum

    Science.gov (United States)

    Müller, Miriam J.; Stokes, Caroline S.; Lammert, Frank; Volmer, Dietrich A.

    2016-02-01

    Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the ‘FDA Guidance for Industry Bioanalytical Method Validation’. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

  20. Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs.

    Science.gov (United States)

    Biermaier, Barbara; Gottschalk, Christoph; Schwaiger, Karin; Gareis, Manfred

    2015-02-01

    Stachybotrys (S.) chartarum is an omnipresent cellulolytic mould which produces secondary metabolites, such as the highly toxic macrocyclic trichothecenes. While it is known to occur in animal feed like hay and straw as well as in water-damaged indoor environments, there is little knowledge about the occurrence of S. chartarum and its secondary metabolites in food. The objective of the present study was to examine selected dried culinary herbs for the presence of S. chartarum chemotype S, to assess the potential risk of a contamination of foods with macrocyclic trichothecenes. In total, 50 Stachybotrys isolates from different types of culinary herbs (n=100) such as marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) were examined by MTT-cell culture test (effect-based bioassay), ELISA, and by liquid chromatography tandem mass spectrometry (LC-MS/MS). Selected toxic and non-toxic isolates (n=15) were genetically characterized by PCR and sequencing. Five isolates (10%) were highly toxic in the MTT-cell culture test, and the production of macrocyclic trichothecenes was proven by ELISA and LC-MS/MS. These five isolates were genetically confirmed as S. chartarum chemotype S. To the best of our knowledge, this is the first report about a contamination of dried culinary herbs with toxigenic S. chartarum.

  1. The genetic toolbox for Leishmania parasites.

    Science.gov (United States)

    Roberts, Sigrid C

    2011-01-01

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

  2. Cytogeography of essential oil chemotypes of Eremophila longifolia F. Muell (Scrophulariaceae).

    Science.gov (United States)

    Sadgrove, Nicholas John; Jones, Graham Lloyd

    2014-09-01

    Previous studies have demonstrated that the widely distributed desert plant Eremophila longifolia has at least six geographically defined essential oil chemotypes. The focus of the present study is to extend and enhance information concerning known chemotypes and to investigate the involvement of cell nuclei ploidy in this variation. Forty field collected specimens of E. longifolia were taken from most of the mainland states of Australia then subjected to hydrodistillation to produce essential oils, which were then chemically characterised. Ploidy was determined using relative fluorescence of cell nuclei stained with propidium iodide, measured in a flow cytometer. Using principal component analysis (PCA), at least three essential oil chemotypes, in addition to the six already described, were identified in the present study. Previously described high yielding essential oil chemotypes were also characterised in terms of diploidy. For the first time diploid populations were identified in New South Wales, correlating with high yielding isomenthone/menthone and karahanaenone chemotypes. Furthermore, the separate diploid population previously described from Western Australia was demonstrated to be the safrole/methyl eugenol type, which is restricted to a small geographic range in far north-west Western Australia (Murchison District). All other chemotypes were shown to be tetraploid, including apparently randomly emerging individuals, representative of chemotypes producing low yields of isomenthone/menthone and karahanaenone similar in composition to the high yielding diploid types.

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

    Science.gov (United States)

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

    2004-02-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  5. Effect of Origanum chemotypes on broiler intestinal bacteria.

    Science.gov (United States)

    Betancourt, Liliana; Rodriguez, Fernando; Phandanouvong, Vienvilay; Ariza-Nieto, Claudia; Hume, Michael; Nisbet, David; Afanador-Téllez, German; Van Kley, Alexandra Martynova; Nalian, Armen

    2014-10-01

    Essential oils have been proposed as alternatives to antibiotic use in food animal production. This study evaluated 3 chemotypes of the Origanum genus, containing varying amounts of secondary metabolites carvacrol, thymol, and sabinene, in the broiler chicken diet. Aerial parts of Origanum vulgare L. (OL), O. vulgare L. ssp. hirtum (OH), and O. majorana (OM) were collected from a greenhouse located in the high altitude Sabana de Bogotá (Savanna of Bogotá) and O. vulgare L. ssp. hirtum (OG) produced and ground in Greece. Oregano essential oils (OEO) from these plants were obtained by steam distillation and analyzed by gas chromatography coupled to a mass spectrometer. Six treatments were evaluated: 200 mg/kg of OEO from OH, OL, and OM, 50 mg/kg of OEO from OG, 500 mg/kg of chlortetracycline, and without additives. Broiler chicks were maintained at 2,600 m above sea level, placed in brooder cages under a completely randomized design. Template DNA was isolated from duodenal, jejunal, ileal, and cecal contents in each group and bacterial 16S rDNA patterns were analyzed by denaturing gradient gel electrophoresis. Dendrograms of denaturing gradient gel electrophoresis band patterns revealed 2 main clusters, OEO-treated chicks and nontreated control chicks, in each intestinal segment. Band patterns from different gut compartments revealed major bacterial population shifts in the foregut (duodenum, jejunum, and ileum) compared with the hindgut (cecum and colon) at all ages evaluated (P < 0.05). The OEO groups showed less shift (62.7% similarity coefficient) between these 2 compartments versus the control groups (53.7% similarity coefficient). A reduction of 59% in mortality from ascites was seen in additive-supplemented groups compared with the control group. This study represents the first work to evaluate the effects of the 3 main chemotypes of Origanum genus in broilers.

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

    Science.gov (United States)

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

    2015-09-15

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

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

    Directory of Open Access Journals (Sweden)

    Syamdas Bandyopadhyay

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

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

    Science.gov (United States)

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

    2013-11-15

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

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

    Directory of Open Access Journals (Sweden)

    Simona eStager

    2012-01-01

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

  10. Diagnostic Antigens of Leishmania.

    Science.gov (United States)

    1994-01-31

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

  11. IDENTIFICATION AND CHARACTERIZATION OF A NEW CHEMOTYPE OF NON-COVALENT SENP INHIBITORS

    Science.gov (United States)

    Madu, Ikenna G.; Namanja, Andrew T.; Su, Yang; Wong, Steven; Li, Yi-Jia; Chen, Yuan

    2013-01-01

    Enzymes called SENPs catalyze both the maturation of small ubiquitin-like modifier (SUMO) precursors and removal of SUMO modifications, which regulate essential cellular functions such as cell cycle progression, DNA damage response and intracellular trafficking. Some members, such as SENP1, are potential targets for developing cancer therapeutics. We searched for small molecule inhibitors of SENPs using in-silico screening in conjunction with biochemical assays, and identified a new chemotype of small molecule inhibitors that non-covalently inhibit SENPs. The inhibitors confer the non-competitive inhibitory mechanism, as shown by nuclear magnetic resonance (NMR) and quantitative enzyme kinetic analysis. The NMR data also provided evidence for substrate-assisted inhibitor binding, which indicates the need for caution in using artificial substrates for compound screening, as the inhibitory effects could be significantly different from using the physiological substrates. This finding also suggests the possibility of designing inhibitors for this class of enzymes that are tuned for substrate-specificity. PMID:23614497

  12. First Phytochemical Evidence of Chemotypes for the Seagrass Zostera noltii

    Directory of Open Access Journals (Sweden)

    Micheline Grignon-Dubois

    2012-09-01

    Full Text Available The variability of the flavonoid content of two populations of Z. noltii from different geographical zones, i.e., the Bay of Arcachon and the Bay of Cadiz, was evaluated. Samples were collected in spring and autumn at the two sites, and extracts were prepared by maceration in water. The phenolic content was fully characterized using Nuclear Magnetic Resonance (NMR, UV and Liquid Chromatography-Mass Spectrometry (LC-MS, and the concentration of the individual phenolic was determined by quantitative High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD. The two populations show a strong geographical differentiation in their flavonoid content. The samples from Cadiz were dominated by apigenin 7-sulfate, which represents 71% (autumn collection and 83% (spring collection of the total flavonoids, whereas the samples from Arcachon were characterized by diosmetin 7-sulfate (85 and 93% of the total flavonoids. Structural elucidation of the individual phenolics was assigned using the complementary information from their spectral evidence. In addition, the results were confirmed by acid hydrolysis of the flavonoid sulfates, and comparison to synthetic standards obtained by sulfation of apigenin, diosmetin and luteolin. The results represent the first experimental evidence of the existence of chemotypes within the species Z. noltii.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major....... Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs....

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ángel Sinagra

    2007-12-01

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

  18. Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes.

    Science.gov (United States)

    Aizpurua-Olaizola, Oier; Soydaner, Umut; Öztürk, Ekin; Schibano, Daniele; Simsir, Yilmaz; Navarro, Patricia; Etxebarria, Nestor; Usobiaga, Aresatz

    2016-02-26

    The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

  19. Chemotype diversity of indigenous Dalmatian sage (Salvia officinalis L.) populations in Montenegro.

    Science.gov (United States)

    Stešević, Danijela; Ristić, Mihailo; Nikolić, Vuko; Nedović, Marijana; Caković, Danka; Šatović, Zlatko

    2014-01-01

    To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild-growing populations was determined by GC-FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis-thujone (16.98-40.35%), camphor (12.75-35.37%), 1,8-cineol (6.40-12.06%), trans-thujone (1.5-10.35%), camphene (2.26-9.97%), borneol (0.97-8.81%), viridiflorol (3.46-7.8%), limonene (1.8-6.47%), α-pinene (1.59-5.46%), and α-humulene (1.77-5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02-P04, P09, and P10 complied with the German Drug Codex. A few of the main essential-oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between α-pinene and camphene, camphene and camphor, as well as between cis-thujone and trans-thujone. Strong negative correlations were evidenced between cis-thujone and α-pinene, cis-thujone and champhene, cis-thujone and camphor, as well as between trans-thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, α-pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and α-pinene. The chemotypes did not significantly differ in the total essential-oil content and the cis/trans-thujone ratio.

  20. Isolation and characterization of polyphenol oxidase from Sardinian poisonous and non-poisonous chemotypes of Ferula communis (L.).

    Science.gov (United States)

    Zucca, Paolo; Sanjust, Enrico; Loi, Martina; Sollai, Francesca; Ballero, Mauro; Pintus, Manuela; Rescigno, Antonio

    2013-06-01

    Ferula communis (L.), a plant belonging to Apiaceae, is widely present in Sardinia, Italy. Currently, interest in F. communis focuses on the presence of two chemotypes in the wild. One chemotype is poisonous to animals, whereas the other chemotype is non-poisonous. Polyphenol oxidase (PPO) has been extracted and partially purified from the two chemotypes of F. communis. The biochemical characterization of the enzymes showed significant differences. In particular, while the two PPOs were not able to use 6- and 7-hydroxycoumarin as substrates, they showed distinct specificity for 6,7- and 7,8-dihydroxycoumarin. Significant differences in the enzyme behavior towards common PPO inhibitors were also observed. In addition, activation energy and activation energy for denaturation were determined, showing significant differences between FP-PPO and FNP-PPO, particularly for denaturation kinetics. The possible roles of the two PPOs in determining differences in composition and toxicity of the two F. communis chemotypes are also discussed.

  1. Identification of the chemotypes of Ocimum forskolei and Ocimum basilicum by NMR spectroscopy.

    Science.gov (United States)

    Fatope, Majekodunmi O; Marwah, Ruchi G; Al Hadhrami, Nabil M; Onifade, Anthony K; Williams, John R

    2008-11-01

    The chemotypes of Ocimum forskolei Benth and Ocimum basilicum L. growing wild in Oman have been established by (13)C-NMR analyses of the vegetative and floral oils of the plants. The chemotypes, estragole for O. forskolei and linalool for O. basilicum, suggested by (13)C-NMR fingerprinting were also confirmed by GC-FID and GC/MS analyses. The oil of O. forskolei demonstrated better activities against bacteria and dermatophytes. The significance of the presence of estragole and linalool in the volatile oils of plants whose fragrances are traditionally inhaled, added to food, or rubbed on the skin are discussed.

  2. Homologous recombination in Leishmania enriettii.

    OpenAIRE

    1991-01-01

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

  3. Analytical discrimination of poisonous and nonpoisonous chemotypes of giant fennel (Ferula communis L.) through their biologically active and volatile fractions.

    Science.gov (United States)

    Rubiolo, Patrizia; Matteodo, Maura; Riccio, Giovanna; Ballero, Mauro; Christen, Philippe; Fleury-Souverain, Sandrine; Veuthey, Jean-Luc; Bicchi, Carlo

    2006-10-04

    Giant fennel (Ferula communis L.) from Sardinia is characterized by two chemotypes with different biological activities. One chemotype is poisonous, due to prenylcoumarins, and responsible for ferulosis, which mainly affects sheep and goats, cattle, and horses; the other chemotype is nonpoisonous and contains daucane esters. The two chemotypes cannot be distinguished botanically. High-performance liquid chromatography-diode array-ultraviolet detection-mass spectrometry (HPLC-DAD-UV-MS) analysis of the composition of the fractions containing the biologically active metabolites and of the volatile fractions, by gas chromatography-mass spectrometry (GC-MS), of both essential oil and headspace sampled by headspace solid-phase microextraction (HS-SPME) are here shown to be effective in discriminating the poisonous and nonpoisonous chemotypes. HS-SPME with CAR/PDMS/DVB in combination with GC-MS has also been found to be a successful, fully automated one-step method for rapid and unequivocal discrimination of the two chemotypes, using aristolene and allohedycaryol as markers of the poisonous and nonpoisonous chemotypes, respectively.

  4. Limited Stability of Microcystins in Oligopeptide Compositions of Microcystis aeruginosa (Cyanobacteria: Implications in the Definition of Chemotypes

    Directory of Open Access Journals (Sweden)

    Ramsy Agha

    2013-06-01

    Full Text Available The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical subpopulations, which have been shown to possess dissimilar ecological traits. However, the stability of chemotypes under changing abiotic conditions is usually assumed and has not been assessed in detail. We monitored oligopeptide patterns of three strains of Microcystis aeruginosa under different nutrient and light conditions. MALDI-TOF MS revealed alterations in the microcystins signatures under N and P poor conditions and high light intensities (150 and 400 μmol photons m−2s−1. Variations in the general oligopeptide composition were caused by a gradual disappearance of microcystins with low relative intensity signals from the fingerprint. The extent of such variations seems to be closely related to physiological stress caused by treatments. Under identical clonal compositions, alterations in the oligopeptide fingerprint may be misinterpreted as apparent shifts in chemotype succession. We discuss the nature of such variations, as well as the consequent implications in the use of cyanobacterial chemotyping in studies at the subpopulation level and propose new guidance for the definition of chemotypes as a consistent subpopulation marker.

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

    OpenAIRE

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

    2014-01-01

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

  6. First molecular detection of Leishmania tarentolae-like DNA in Sergentomyia minuta in Spain.

    Science.gov (United States)

    Bravo-Barriga, Daniel; Parreira, Ricardo; Maia, Carla; Blanco-Ciudad, Juan; Afonso, Maria Odete; Frontera, Eva; Campino, Lenea; Pérez-Martín, Juan Enrique; Serrano Aguilera, Francisco Javier; Reina, David

    2016-03-01

    Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

  7. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Directory of Open Access Journals (Sweden)

    Dirlei Nico

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

  8. Leukodepletion Filters for Prevention of Transfusion Transmission Of Leishmania

    Science.gov (United States)

    2006-11-01

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

  9. Leishmania Exosomes Deliver Preemptive Strikes to Create an Environment Permissive for Early Infection

    Directory of Open Access Journals (Sweden)

    Judith Maxwell Silverman

    2012-01-01

    Full Text Available Herein, we review evidence supporting a role for leishmania exosomes during early infection. We suggest a model in which leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, ; wild-type (WT exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α by leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The leishmania proteins GP63 and elongation factor-1α (EF-1α are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein tyrosine phosphatases (PTPs. Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell

  10. Leishmania exosomes deliver preemptive strikes to create an environment permissive for early infection.

    Science.gov (United States)

    Silverman, Judith Maxwell; Reiner, Neil E

    2011-01-01

    Herein, we review evidence supporting a role for Leishmania exosomes during early infection. We suggest a model in which Leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for Leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, wild-type (WT) exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) by Leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The Leishmania proteins GP63 and elongation factor-1α (EF-1α) are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein-tyrosine phosphatases (PTPs). Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that Leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell signaling pathways.

  11. Discovery of new antimalarial chemotypes through chemical methodology and library development.

    Science.gov (United States)

    Brown, Lauren E; Chih-Chien Cheng, Ken; Wei, Wan-Guo; Yuan, Pingwei; Dai, Peng; Trilles, Richard; Ni, Feng; Yuan, Jing; MacArthur, Ryan; Guha, Rajarshi; Johnson, Ronald L; Su, Xin-zhuan; Dominguez, Melissa M; Snyder, John K; Beeler, Aaron B; Schaus, Scott E; Inglese, James; Porco, John A

    2011-04-26

    In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

  12. Ligand-Based Virtual Screening in a Search for Novel Anti-HIV-1 Chemotypes.

    Science.gov (United States)

    Kurczyk, Agata; Warszycki, Dawid; Musiol, Robert; Kafel, Rafał; Bojarski, Andrzej J; Polanski, Jaroslaw

    2015-10-26

    In a search for new anti-HIV-1 chemotypes, we developed a multistep ligand-based virtual screening (VS) protocol combining machine learning (ML) methods with the privileged structures (PS) concept. In its learning step, the VS protocol was based on HIV integrase (IN) inhibitors fetched from the ChEMBL database. The performances of various ML methods and PS weighting scheme were evaluated and applied as VS filtering criteria. Finally, a database of 1.5 million commercially available compounds was virtually screened using a multistep ligand-based cascade, and 13 selected unique structures were tested by measuring the inhibition of HIV replication in infected cells. This approach resulted in the discovery of two novel chemotypes with moderate antiretroviral activity, that, together with their topological diversity, make them good candidates as lead structures for future optimization.

  13. Chemical Composition and Antimicrobial Activity of a New Chemotype of Hyptis suaveolens (Poit from Nigeria

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    B.A. Iwalokun

    2012-04-01

    Full Text Available Hyptis suaveolens is one of the aromatic plants credited for substantial medicinal values in the tropics with three chemotypes previously reported in Nigeria. This study provided biological and chemical evidence for a new chemotype of Hyptis suaveolens in Lagos. Hydrodistillation of the dried leaves of the plant produced volatile oil with a yield of 0.31% and subsequent analyses by GC-MS identified 28 volatile compounds that accounted for 99.1% of the total oil composition. Although the oil was monoterpenoid dominated and has comparable levels of sabinene (25.8 vs. 13.2-30.1%, a-thujene (1.1 vs. 0.9-1.2%, and 4- terpineol (8.4-9.8 vs. 11.4%, it elicited a moderate level of α-pinene (4.7 vs. 1.8-13.6, higher levels of β- pinene (9.7 vs. 0-4.4%, limonene (2.3 vs. 0-0.8%, 1,8-cineole (4.8 vs. 0-1.2%, γ-terpinene (9.3 vs. 1.6-4.2% and terpinolene (8.4 vs. 5.6-6.3 and the presence of new compounds: aromadendrene (0.3%, camphor (0.3%, germacrene B (0.4% and himachalol (0.1% when compared with the previous chemotypes. In vitro, the oil was found by agar diffusion assay to elicit antibacterial activity against E. coli ATCC25922, and S. aureus ATCC25923 and antifungal activity with C. albicans showing higher sensitivity (MFC = 53.3 μL/mL and Aspergillus niger and Trichophyton rubrum displaying moderate to low sensitivity. Biological effect of the oil at sub-MIC on E. coli ATCC25922 was characterized by dose-dependent loss of outer membrane proteins. These findings provide evidence for a new chemotype of Hyptis suaveolens in Nigeria.

  14. Intensive sampling identifies previously unknown chemotypes, population divergence and biosynthetic connections among terpenoids in Eucalyptus tricarpa.

    Science.gov (United States)

    Andrew, Rose L; Keszei, Andras; Foley, William J

    2013-10-01

    Australian members of the Myrtaceae produce large quantities of ecologically and economically important terpenes and display abundant diversity in both yield and composition of their oils. In a survey of the concentrations of leaf terpenes in Eucalyptus tricarpa (L.A.S. Johnson) L.A.S. Johnson & K.D. Hill, which were previously known from few samples, exceptional variability was found in composition. The aim was to characterize the patterns of variation and covariation among terpene components in this species and to use this information to enhance our understanding of their biosynthesis. There were marked discontinuities in the distributions of numerous compounds, including the overall proportions of mono- and sesquiterpenes, leading us to delineate three distinct chemotypes. Overall, positive covariation predominated, but negative covariation suggested competitive interactions involved in monoterpene synthesis. Two groups of covarying monoterpenes were found, each of which was positively correlated with a group of sesquiterpenes and negatively correlated with the alternate sesquiterpene group. These results imply substantial cross-talk between mono- and sesquiterpene biosynthesis pathways. However, only those compounds hypothesized to share final carbocation intermediates or post-processing steps were strongly positively correlated within chemotypes. This suggests that the broader patterns of covariation among groups of compounds may result from co-regulation of multiple biosynthetic genes, controlling the complex terpene profiles of the chemotypes of Eucalyptus.

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

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    Abhishek Subramanian

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

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

    Science.gov (United States)

    Vanhollebeke, Benoit; Caljon, Guy; Wolfe, Alan R.; McKerrow, James; Dujardin, Jean-Claude

    2016-01-01

    Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy. PMID:28036391

  17. A 3D similarity method for scaffold hopping from known drugs or natural ligands to new chemotypes.

    Science.gov (United States)

    Jenkins, Jeremy L; Glick, Meir; Davies, John W

    2004-12-01

    A primary goal of 3D similarity searching is to find compounds with similar bioactivity to a reference ligand but with different chemotypes, i.e., "scaffold hopping". However, an adequate description of chemical structures in 3D conformational space is difficult due to the high-dimensionality of the problem. We present an automated method that simplifies flexible 3D chemical descriptions in which clustering techniques traditionally used in data mining are exploited to create "fuzzy" molecular representations called FEPOPS (feature point pharmacophores). The representations can be used for flexible 3D similarity searching given one or more active compounds without a priori knowledge of bioactive conformations or pharmacophores. We demonstrate that similarity searching with FEPOPS significantly enriches for actives taken from in-house high-throughput screening datasets and from MDDR activity classes COX-2, 5-HT3A, and HIV-RT, while also scaffold or ring-system hopping to new chemical frameworks. Further, inhibitors of target proteins (dopamine 2 and retinoic acid receptor) are recalled by FEPOPS by scaffold hopping from their associated endogenous ligands (dopamine and retinoic acid). Importantly, the method excels in comparison to commonly used 2D similarity methods (DAYLIGHT, MACCS, Pipeline Pilot fingerprints) and a commercial 3D method (Pharmacophore Distance Triplets) at finding novel scaffold classes given a single query molecule.

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

    Science.gov (United States)

    Chrobak, Mareike; Förster, Sabine; Meisel, Sarah; Pfefferkorn, Roxana; Förster, Frank; Clos, Joachim

    2012-04-01

    Genes for HslVU-type peptidases are found in bacteria and in a few select Eukaryota, among those such important pathogens as Plasmodium spp. and Leishmania spp. In this study, we performed replacements of all three HslV/HslU gene homologues and found one of those, HslV, to be essential for Leishmania donovani viability. The Leishmania HslV gene can also partially relieve the thermosensitive phenotype of a combined HslVU/Lon/ClpXP knockout mutant of Escherichia coli, indicating a conserved function. However, we found that the role and function of the two Leishmania HslU genes has diverged since neither of those interacts stably with HslV. The latter forms a dodecameric complex by itself and shows a punctate distribution. We conclude that whilst the basic function of HslV may be conserved in Leishmania, its organisation and interaction with its canonical complex partner HslU is not. Nevertheless, given the absence of HslV from the proteome of mammals and its essential role in Leishmania viability, HslV is a promising target for intervention.

  19. Detection of Leishmania RNA Virus in Leishmania Parasites

    Science.gov (United States)

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

    2013-01-01

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

  20. 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.

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

    Science.gov (United States)

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

    2016-08-01

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

  2. Phylogenetic and chemotypic diversity of Periglandula species in eight new morning glory hosts (Convolvulaceae).

    Science.gov (United States)

    Beaulieu, Wesley T; Panaccione, Daniel G; Ryan, Katy L; Kaonongbua, Wittaya; Clay, Keith

    2015-01-01

    Periglandula ipomoeae and P. turbinae (Ascomycota, Clavicipitaceae) are recently described fungi that form symbiotic associations with the morning glories (Convolvulaceae) Ipomoea asarifolia and Turbina corymbosa, respectively. These Periglandula species are vertically transmitted and produce bioactive ergot alkaloids in seeds of infected plants and ephemeral mycelia on the adaxial surface of young leaves. Whether other morning glories that contain ergot alkaloids also are infected by Periglandula fungi is a central question. Here we report on a survey of eight species of Convolvulaceae (Argyreia nervosa, I. amnicola, I. argillicola, I. gracilis, I. hildebrandtii, I. leptophylla, I. muelleri, I. pes-caprae) for ergot alkaloids in seeds and associated clavicipitaceous fungi potentially responsible for their production. All host species contained ergot alkaloids in four distinct chemotypes with concentrations of 15.8-3223.0 μg/g. Each chemotype was a combination of four or five ergot alkaloids out of seven alkaloids detected across all hosts. In addition, each host species exhibited characteristic epiphytic mycelia on adaxial surfaces of young leaves with considerable interspecific differences in mycelial density. We sequenced three loci from fungi infecting each host: the nuclear rDNA internal transcribed spacer region (ITS), introns of the translation factor 1-α gene (tefA) and the dimethylallyl-tryptophan synthase gene (dmaW), which codes for the enzyme that catalyzes the first step in ergot alkaloid biosynthesis. Phylogenetic analyses confirmed that these fungi are in the family Clavicipitaceae and form a monophyletic group with the two described Periglandula species. This study is the first to report Periglandula spp. from Asian, Australian, African and North American species of Convolvulaceae, including host species with a shrub growth form and host species occurring outside of the tropics. This study demonstrates that ergot alkaloids in morning glories

  3. Chemical composition of teas from two cultivated chemotypes of Egletes viscosa ('Macela-da-terra')

    Energy Technology Data Exchange (ETDEWEB)

    Vieira, Gizelle Angela B.; Lima, Anne S.; Silveira, Edilberto R. [Ceara Univ., Fortaleza, CE (Brazil). Dept. de Quimica Organica e Inorganica. Curso de Pos-Graduacao em Quimica Organica]. E-mail: edil@ufc.br; Bezerra, Antonio Marcos E. [Ceara Univ., Fortaleza, CE (Brazil). Dept. de Fitotecnia

    2006-01-15

    Phytochemical analysis of flower buds infusion from two cultivated chemotypes of Egletes viscosa Less was accomplished. The new diterpene 12-acetoxy-7-hydroxy-3,13(14)- clerodandien-18,19:15,16-diolide, ternatin, centipedic acid and 12-acetoxy-hawtriwaic lactone were isolated from the chemotype trans-pinocarveyl acetate. Analysis of the chemotype cisisopinocarveyl acetate yielded 12-acetoxy-7-hydroxy-3,13(14)-clerodandien-18,19:15,16- diolide, 12-epi-bacchotricuneatin, ternatin and scopoletin. Structural elucidation of the isolated compounds was established on the basis of spectral data through the use of 1D NMR and several 2D shift correlated NMR pulse sequences and comparison with literature data. (author)

  4. Natural infection of Algerian hedgehog, Atelerix algirus (Lereboullet 1842) with Leishmania parasites in Tunisia.

    Science.gov (United States)

    Chemkhi, Jomaa; Souguir, Hejer; Ali, Insaf Bel Hadj; Driss, Mehdi; Guizani, Ikram; Guerbouj, Souheila

    2015-10-01

    In Tunisia, Leishmania parasites are responsible of visceral leishmaniasis, caused by Leishmania infantum species while three cutaneous disease forms are documented: chronic cutaneous leishmaniasis due to Leishmania killicki, sporadic cutaneous form (SCL) caused by L. infantum and the predominant zoonotic cutaneous leishmanaisis (ZCL) due to Leishmania major. ZCL reservoirs are rodents of the Psammomys and Meriones genera, while for SCL the dog is supposed to be a reservoir. Ctenodactylus gundii is involved in the transmission of L. killicki. However, other mammals could constitute potential reservoir hosts in Tunisia and other North African countries. In order to explore the role of hedgehogs as potential reservoirs of leishmaniasis, specimens (N=6) were captured during July-November period in 2011-2013 in an SCL endemic area in El Kef region, North-Western Tunisia. Using morphological characteristics, all specimens were described and measured. Biopsies from liver, heart, kidney and spleen of each animal were used to extract genomic DNA, which was further used in PCR assays to assess the presence of Leishmania parasites. Different PCRs targeting kinetoplast minicircles, ITS1, mini-exon genes and a repetitive Leishmania- specific sequence, were applied. To further identify Leishmania species involved, RFLP analysis of amplified fragments was performed with appropriate restriction enzymes. Using morphological characters, animals were identified as North African hedgehogs, also called Algerian hedgehogs, that belong to the Erinaceidae family, genus Atelerix Pomel 1848, and species algirus (Lereboullet, 1842). PCR results showed in total that all specimens were Leishmania infected, with different organs incriminated, mainly liver and spleen. Results were confirmed by direct sequencing of amplified fragments. Species identification showed that all specimens were infected with L. major, three of which were additionally co-infected with L. infantum. The present study

  5. Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

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    Oliva Glaucius

    2007-09-01

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

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

    Science.gov (United States)

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

    2008-09-01

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

  7. Sesquiterpene lactones in Arnica montana: helenalin and dihydrohelenalin chemotypes in Spain.

    Science.gov (United States)

    Perry, Nigel B; Burgess, Elaine J; Rodríguez Guitián, Manuel A; Romero Franco, Rosa; López Mosquera, Elvira; Smallfield, Bruce M; Joyce, Nigel I; Littlejohn, Roger P

    2009-05-01

    An analytical RPLC method for sesquiterpene lactones in Arnica montana has been extended to include quantitative analyses of dihydrohelenalin esters. LC-ESI-MS-MS distinguished the isomeric helenalin and dihydrohelenalin esters. The dihydrohelenalin esters have lower response factors for UV detection than do helenalin esters, which must be taken into account for quantitative analyses. Analyses of flowers from 16 different wild populations of A. montana in Spain showed differing proportions of helenalin and dihydrohelenalin esters. For the first time a chemotype with high levels of helenalin esters (total helenalins 5.2-10.3 mg/g dry weight) is reported in Spanish A. montana. These samples were from heath lands at high altitude (1330-1460 m), whereas samples from meadows and peat bogs at lower altitudes were the expected chemotype with high levels of dihydrohelenalin esters (total dihydrohelenalins 10.9-18.2 mg/g). The phenolic compounds, both flavonoid glycosides and caffeoylquinic acids, in Spanish A. montana are reported for the first time. The levels of several of these compounds differed significantly between samples from heath lands and samples from peat bogs or meadows, with the heath land samples being most similar to central European A. montana in their phenolic composition.

  8. Selection of new clones of linalool chemotype from genetic recombination in Lippia alba

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    Elcio Rodrigo Rufino

    2012-01-01

    Full Text Available The aromatic and medicinal species Lippia alba is vigorous and rugged native to the South America (Atlantic Rainforest. Because it is an allogamous and self-incompatible species, natural populations have high morphological and chemical variability. This work had as objective to conduct a preliminary screening to identify new promising clones from a novel (recombinant base population of Lippia alba with regard to its agronomic and phytochemical traits, using the linalool oil or chemotype as model. The two superior linalool clones, obtained by collection, were used as controls. Traits evaluated included: dry mass of leaves (DML, oil yield percentage (EOY%, oil production per plant (OP, and linalool percentage (LN%. Forty linalool chemotype clones were evaluated in three experiments, in a random block design with four replicates and four cuttings (clones per plot. Besides means comparisons, multivariate analysis was used in order to aid in the preliminary selection of clones. There were positive correlations from moderate to strong for DML vs. EOY%, OP vs. EOY% and DML vs. OP. Linalool clones superior or similar to both controls were identified for the DML, EOY%, OP, and LN% traits (univariate analyses, aimed at further validating experimentation. Five distinct groups were defined in the cluster analysis (UPGMA, each containing subgroups as well.

  9. Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

    Science.gov (United States)

    Infante, Paola; Alfonsi, Romina; Ingallina, Cinzia; Quaglio, Deborah; Ghirga, Francesca; D'Acquarica, Ilaria; Bernardi, Flavia; Di Magno, Laura; Canettieri, Gianluca; Screpanti, Isabella; Gulino, Alberto; Botta, Bruno; Mori, Mattia; Di Marcotullio, Lucia

    2016-01-01

    Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance. PMID:27899820

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

    Science.gov (United States)

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

    2012-11-01

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

  11. 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

    2016-01-01

    Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival. PMID:27548150

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

    Science.gov (United States)

    Corrales, Rosa Milagros; Sereno, Denis; Mathieu-Daudé, Françoise

    2010-02-01

    Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis. Survival and transmission of these parasites in their different hosts require membrane-bound or extracellular factors to interact with and modify their host environments. Over the last decade, several approaches have been applied to study all the extracellular proteins exported by an organism at a particular time or stage in its life cycle and under defined conditions, collectively termed the secretome or the exoproteome. In this review, we focus on emerging data shedding light on the secretion mechanisms involved in the production of the Leishmania exoproteome. We also describe other methodologies currently available that could be used to analyse the Leishmania exoproteome. Understanding the complexity of the Leishmania exoproteome is a key component to elucidating the mechanisms used by these parasites for exporting proteins to the extracellular space during its life cycle. Given the importance of extracellular factors, a detailed knowledge of the Leishmania exoproteome may provide novel targets for rational drug design and/or a source of antigens for vaccine development.

  13. In vitro antifungal activity of four chemotypes of Lippia alba (Verbenaceae essential oils against Alternaria solani (Pleosporeaceae isolates

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    ELISA Z. TOMAZONI

    2016-06-01

    Full Text Available Several volatile natural compounds produced by plant secondary metabolism have been proven to present antimicrobial action, enabling their use in phytopathogen control. They also present low environmental impact when compared to conventional pesticides. Essential oils contain these compounds and can be found in several plant species, such as Lippia alba (Mill. N.E. Brown (Verbenaceae. Essential oils of four chemotypes of L. alba, characterized by their major compounds, namely camphor, citral, linalool and camphor/1,8-cineole, were tested against the phytopathogen Alternaria solani Sorauer (Pleosporaceae, which causes early blight on tomatoes and is responsible for great economic losses regarding production. Essential oils antifungal action was tested in vitro using potato dextrose agar medium with essential oil concentrations at 0.1, 0.5, 1.0, 1.5 and 2.0 µL mL-1. The chemotype that had the best performance was citral, showing significant inhibition compared to the others, starting at the 0.5 µL mL-1 concentration. The essential oil belonging to the linalool chemotype was efficient starting at the 1.5 µL mL-1 concentration. Conversely, the camphor chemotype did not show any action against the phytopathogen. Moreover, the essential oils had no remarkable effect on tomato germination and growth. In conclusion, these essential oils presented fungicidal action against A. solani.

  14. In vitro antifungal activity of four chemotypes of Lippia alba (Verbenaceae) essential oils against Alternaria solani (Pleosporeaceae) isolates.

    Science.gov (United States)

    Tomazoni, Elisa Z; Pansera, Márcia R; Pauletti, Gabriel F; Moura, Sidnei; Ribeiro, Rute T S; Schwambach, Joséli

    2016-05-31

    Several volatile natural compounds produced by plant secondary metabolism have been proven to present antimicrobial action, enabling their use in phytopathogen control. They also present low environmental impact when compared to conventional pesticides. Essential oils contain these compounds and can be found in several plant species, such as Lippia alba (Mill.) N.E. Brown (Verbenaceae). Essential oils of four chemotypes of L. alba, characterized by their major compounds, namely camphor, citral, linalool and camphor/1,8-cineole, were tested against the phytopathogen Alternaria solani Sorauer (Pleosporaceae), which causes early blight on tomatoes and is responsible for great economic losses regarding production. Essential oils antifungal action was tested in vitro using potato dextrose agar medium with essential oil concentrations at 0.1, 0.5, 1.0, 1.5 and 2.0 µL mL-1. The chemotype that had the best performance was citral, showing significant inhibition compared to the others, starting at the 0.5 µL mL-1 concentration. The essential oil belonging to the linalool chemotype was efficient starting at the 1.5 µL mL-1 concentration. Conversely, the camphor chemotype did not show any action against the phytopathogen. Moreover, the essential oils had no remarkable effect on tomato germination and growth. In conclusion, these essential oils presented fungicidal action against A. solani.

  15. Homologous recombination in Leishmania enriettii.

    Science.gov (United States)

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

    1991-02-01

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

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

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    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

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

    Science.gov (United States)

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

    2004-01-01

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

  18. MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

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    Vanessa Cristina Fitipaldi Veloso Guimarães

    2014-07-01

    Full Text Available Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600. Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

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

    Science.gov (United States)

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

    2017-01-01

    Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (Tm) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the Tm values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate.

  20. Exploring the 3-piperidin-4-yl-1H-indole scaffold as a novel antimalarial chemotype.

    Science.gov (United States)

    Santos, Sofia A; Lukens, Amanda K; Coelho, Lis; Nogueira, Fátima; Wirth, Dyann F; Mazitschek, Ralph; Moreira, Rui; Paulo, Alexandra

    2015-09-18

    A series of 3-piperidin-4-yl-1H-indoles with building block diversity was synthesized based on a hit derived from an HTS whole-cell screen against Plasmodium falciparum. Thirty-eight compounds were obtained following a three-step synthetic approach and evaluated for anti-parasitic activity. The SAR shows that 3-piperidin-4-yl-1H-indole is intolerant to most N-piperidinyl modifications. Nevertheless, we were able to identify a new compound (10d) with lead-like properties (MW = 305; cLogP = 2.42), showing antimalarial activity against drug-resistant and sensitive strains (EC50 values ∼ 3 μM), selectivity for malaria parasite and no cross-resistance with chloroquine, thus representing a potential new chemotype for further optimization towards novel and affordable antimalarial drugs.

  1. Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes.

    Science.gov (United States)

    El Maatougui, Abdelaziz; Azuaje, Jhonny; González-Gómez, Manuel; Miguez, Gabriel; Crespo, Abel; Carbajales, Carlos; Escalante, Luz; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2016-03-10

    Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A, and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.

  2. Laboratory and field measurements of enantiomeric monoterpene emissions as a function of chemotype, light and temperature

    Science.gov (United States)

    Song, W.; Staudt, M.; Bourgeois, I.; Williams, J.

    2014-03-01

    Plants emit significant amounts of monoterpenes into the earth's atmosphere, where they react rapidly to form a multitude of gas phase species and particles. Many monoterpenes exist in mirror-image forms or enantiomers. In this study the enantiomeric monoterpene profile for several representative plants (Quercus ilex L., Rosmarinus officinalis L., and Pinus halepensis Mill.) was investigated as a function of chemotype, light and temperature both in the laboratory and in the field. Analysis of enantiomeric monoterpenes from 19 Quercus ilex individuals from Southern France and Spain revealed four regiospecific chemotypes (genetically fixed emission patterns). In agreement with previous work, only Quercus ilex emissions increased strongly with light. However, for all three plant species no consistent enantiomeric variation was observed as a function of light, and the enantiomeric ratio of α-pinene was found to vary by less than 20% from 100 and 1000 μmol m-2 s-1 PAR (photosynthetically active radiation). The rate of monoterpene emission increased with temperature from all three plant species, but little variation in the enantiomeric distribution of α-pinene was observed with temperature. There was more enantiomeric variability between individuals of the same species than could be induced by either light or temperature. Field measurements of α-pinene enantiomer mixing ratios in the air, taken at a Quercus ilex forest in Southern France, and several other previously reported field enantiomeric ratio diel cycle profiles are compared. All show smoothly varying diel cycles (some positive and some negative) even over changing wind directions. This is surprising in comparison with variations of enantiomeric emission patterns shown by individuals of the same species.

  3. Genotypic and chemotypic diversity of Neotyphodium endophytes in tall fescue from Greece.

    Science.gov (United States)

    Takach, Johanna E; Mittal, Shipra; Swoboda, Ginger A; Bright, Sherrita K; Trammell, Michael A; Hopkins, Andrew A; Young, Carolyn A

    2012-08-01

    Epichloid endophytes provide protection from a variety of biotic and abiotic stresses for cool-season grasses, including tall fescue. A collection of 85 tall fescue lines from 15 locations in Greece, including both Continental and Mediterranean germplasm, was screened for the presence of native endophytes. A total of 37 endophyte-infected lines from 10 locations were identified, and the endophytes were classified into five distinct groups (G1 to G5) based on physical characteristics such as colony morphology, growth rate, and conidial morphology. These classifications were supported by phylogenetic analyses of housekeeping genes tefA and tubB, and the endophytes were further categorized as Neotyphodium coenophialum isolates (G1, G4, and G5) or Neotyphodium sp. FaTG-2 (Festuca arundinacea taxonomic group 2 isolates (G2 and G3). Analyses of the tall fescue matK chloroplast genes indicated a population-wide, host-specific association between N. coenophialum and Continental tall fescue and between FaTG-2 and Mediterranean tall fescue that was also reflected by differences in colonization of host tillers by the native endophytes. Genotypic analyses of alkaloid gene loci combined with chemotypic (chemical phenotype) profiles provided insight into the genetic basis of chemotype diversity. Variation in alkaloid gene content, specifically the presence and absence of genes, and copy number of gene clusters explained the alkaloid diversity observed in the endophyte-infected tall fescue, with one exception. The results from this study provide insight into endophyte germplasm diversity present in living tall fescue populations.

  4. Depicting the Discrepancy between Tri Genotype and Chemotype on the Basis of Strain CBS 139514 from a Field Population of F. graminearum Sensu Stricto from Argentina

    Science.gov (United States)

    Kulik, Tomasz; Buśko, Maciej; Bilska, Katarzyna; Ostrowska-Kołodziejczak, Anna; van Diepeningen, Anne D.; Perkowski, Juliusz; Stenglein, Sebastian

    2016-01-01

    Recent studies on a field population of F. graminearum sensu stricto from Argentina revealed an atypical panel of strains identified through PCR genotyping as 15ADON genotypes, but producing high levels of 3ADON. Based on representative strain CBS 139514, we asked if the discrepancy between the trichothecene genotype and chemotype might result from an inter-chemotype recombination of the chemotype-determining genes. To answer this, we sequenced the complete core Tri gene cluster (around 30,200 bp) from this strain and compared its sequence to sequence data of typical type B trichothecene genotypes/chemotypes. Sequence alignment showed that CBS 139514 has an identical sequence within the entire core Tri cluster to the 15ADON genotype. The revealed discrepancy underlines the need for using both molecular and chemical methods for reliable characterization of toxigenic strains of Fusarium. PMID:27845742

  5. Depicting the Discrepancy between Tri Genotype and Chemotype on the Basis of Strain CBS 139514 from a Field Population of F. graminearum Sensu Stricto from Argentina

    Directory of Open Access Journals (Sweden)

    Tomasz Kulik

    2016-11-01

    Full Text Available Recent studies on a field population of F. graminearum sensu stricto from Argentina revealed an atypical panel of strains identified through PCR genotyping as 15ADON genotypes, but producing high levels of 3ADON. Based on representative strain CBS 139514, we asked if the discrepancy between the trichothecene genotype and chemotype might result from an inter-chemotype recombination of the chemotype-determining genes. To answer this, we sequenced the complete core Tri gene cluster (around 30,200 bp from this strain and compared its sequence to sequence data of typical type B trichothecene genotypes/chemotypes. Sequence alignment showed that CBS 139514 has an identical sequence within the entire core Tri cluster to the 15ADON genotype. The revealed discrepancy underlines the need for using both molecular and chemical methods for reliable characterization of toxigenic strains of Fusarium.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Advanced Developement of Leishmania Tropical Skin Test Antigen

    Science.gov (United States)

    2011-09-01

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

  8. A COMPARISON OF DIFFERENT LIPOPOLYSACCHARIDE CHEMOTYPES FROM ESCHERICHIA COLI AND SALMONELLA UPON SYNTHESIS OF TNFα AND IL-6 BY MACROPHAGE-LIKE THP-1 CELLS

    Directory of Open Access Journals (Sweden)

    E. V. Voloshina

    2009-01-01

    Full Text Available Abstract. Present study was performed to investigate the influence of polysaccharide fragment or lipid A upon induction of TNFα and IL-6 cytokines. The study was performed with human THP-1 monocytic leukemia cells that were induced to differentiate into macrophage-like cells using PMA treatment. Bacterial lipopolysaccharides from S. typhimurium (S-chemotype form, S. typhimurium SL1181 (R-chemotype, Re-mutant, E. coli O55:B5 (S-chemotype, and E. coli JM103 (R-chemotype, Re-mutant were used in this study. A decreased molar ratio for lipid A-KDO in S-form of LPS from E. coli is accompanied by diminished TNFα and IL-6 expression. By the contrast, for S-form of LPS from Salmonella, a decrease in lipid A-KDO molar ratio did cause a sufficient enhancement of TNFα expression. A contribution of lipid A structure into biological activity of LPS is more significant for Re-chemotype than for S-chemotype, independently on bacterial species.

  9. Composition and Bioactivities of an (E)-β-Farnesene Chemotype of Chamomile (Matricaria chamomilla) Essential Oil from Nepal.

    Science.gov (United States)

    Satyal, Prabodh; Shrestha, Samon; Setzer, William N

    2015-08-01

    The essential oil of Matricaria chamomilla, collected from Nepal, was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The major components in Nepalese chamomile oil were (E)-β-famesene (42.2%), α-bisabolol oxide A (22.3%), (E,E)-α-famesene (8.3%), cis-bicycloether (5.0%), α-bisabolol oxide B (4.5%), and α-bisabolone oxide A (4.0%). A cluster analysis based on the chemical compositions of 48 samples of chamomile oil reported in the literature has revealed seven chemotypes, and the oil from Nepal represents the (E)-β-farnesene chemotype. The chamomile oil was screened for antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger, and toxicity toward MCF-7 breast tumor cells, Artemia salina, Chaoborus plumicornis, Caenorhabditis elegans, and Drosophila melanogaster.

  10. Intraspecific variation in essential oil composition of the medicinal plant Lippia integrifolia (Verbenaceae). Evidence for five chemotypes.

    Science.gov (United States)

    Marcial, Guillermo; de Lampasona, Marina P; Vega, Marta I; Lizarraga, Emilio; Viturro, Carmen I; Slanis, Alberto; Juárez, Miguel A; Elechosa, Miguel A; Catalán, César A N

    2016-02-01

    The aerial parts of Lippia integrifolia (incayuyo) are widely used in northwestern and central Argentina for their medicinal and aromatic properties. The essential oil composition of thirty-one wild populations of L. integrifolia covering most of its natural range was analyzed by GC and GC-MS. A total of one hundred and fifty two terpenoids were identified in the essential oils. Sesquiterpenoids were the dominant components in all but one of the collections analyzed, the only exception being a sample collected in San Juan province where monoterpenoids amounted to 51%. Five clearly defined chemotypes were observed. One possessed an exquisite and delicate sweet aroma with trans-davanone as dominant component (usually above 80%). Another with an exotic floral odour was rich in oxygenated sesquiterpenoids based on the rare lippifoliane and africanane skeletons. The trans-davanone chemotype is the first report of an essential oil containing that sesquiterpene ketone as the main constituent. The absolute configuration of trans-davanone from L. integrifolia was established as 6S, 7S, 10S, the enantiomer of trans-davanone from 'davana oil' (Artemisia pallens). Wild plants belonging to trans-davanone and lippifolienone chemotypes were propagated and cultivated in the same parcel of land in Santa Maria, Catamarca. The essential oil compositions of the cultivated plants were essentially identical to the original plants in the wild, indicating that the essential oil composition is largely under genetic control. Specimens collected near the Bolivian border that initially were identified as L. boliviana Rusby yielded an essential oil practically identical to the trans-davanone chemotype of L. integrifolia supporting the recent view that L. integrifolia (Gris.) Hieron. and L. boliviana Rusby are synonymous.

  11. Phytochemical Analysis and Antimicrobial, Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes of Pimenta pseudocaryophyllus (Myrtaceae).

    Science.gov (United States)

    de Paula, Joelma Abadia Marciano; Silva, Maria do Rosário Rodrigues; Costa, Maysa P; Diniz, Danielle Guimarães Almeida; Sá, Fabyola A S; Alves, Suzana Ferreira; Costa, Elson Alves; Lino, Roberta Campos; de Paula, José Realino

    2012-01-01

    Preparations from Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) have been widely used in Brazilian folk medicine. This study aims to evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus and to perform the antinociceptive and anti-inflammatory screening. The ethanol extracts were purified by column chromatography and main compounds were spectrally characterised (1D and 2D (1)H and (13)C NMR). The essential oils constituents were identified by GC/MS. The broth microdilution method was used for testing the antimicrobial activity. The abdominal contortions induced by acetic acid and the ear oedema induced by croton oil were used for screening of antinociceptive and anti-inflammatory activities, respectively. The phytochemical analysis resulted in the isolation of pentacyclic triterpenes, flavonoids, and phenol acids. The oleanolic acid showed the best profile of antibacterial activity for Gram-positive bacteria (31.2-125 μg mL(-1)), followed by the essential oil of the citral chemotype (62.5-250 μg mL(-1)). Among the semipurified substances, Ppm5, which contained gallic acid, was the most active for Candida spp. (31.2 μg mL(-1)) and Cryptococcus spp. (3.9-15.6 μg mL(-1)). The crude ethanol extract and fractions from citral chemotype showed antinociceptive and anti-inflammatory effects.

  12. Metabolic profiling for studying chemotype variations in Withania somnifera (L.) Dunal fruits using GC-MS and NMR spectroscopy.

    Science.gov (United States)

    Bhatia, Anil; Bharti, Santosh K; Tewari, Shri K; Sidhu, Om P; Roy, Raja

    2013-09-01

    Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plant with several pharmaceutical and nutraceutical applications. Metabolic profiling was performed by GC-MS and NMR spectroscopy on the fruits obtained from four chemotypes of W. somnifera. A combination of (1)H NMR spectroscopy and GC-MS identified 82 chemically diverse metabolites consisting of organic acids, fatty acids, aliphatic and aromatic amino acids, polyols, sugars, sterols, tocopherols, phenolic acids and withanamides in the fruits of W. somnifera. The range of metabolites identified by GC-MS and NMR of W. somnifera fruits showed various known and unknown metabolites. The primary and secondary metabolites observed in this study represent MVA, DOXP, shikimic acid and phenylpropanoid biosynthetic metabolic pathways. Squalene and tocopherol have been rated as the most potent naturally occurring compounds with antioxidant properties. These compounds have been identified by us for the first time in the fruits of W. somnifera. Multivariate principal component analysis (PCA) on GC-MS and NMR data revealed clear distinctions in the primary and secondary metabolites among the chemotypes. The variation in the metabolite concentration among different chemotypes of the fruits of W. somnifera suggest that specific chemovars can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.

  13. Molecular detection of Leishmania in phlebotomine sand flies (Diptera: Psychodidae) from a cutaneous leishmaniasis focus atXakriabá Indigenous Reserve, Brazil.

    Science.gov (United States)

    Rêgo, Felipe Dutra; Rugani, Jeronimo Marteleto Nunes; Shimabukuro, Paloma Helena Fernandes; Tonelli, Gabriel Barbosa; Quaresma, Patrícia Flávia; Gontijo, Célia Maria Ferreira

    2015-01-01

    Autochthonous cases of American cutaneous leishmaniasis (ACL) have been reported since 2001 in the Xakriabá Indigenous Reserve located in the municipality of São João das Missões in northern Minas Gerais state, Brazil. In order to study the presence of Leishmania DNA in phlebotomine sand flies, six entomological collections were carried out from July 2008 through July 2009, using 40 light traps placed in peridomicile areas of 20 randomly selected houses. From October 2011 through August 2012, another six collections were carried out with 20 light traps distributed among four trails (five traps per trail) selected for a previous study of wild and synanthropic hosts of Leishmania. A total of 4,760 phlebotomine specimens were collected belonging to ten genera and twenty-three species. Single female specimens or pools with up to ten specimens of the same locality, species and date, for Leishmania detection by molecular methods. Species identification of parasites was performed with ITS1 PCR-RFLP using HaeIII enzyme and genetic sequencing for SSU rRNA target. The presence of Leishmania DNA was detected in eleven samples from peridomicile areas: Lu. longipalpis (two), Nyssomyia intermedia (four), Lu. renei (two), Lu. ischnacantha, Micropygomyia goiana and Evandromyia lenti (one pool of each specie). The presence of Leishmania DNA was detected in twelve samples from among the trails: Martinsmyia minasensis (six), Ny. intermedia (three), Mi. peresi (two) and Ev. lenti (one). The presence of Leishmania infantum DNA in Lu. longipalpis and Leishmania braziliensis DNA in Ny. intermediasupport the epidemiological importance of these species of sand flies in the cycle of visceral and cutaneous leishmaniasis, respectively. The results also found other species associated with Leishmania DNA, such as Mt. minasensis and Ev. lenti, which may participate in a wild and/or synanthropic cycle of Leishmania transmission in the studied area.

  14. Mechanisms of pathogenesis: differences amongst Leishmania species.

    Science.gov (United States)

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

    2002-04-01

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

  15. Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

    Directory of Open Access Journals (Sweden)

    Patricia Barrera

    2013-01-01

    Full Text Available Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host’s cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc, dehydroleucodine (DhL, psilostachyin (Psi, and, at lesser extent, psilostachyin C (Psi C is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.

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

    Science.gov (United States)

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

    2012-04-01

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

  17. Innate Immunity to Leishmania Infection: Within Phagocytes

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    Marcela Freitas Lopes

    2014-01-01

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

  18. First report on naturalLeishmania infection ofPhlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran

    Institute of Scientific and Technical Information of China (English)

    Aghaei Afshar A; Rassi Y; Sharifi I; Vatandoost H; Mollaie HR; Oshaghi MA; Abai MR; Rafizadeh S

    2014-01-01

    Objective:To identify the Leishmaniaspecies in infected sand flies byReal-timePCR coupled withHRM analysis.Methods:Real-timePCR coupled withHRM analysis targeting the first internal transcribed spacer(ITS1) of nuclear ribosomalDNA as the genetic marker was used to identify and distinguish Leishmania species in sand flies specimens.Results:Three out of115 females ofPhlebotomus sergenti(P. sergenti)(2.6%) were positive toLeishmania tropica(L. tropica). Conclusions:This is the first report onP. sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis inDehbakriCounty usingReal-timePCR coupled withHRM analysis. This method is rapid, sensitive and specific for diagnosing of parasites in infectedSand flies and ideal for large scale genotyping projects.

  19. Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance [version 1; referees: 5 approved

    Directory of Open Access Journals (Sweden)

    Marie-Claude N. Laffitte

    2016-09-01

    Full Text Available Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV. CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.

  20. Isolation and molecular identification of Leishmania ( Viannia peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae in the Peruvian Andes

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    J Enrique Perez

    2007-08-01

    Full Text Available Leishmania (Viannia peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru. Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia sp. was determined by polymerase chain reaction (PCR, and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V. peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

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

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    BL Travi

    2002-09-01

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

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

    Science.gov (United States)

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

    2014-06-30

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

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

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    Prasad K Padmanabhan

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  5. Developmentally regulated sphingolipid degradation in Leishmania major.

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    Ou Zhang

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

  6. Comorbidity of Leishmania major with cutaneous sarcoidosis

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    Hamideh Moravvej

    2014-01-01

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

  7. Antimony Resistance in Leishmania, Focusing on Experimental Research

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    Fakhri Jeddi

    2011-01-01

    Full Text Available Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases. There are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions. However, resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules. In this paper, we reviewed the studies carried out on antimonial resistance in Leishmania. Several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related. Afterwards, phenotypic determination of resistance is described, then confronted to clinical outcome. Finally, we detail molecular mechanisms and targets involved in resistance and already identified in vitro within selected mutant strains or in clinical isolates.

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

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    Venkateswara Reddy Gogulamudi

    2015-10-01

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

  9. ¹H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification.

    Science.gov (United States)

    Peschel, Wieland; Politi, Matteo

    2015-08-01

    The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ∆9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of (1)H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide together with two new validated HPLC/DAD methods used for identification and extract profiling based on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, cannabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid acids in non-heated extracts suggest their consideration for total values in chemotype distinction and specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile. Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2-4 lead cannabinoids are considered essential. The suitability of analytical methods and the range of compound groups summarized in group and ratio markers are discussed regarding plant classification and pharmaceutical specification.

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

    Directory of Open Access Journals (Sweden)

    Marcela Orsini

    2002-06-01

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

  11. Image Annotation and Database Mining to Create a Novel Screen for the Chemotype-Dependent Crystallization of HCV NS3 Protease

    Energy Technology Data Exchange (ETDEWEB)

    H Klei; K Kish; M Russo; S Michalczyk; M Cahn; J Tredup; C Chang; J Khan; E Baldwin

    2011-12-31

    An effective process for screening, imaging, and optimizing crystallization trials using a combination of external and internal hardware and software has been deployed. The combination of this infrastructure with a vast annotated crystallization database enables the creation of custom crystallization screening strategies. Because of the strong chemotype-dependent crystallization observed with HCV NS3 protease (HCVPr), this strategy was applied to a chemotype resistant to all prior crystallization efforts. The crystallization database was mined for ingredients used to generate earlier HCVPr/inhibitor co-crystals. A random screen was created from the most prolific ingredients. A previously untested combination of proven ingredients was identified that led to a successful crystallization condition for the resistant chemotype.

  12. Advanced Development of Leishmania Topical Skin Test Antigen

    Science.gov (United States)

    2012-09-28

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

  13. Development and Production of a Leishmania Skin Test

    Science.gov (United States)

    2009-03-01

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

  14. High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase.

    Directory of Open Access Journals (Sweden)

    Roderick G Walker

    target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

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

    Science.gov (United States)

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

    2013-01-01

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

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

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    Teklu Kuru Gerbaba

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

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

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    Roger Magno Macedo-Silva

    2014-02-01

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

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

    Science.gov (United States)

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

    2014-06-24

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

  19. Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

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    Coelho Adriano C.

    2004-01-01

    Full Text Available Pentamidine (PEN is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

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

    Directory of Open Access Journals (Sweden)

    Andrea Niño

    2005-05-01

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

  1. Computational prediction of protein-protein interactions in Leishmania predicted proteomes.

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    Antonio M Rezende

    Full Text Available The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks

  2. Comparative Analysis of Deoxynivalenol Biosynthesis Related Gene Expression among Different Chemotypes of Fusarium graminearum in Spring Wheat

    Science.gov (United States)

    Amarasinghe, Chami C.; Fernando, W. G. Dilantha

    2016-01-01

    Fusarium mycotoxins, deoxynivalenol (DON) and nivalenol (NIV) act as virulence factors and are essential for symptom development after initial infection in wheat. To date, 16 genes have been identified in the DON biosynthesis pathway. However, a comparative gene expression analysis in different chemotypes of Fusarium graminearum in response to Fusarium head blight infection remains to be explored. Therefore, in this study, nine genes that involved in trichothecene biosynthesis were analyzed among 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol producing F. graminearum strains in a time course study. Quantitative reverse transcription polymerase chain reaction revealed that the expression of all examined TRI gene transcripts initiated at 2 days post-inoculation (dpi), peaked at three to four dpi and gradually decreased at seven dpi. The early induction of TRI genes indicates that presence of high levels of TRI gene transcripts at early stages is important to initiate the biosynthetic pathway of DON and NIV. Comparison of gene expression among the three chemotypes showed that relative expression of TRI genes was higher in 3-ADON producing strains compared with 15-ADON and NIV strains. Comparatively higher levels of gene expression may contribute to the higher levels of DON produced by 3-ADON strains in infected grains. PMID:27550207

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

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    Elizaide L. A. Yoshida

    1993-09-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Visceral leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self-healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human DCs’ biology for better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24 h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of DCs after 24 h. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model. PMID:27536300

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

    Directory of Open Access Journals (Sweden)

    Sarah Athayde Couto Falcão

    2016-08-01

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

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

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    Imen Mkada-Driss

    Full Text Available Visceral leishmaniasis (VL is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5' end transversions, and presence of inter- and intra- taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers

  7. Leishmania major: Parasite Interactions Suggesting Sexuality

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    Sousa Maria Auxiliadora de

    1997-01-01

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

  8. 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.

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

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    Loiseau P.M.

    2000-06-01

    Full Text Available lr-(COD-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 μM. In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9 /Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 μmoles/ kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23 % of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 μmoles/ kg/day x 5 (54 % of parasite suppression, the iridium complex exhibited 32 % of parasite suppression after a treatment at 76 μmoles/ kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

  10. Testing of Experimental Compounds for Efficacy against Leishmania.

    Science.gov (United States)

    1987-02-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Comparative inhibitory effect of prenylated coumarins, ferulenol and ferprenin, contained in the 'poisonous chemotype' of Ferula communis on mammal liver microsomal VKORC1 activity.

    Science.gov (United States)

    Louvet, Marie-Sophie; Gault, Gilbert; Lefebvre, Sébastien; Popowycz, Florence; Boulven, Manon; Besse, Stéphane; Benoit, Etienne; Lattard, Virginie; Grancher, Denis

    2015-10-01

    Two distinguishable chemotypes of Ferula communis have been described: the 'nonpoisonous' chemotype, containing as main constituents the daucane esters; and the 'poisonous' chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to 'poisonous' chemotypes of F. communis.

  13. Chemical composition, olfactory analysis and antibacterial activity of Thymus vulgaris chemotypes geraniol, 4-thujanol/terpinen-4-ol, thymol and linalool cultivated in southern France.

    Science.gov (United States)

    Schmidt, Erich; Wanner, Jürgen; Hiiferl, Martina; Jirovetz, Leopold; Buchbauer, Gerhard; Gochev, Velizar; Girova, Tania; Stoyanova, Albena; Geissler, Margit

    2012-08-01

    The essential oils of four chemotypes of Thymus vulgaris L. (Lamiaceae) were analyzed for their composition and antibacterial activity to assess their different properties. GC-MS and GC-FID analyses revealed that the essentials oils can be classified into the chemotypes thymol (41.0% thymol), geraniol (26.4% geraniol), linalool (72.5% linalool) and 4-thujanol/terpinen-4-ol (42.2% cis- and 7.3% trans-sabinene hydrate, 6.5 % terpinen-4-ol). The olfactory examination confirmed the explicit differences between these chemotypes. Furthermore, antibacterial activity was investigated against several strains of two Gram-positive (Brochothrix thermosphacta and Staphylococcus aureus) and four Gram-negative food-borne bacteria (Escherichia coli, Salmonella abony, Pseudomonas aeruginosa and P. fragi). All essential oil samples were demonstrated to be highly effective against Gram-positive strains, whereas the impact on Gram-negative microorganisms was significantly smaller, but still considerable. The results obtained indicate that, despite their different properties, the essential oils of selected T. vulgaris chemotypes are potent antimicrobials to be employed as useful additives in food products as well as for therapeutic applications.

  14. Molecular and phenotypic description of Stachybotrys chlorohalonata sp. nov and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

    DEFF Research Database (Denmark)

    Andersen, Birgitte; Nielsen, Kristian Fog; Thrane, Ulf;

    2003-01-01

    Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, rising morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differ...

  15. A Comparison of Aggressiveness and Deoxynivalenol Production Between Canadian Fusarium graminearum Isolates with 3-Acetyl and 15-Acetyldeoxynivalenol Chemotypes in Field-Grown Spring Wheat

    Science.gov (United States)

    Twenty four isolates of Fusarium graminearum, half of which were 3- acetyldeoxynivalenol (3-ADON) and half 15-acetyldeoxynivalenol (15-ADON) chemotypes, were tested for their ability to produce deoxynivalenol and to cause Fusarium head blight (FHB), in spring wheat cultivars. The objectives of this...

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

    Science.gov (United States)

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

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

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

    Science.gov (United States)

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

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

  18. Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco

    Directory of Open Access Journals (Sweden)

    Nargys Es-Sette

    2014-04-01

    Full Text Available Background & objectives: An entomological study using CDC miniature light-traps was performed in El Hanchane locality, where cutaneous leishmaniasis (CL was emerging during the summer of 2011. The aim of this study is to identify the vectors of Leishmania and of phleboviruses. Methods: In the field, a total of 643 sandfly specimens were collected, identified by morphological keys and categorized by sex and species. A total of nine distinct species were morphologically identified where seven belonged to the Phlebotomus genus and two species to the Sergentomyia genus. Phlebotomus sergenti was the most abundant species (76%. Phleboviruses were detected by nested RT-PCR using 30 pooled sandflies while P. sergenti females were tested individually for infections of Leishmania species. Results: By using ITS1-PCR-RFLP approach, Leishmania tropica DNA was detected in 10 females, caught in this emerging focus, and provide additional evidence in favour of the role of P. sergenti as vector of L. tropica in Morocco. Real-time PCR screening for phlebovirus RNA, using an assay targeting the polymerase gene, showed positive result in one pool of male P. sergenti. Interpretation & conclusion: In this study, P. sergenti were infected by L. tropica and Toscana virus. To our knowledge, actually this is the first time that Toscana virus has been detected in P. sergenti.

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2009-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Ralph Lainson

    1997-05-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

  3. Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill. N.E. Brown: composition, cytotoxicity and antifungal activity

    Directory of Open Access Journals (Sweden)

    Ana Cecilia Mesa-Arango

    2009-09-01

    Full Text Available Two essential oils of Lippia alba (Mill. N.E. Brown (Verbenacea, the carvone and citral chemotypes and 15 of their compounds were evaluated to determine cytotoxicity and antifungal activity. Cytotoxicity assays for both the citral and carvone chemotypes were carried out with tetrazolium-dye, which showed a dose-dependent cytotoxic effect against HeLa cells. Interestingly, this effect on the evaluated cells (HeLa and the non-tumoural cell line, Vero was lower than that of commercial citral alone. Commercial citral showed the highest cytotoxic activity on HeLa cells. The antifungal activity was evaluated against Candida parapsilosis, Candida krusei, Aspergillus flavus and Aspergillus fumigatus strains following the standard protocols, Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing and CLSI M38-A. Results demonstrated that the most active essential oil was the citral chemotype, with geometric means-minimal inhibitory concentration (GM-MIC values of 78.7 and 270.8 μg/mL for A. fumigatus and C. krusei, respectively. Commercial citral showed an antifungal activity similar to that of the citral chemotype (GM-MIC values of 62.5 μg/mL for A. fumigatus and 39.7 μg/mL for C. krusei. Although the citronellal and geraniol were found in lower concentrations in the citral chemotype, they had significant antifungal activity, with GM-MIC values of 49.6 μg/mL for C. krusei and 176.8 μg/mL for A. fumigatus.

  4. Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown: composition, cytotoxicity and antifungal activity.

    Science.gov (United States)

    Mesa-Arango, Ana Cecilia; Montiel-Ramos, Jehidys; Zapata, Bibiana; Durán, Camilo; Betancur-Galvis, Liliana; Stashenko, Elena

    2009-09-01

    Two essential oils of Lippia alba (Mill.) N.E. Brown (Verbenacea), the carvone and citral chemotypes and 15 of their compounds were evaluated to determine cytotoxicity and antifungal activity. Cytotoxicity assays for both the citral and carvone chemotypes were carried out with tetrazolium-dye, which showed a dose-dependent cytotoxic effect against HeLa cells. Interestingly, this effect on the evaluated cells (HeLa and the non-tumoural cell line, Vero) was lower than that of commercial citral alone. Commercial citral showed the highest cytotoxic activity on HeLa cells. The antifungal activity was evaluated against Candida parapsilosis, Candida krusei, Aspergillus flavus and Aspergillus fumigatus strains following the standard protocols, Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing and CLSI M38-A. Results demonstrated that the most active essential oil was the citral chemotype, with geometric means-minimal inhibitory concentration (GM-MIC) values of 78.7 and 270.8 microg/mL for A. fumigatus and C. krusei, respectively. Commercial citral showed an antifungal activity similar to that of the citral chemotype (GM-MIC values of 62.5 microg/mL for A. fumigatus and 39.7 microg/mL for C. krusei). Although the citronellal and geraniol were found in lower concentrations in the citral chemotype, they had significant antifungal activity, with GM-MIC values of 49.6 microg/mL for C. krusei and 176.8 microg/mL for A. fumigatus.

  5. Cystathionine γ-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp.

    Science.gov (United States)

    Giordana, Lucila; Mantilla, Brian Suárez; Santana, Marianela; Silber, Ariel M; Nowicki, Cristina

    2014-01-01

    Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine β-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 μM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.

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

    Science.gov (United States)

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

    2014-10-01

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

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

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    Wai-Lok Yau

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

  8. Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

    Science.gov (United States)

    Hajjaran, Homa; Mohebali, Mehdi; Teimouri, Aref; Oshaghi, Mohammad Ali; Mirjalali, Hamed; Kazemi-Rad, Elham; Shiee, Mohammad Reza; Naddaf, Saied Reza

    2014-08-01

    The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates

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

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

    2015-09-01

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

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

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    Jarina Pena DaMata

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

  11. Augmented Oxygen-Dependent Killing of Leishmania.

    Science.gov (United States)

    1992-06-30

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

  12. Novel Leishmania and Malaria Potassium Channels: Candidate Therapeutic Targets

    Science.gov (United States)

    2005-08-01

    3C 0 C. 0 0o o -a .0 ~ 00 Cu C 0 I -u-- C~. ~~rI viE Lo ~r~ fiin If III FITC DARI Phase A anfi-PfKl F4W ring fing tropfiozaite schizont schizont FITO ...DARI Phase B anti-PfK2 F4 ring ring tro phozo ite trop hozo lie schlizont schizont FITO DARI Phase C anti-KAHRP ring trciphozoite schi zo ni D anti

  13. 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.

  14. Effects of Brazilian propolis on Leishmania amazonensis

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    Diana Copi Ayres

    2007-03-01

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

  15. The leishmania ARL-1 and Golgi traffic.

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    Annelise Sahin

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

  16. Cloning of Leishmania Major P4 Gene

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    Minoo Shaddel

    2008-01-01

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

  17. Cyclic nucleotide specific phosphodiesterases of Leishmania major

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    Linder Markus

    2006-03-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    Magyda AA Dahroug

    2011-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

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

    2011-06-01

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

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

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    Jorge Augusto de Oliveira Guerra

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    OpenAIRE

    Chaara, Dhekra; Ravel, Christophe; Bañuls, Anne- Laure; Haouas, Najoua; Lami, Patrick; Talignani, Loïc; El Baidouri, Fouad; Jaouadi, Kaouther; Harrat, Zoubir; Dedet, Jean-Pierre; Babba, Hamouda; Pratlong, Francine

    2015-01-01

    Background: The taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica. Methods: Thirty-five L. killicki and 25 L. tropica strain...

  7. Implications of a Neotropical Origin of the Genus Leishmania

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    Noyes Harry

    1998-01-01

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

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

    OpenAIRE

    2009-01-01

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

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

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    Mansour Dabirzadeh

    2016-04-01

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

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

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    Kárita Cláudia Freitas-Lidani

    2014-07-01

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

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

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    Letícia da Cruz Sanches

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

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

    Science.gov (United States)

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

    2011-01-10

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

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

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    Ryuichi Miura

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

  14. The genome of the kinetoplastid parasite, Leishmania major

    OpenAIRE

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

    2005-01-01

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

  15. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    OpenAIRE

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

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly infl...

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

    OpenAIRE

    REZVAN, Hossein; MOAFI, Mohammad

    2015-01-01

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

  17. Molecular identification of Lutzomyia migonei (Diptera: Psychodidae) as a potential vector for Leishmania infantum (Kinetoplastida: Trypanosomatidae).

    Science.gov (United States)

    Rodrigues, Ana Caroline Moura; Melo, Luciana Magalhães; Magalhães, Rafaela Damasceno; de Moraes, Nélio Batista; de Souza Júnior, Antônio Domingos; Bevilaqua, Claudia Maria Leal

    2016-04-15

    Visceral leishmaniasis (VL) in Brazil is caused by the protozoan Leishmania infantum. This parasite is transmitted by the bite of a female sand fly. The most important sand fly species in VL transmission is Lutzomyia longipalpis. In Fortaleza, the capital of Ceará State, Brazil, the simultaneous occurrence of Lutzomyia migonei and L. longipalpis was detected in localities where VL transmission is observed. The purpose of this study was to determine conclusively if L. migonei can be found naturally infected with L. infantum in key focus in Fortaleza. Using a CDC traps we performed phlebotomine capture during one year. External morphological features and qPCR targeting species-specific gene sequences of Lutzomyia species were used to identify the female phlebotomine sand flies. The molecular identification of the Leishmania species was performed using qPCR targeting species-specific gene sequences of L. infantum and Leishmania braziliensis. The males L. migonei abundance was higher in the rainy season. Humidity and rainfall positively correlated with males L. migonei abundance, while temperature showed a negative correlation. The correlation between the density of L. migonei female with rainfall, relative air humidity, and temperature were not statistically significant. According to the molecular data produced by qPCR amplifications, three positive sand flies were identified as L. longipalpis, and one was identified as L. migonei. The infection rate was 0.35% and 0.18%, respectively. The parasite load was 32,492±2572 L. infantum in L. migonei while the L. longipalpis had parasite loads between 2,444,964.6±116,000 and 6,287,130±124,277. Our findings confirm L. migonei as a potential vector of VL in Fortaleza at a molecular level.

  18. Characterization and functional analysis of eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L.

    Science.gov (United States)

    Renu, Indu Kumari; Haque, Inamul; Kumar, Manish; Poddar, Raju; Bandopadhyay, Rajib; Rai, Amit; Mukhopadhyay, Kunal

    2014-03-01

    Eugenol-O-methyltransferase (EOMT) catalyzes the conversion of eugenol to methyleugenol in one of the final steps of phenylpropanoid pathway. There are no comprehensive reports on comparative EOMT gene expression and developmental stage specific accumulation of phenylpropenes in Ocimum tenuiflorum. Seven chemotypes, rich in eugenol and methyleugenol, were selected by assessment of volatile metabolites through multivariate data analysis. Isoeugenol accumulated in higher levels during juvenile stage (36.86 ng g(-1)), but reduced sharply during preflowering (8.04 ng g(-1)), flowering (2.29 ng g(-1)) and postflowering stages (0.17 ng g(-1)), whereas methyleugenol content gradually increased from juvenile (12.25 ng g(-1)) up to preflowering (16.35 ng g(-1)) and then decreased at flowering (7.13 ng g(-1)) and post flowering (5.95 ng g(-1)) from fresh tissue. Extreme variations of free intracellular and alkali hydrolysable cell wall released phenylpropanoid compounds were observed at different developmental stages. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90 bp intron. The translated proteins had eight catalytic domains, the major two being dimerisation superfamily and methyltransferase_2 superfamily. A validated 3D structure of EOMT protein was also determined. The chemotype Ot7 had a reduced reading frame that lacked both dimerisation domains and one of the two protein-kinase-phosphorylation sites; this was also reflected in reduced accumulation of methyleugenol compared to other chemotypes. EOMT transcripts showed enhanced expression in juvenile stage that increased further during preflowering but decreased at flowering and further at postflowering. The expression patterns may possibly be compared and correlated to the amounts of eugenol/isoeugenol and methyleugenol in different developmental stages of all chemotypes.

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

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    Nour Hammoudeh

    2014-12-01

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

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

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    Tiago Donatelli Serafim

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

  1. Canine inflammatory myopathy associated with Leishmania Infantum infection.

    Science.gov (United States)

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

    2009-02-01

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

  2. 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

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

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    Matheus Balduíno Goncalves dos Reis

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

  4. Chemical Composition, and Antibacterial and Free-Radical-Scavenging Activities of the Essential Oils of a Citronellol Producing New Chemotype of Thymus pubescens Boiss. & Kotschy ex Celak

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    Satyajit D. Sarker

    2011-01-01

    Full Text Available The composition of the essential oils obtained from the flowering aerial parts of two populations of Thymus pubescens, collected from Mishov-Dagh, was determined by the GC-MS analyses. A total of 18 compounds, representing about 95% of the total oils, were identified in both samples of the essential oils. The essential oils of these two populations showed the presence of high amounts of citronellol (42.0% and 42.6%, geranyl acetate (14.0 and 14.0%, geraniol (13.0 and 13.1%, citronellyl acetate (3.9 and 3.8%, L-linalool (7.8% and 7.9%, cis-nerodiol (5.9% and 5.5% and citronellyl acetate (3.9% and 3.8%. However, in the published literature, carvacrol, thymol and p-cymene were reported to be the major compounds in T. pubescens. This significant difference in the composition of the essential oils was a clear evidence of chemical polymorphism with in the T. pubescens taxon, suggesting that these two populations of T. pubescens were in deed a new chemotype of this species, and the name Thymus pubescens Boiss. & Kotschy ex Celak chemotype Citronellol for this new chemotype has been proposed . The antibacterial and free-radical-scavenging properties of the essential oils of T. pubescens have also been evaluated.

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

    OpenAIRE

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2011-06-01

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

  7. Delayed culture of Leishmania in skin biopsies.

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

  9. Do co-occurring plant species adapt to one another? The response of Bromus erectus to the presence of different Thymus vulgaris chemotypes.

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    Ehlers, Bodil K; Thompson, John

    2004-11-01

    Local modification of the soil environment by individual plants may affect the performance and composition of associated plant species. The aromatic plant Thymus vulgaris has the potential to modify the soil through leaching of water-soluble compounds from leaves and litter decomposition. In southern France, six different thyme chemotypes can be distinguished based on the dominant monoterpene in the essential oil, which is either phenolic or non-phenolic in structure. We examine how soils from within and away from thyme patches in sites dominated by either phenolic or non-phenolic chemotypes affect germination, growth and reproduction of the associated grass species Bromus erectus. To do so, we collected seeds of B. erectus from three phenolic and three non-phenolic sites. Seeds and seedlings were grown on soils from these sites in a reciprocal transplant type experiment in the glasshouse. Brome of non-phenolic origin performed significantly better on its home soil than on soil from a different non-phenolic or a phenolic site. This response to local chemotypes was only observed on soil collected directly underneath thyme plants and not on soil in the same site (<5 m away) but where no thyme plants were present. This is preliminary evidence that brome plants show an adaptive response to soil modifications mediated by the local thyme chemotypes. Reproductive effort was consistently higher in brome of phenolic origin than in brome of non-phenolic origin (on both thyme- and grass-soil), indicating that life-history variation may be related to environmental factors which also contribute to the spatial differentiation of thyme chemotypes. Moreover, we found that brome growing on thyme-soil in general was heavier than when growing on grass-soil, regardless of the origin of the brome plants. This is concordant with thyme-soil containing higher amounts of organic matter and nitrogen than grass-soil. Our results indicate that patterns of genetic differentiation and local

  10. Sterol biosynthesis is required for heat resistance but not extracellular survival in leishmania.

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    Wei Xu

    2014-10-01

    Full Text Available Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm(- were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm(- mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm(- causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

  11. Sterol biosynthesis is required for heat resistance but not extracellular survival in leishmania.

    Science.gov (United States)

    Xu, Wei; Hsu, Fong-Fu; Baykal, Eda; Huang, Juyang; Zhang, Kai

    2014-10-01

    Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm(-)) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm(-) mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm(-) causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

  12. Quantitative HPLC analysis of sesquiterpene lactones and determination of chemotypes in Eremanthus seidelii MacLeish and Schumacher (Asteraceae)

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    Sakamoto, Humberto T. [Sao Paulo Univ., Ribeirao Preto, SP (Brazil). Faculdade de Filosofia, Ciencias e Letras. Dept. de Quimica; Gobbo-Neto, Leonardo; Lopes, Norberto P.; Lopes, Joao L.C. [Sao Paulo Univ., Ribeirao Preto, SP (Brazil). Faculdade de Ciencias Farmaceuticas. Dept. de Fisica e Quimica]. E-mail: joaoluis@usp.br; npelopes@fcfrp.usp.br; Cavalheiro, Alberto J. [UNESP, Araraquara, SP (Brazil). Inst. de Quimica

    2005-11-15

    anthus seidelii MacLeish and Schumacher has a restricted occurrence to the Brazilian 'cerrado' surrounding the Furnas (MG) reservoir, in environments that have been seriously damaged by human activity. The present phytochemical investigation reveals that the sesquiterpene lactones (SL) 4{beta},5-dihydro-2',3'-dihydroxy-15-desoxy-goyazensolide (1) and 4{beta},5-dihydro-1',2'-epoxy-eremantholide-C (2) are the major secondary metabolites in E. seidelii leaves, and an HPLC method was developed for their quantitative analysis. HPLC analysis showed no significant seasonal variation in the concentrations of both SL. No qualitative differences were found in the SL patterns of all individuals sampled. However, there is a different SL quantitative pattern among the plants analyzed, pointing to the existence of three quantitative chemotypes of this species, with differences possibly originating from the activity of the enzymes that cyclize the goyazensolide type SL (1) to a eremantholide type SL (2). (author)

  13. 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

    sand fly species, including Lu. trapidoi, in which it frequently co-occurred with Leishmania. CONCLUSIONS: Both morphological and molecular methods were used to examine an assemblage of 20 sand fly species occurring in the forests of the Panama Canal area. Two of these species, members of separate clades, were found to carry Leishmania at high frequency and hence are likely vectors of leishmaniasis to humans or other mammal species. A single Leishmania species, identified with high confidence as Le. naiffi, was carried by both species. That Le. naiffi is known to cause cutaneous lesions in South America but has hitherto not been reported or implicated in Panama opens the possibility that its range has recently expanded to include the Isthmus or that it occurs as a recent introduction. The occurrence of Leishmania and Wolbachia in Lu. trapidoi identifies one important vector of the disease as a potential target for gene introductions using Wolbachia population sweeps.

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

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    Mohammad Karimian Shirazi

    2014-08-01

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

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

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    Saldarriaga, Omar A.; Castellanos-Gonzalez, Alejandro; Porrozzi, Renato; Baldeviano, Gerald C.; Lescano, Andrés G.; de Los Santos, Maxy B.; Fernandez, Olga L.; Saravia, Nancy G.; Costa, Erika; Melby, Peter C.; Travi, Bruno L.

    2016-01-01

    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. PMID:27115155

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

    Science.gov (United States)

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

    2013-01-01

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

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

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    Sara Teixeira de Macedo-Silva

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

  18. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

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

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    Epco Hasker

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

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

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    Schwenkenbecher Jan

    2008-06-01

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

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

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    Hide, M; Bañuls, A L

    2008-02-01

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

  3. Cryptic Leishmania infantum infection in Italian HIV infected patients

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    Rubino Raffaella

    2009-12-01

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

  4. The crystal structure of the Leishmania major deoxyuridine triphosphate nucleotidohydrolase in complex with nucleotide analogues, dUMP, and deoxyuridine.

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    Hemsworth, Glyn R; Moroz, Olga V; Fogg, Mark J; Scott, Benjamin; Bosch-Navarrete, Cristina; González-Pacanowska, Dolores; Wilson, Keith S

    2011-05-06

    Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.

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

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

    2017-01-01

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

  6. Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis.

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    Diana Moreira

    2015-03-01

    Full Text Available Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.

  7. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

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    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4+ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis. PMID:25801301

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

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    Mary Dan-Goor

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

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    Marcelo Rosandiski LYRA

    2015-10-01

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

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

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

    2015-01-01

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

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

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

    2011-02-01

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

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

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

    2014-01-01

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

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

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    Andrade-Narvaez Fernando J

    2003-01-01

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

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

    Science.gov (United States)

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

    2003-12-01

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

  16. 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.

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

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    Andrade-Narváez Fernando J

    2001-01-01

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

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

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    Gülnaz Culha

    2014-06-01

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

  19. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    Science.gov (United States)

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

  20. Molecular crosstalks in Leishmania-sandfly-host relationships

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    Volf P.

    2008-09-01

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

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

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    Marilia S. Faria

    2012-01-01

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

  2. Methods of Control of the Leishmania infantum Dog Reservoir: State of the Art

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    Michele Podaliri Vulpiani

    2011-01-01

    Full Text Available Leishmania infantum is a protozoan parasite causing severe vector-borne visceral diseases both in humans and dogs. The latter are the most important natural reservoir and therefore should be the main target of control measures. The real efficacy of seropositive dogs culling as a direct control method is still debated, and the new sensitivity of large part of population considers ethically unacceptable this kind of approach. Treatment of infectious dogs with one of the available therapeutic protocols is recommendable as it allows to reduce parasite burdens and therefore the possibility of transmission of Leishmania infantum to vectors. Vaccination has been proven to be a very effective control tool, but the absence of a commonly recognized diagnostic method able to distinguish vaccinate from seropositive individuals is still an important limit. Concerning indirect control methods, a number of studies have demonstrated the efficacy of topical insecticides treatment (collars, spot-on, and sprays in reducing incidence and prevalence of L. infantum. Also, the reduction of the odds of seroconversion in humans in endemic areas has been reported after the application of indirect control measures on dogs. The contemporary use of direct and indirect methods is even more effective in reducing seroprevalence in dogs.

  3. 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

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

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    Miriam Berzunza-Cruz

    2015-01-01

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

  5. Diel cycles of isoprenoids in the emissions of Norway spruce, four Scots pine chemotypes, and in Boreal forest ambient air during HUMPPA-COPEC-2010

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    N. Yassaa

    2012-08-01

    Full Text Available Branch enclosure based emission rates of monoterpenes and sesquiterpenes from four Scots pines (Pinus sylvestris and one Norway spruce (Picea abies, as well as the ambient mixing ratios of monoterpenes were determined during the HUMPPA-COPEC 2010 summer campaign. Differences in chemical composition and in emission strength were observed between the different trees, which confirmed that they represented different chemotypes. The chemotypes of Scots pine can be classified according to species with high, no and intermediate content of Δ-3-carene. The "non-Δ-3-carene" chemotype was found to be the strongest emitter of monoterpenes. From this chemotype, β-myrcene, a very reactive monoterpene, was the dominant species accounting for more than 32 % of the total emission rates of isoprenoids followed by β-phellandrene (~27%. Myrcene fluxes ranged from 0.8 to 24 μg g−1 (dw h−1. α-Farnesene was the dominant sesquiterpene species, with average emission rates of 318 ng g−1 (dw h−1. In the high Δ-3-carene chemotype, more than 48% of the total monoterpene emission was Δ-3-carene. The average Δ-3-carene emission rate (from chemotype 3, circa 609 ng g−1 (dw h−1 reported here is consistent with the previously reported summer season value. Daily maximum temperatures varied between 20 and 35 °C during the measurements. The monoterpene emissions from spruce were dominated by limonene (35%, β-phellandrene (15%, α-pinene (14% and eucalyptol (9%. Total spruce monoterpene emissions ranged from 0.55 up to 12.2 μg g−1 (dw h−1. Overall the total terpene flux (monoterpenes + sesquiterpenes from all studied tree species varied from 230 ng g−1 (dw h−1 up to 66 μg g−1 (dw h−1. Total ambient monoterpenes (including α-pinene, Δ-3-carene, β-pinene and β-myrcene measured during the campaign

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

    NARCIS (Netherlands)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

    This work describes natural infection by Leishmania in a domestic cat where amastigote forms of the parasite were observed in the popliteal lymph node imprint. Positive and negative serological reactions were observed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA), respectively. Polymerase chain reaction (PCR) revealed that the nucleotide sequence of the sample was identical to Leishmania (L.) chagasi. This is the first report of the disease in felines of the city of Andradina, SP, an area considered endemic for canine and human visceral leishmaniasis.

  8. MicroRNA expression profile in human macrophages in response to Leishmania major infection.

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    Julien Lemaire

    Full Text Available BACKGROUND: Leishmania (L. are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs, an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. METHODOLOGY/PRINCIPAL FINDINGS: We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h. We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major

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

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    Nicole R Van Wynsberghe

    2000-10-01

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

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    Kendi Okuda

    2016-06-01

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

  12. Molecular detection of Leishmania infantum in naturally infected

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

    2009-12-01

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

  13. Sand fly evolution and its relationship to Leishmania transmission

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    PD Ready

    2000-08-01

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

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

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    Nathan C Peters

    2009-06-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

    NARCIS (Netherlands)

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

    2009-01-01

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

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

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    Dostálová Anna

    2012-12-01

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

  18. Survey of foliar monoterpenes across the range of jack pine reveal three widespread chemotypes: implications to host expansion of invasive mountain pine beetle

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    Spencer eTaft

    2015-05-01

    Full Text Available The secondary compounds of pines (Pinus can strongly affect the physiology, ecology and behaviors of the bark beetles (Coleoptera: Curculionidae, Scolytinae that feed on sub-cortical tissues of hosts. Jack pine (Pinus banksiana has a wide natural distribution range in North America (Canada and USA and thus variations in its secondary compounds, particularly monoterpenes, could affect the host expansion of invasive mountain pine beetle (Dendroctonus ponderosae, which has recently expanded its range into the novel jack pine boreal forest. We investigated monoterpene composition of 601 jack pine trees from natural and provenance forest stands representing 63 populations from Alberta to the Atlantic coast. Throughout its range, jack pine exhibited three chemotypes characterized by high proportions of α-pinene, β-pinene, or limonene. The frequency with which the α-pinene and β-pinene chemotypes occurred at individual sites was correlated to climatic variables, such as continentality and mean annual precipitation, as were the individual α-pinene and β-pinene concentrations. However, other monoterpenes were generally not correlated to climatic variables or geographic distribution. Finally, while the enantiomeric ratios of β-pinene and limonene remained constant across jack pine’s distribution, (‒:(+-α-pinene exhibited two separate trends, thereby delineating two α-pinene phenotypes, both of which occurred across jack pine’s range. These significant variations in jack pine monoterpene composition may have cascading effects on the continued eastward spread and success of D. ponderosae in the Canadian boreal forest.

  19. Additive antimicrobial [corrected] effects of the active components of the essential oil of Thymus vulgaris--chemotype carvacrol.

    Science.gov (United States)

    Iten, Felix; Saller, Reinhard; Abel, Gudrun; Reichling, Jürgen

    2009-09-01

    Herbal remedies are multicomponent mixtures by their nature as well as by pharmaceutical definition. Being a multicomponent mixture is not only a crucial property of herbal remedies, it also represents a precondition for interactions such as synergism or antagonism. Until now, only a few phytomedicines are accurately described concerning the interactions of their active components. The aim of this study was to search for interactions within such a naturally given multi-component mixture and to discuss the pharmaceutical and clinical impacts. The thyme oil chosen for the examination belongs to the essential oils with the most pronounced antimicrobial activity. Antibiotic activity of thyme oil and single active components were tested against six different strains of microorganisms. The checkerboard assay was used to search for interactions. The time-kill assay was used to verify the observed effects and to get information about the temporal resolution of the antimicrobial activity. The degree of the detected interactions corresponded with the demarcating FICI measure of 0.5, which separates the additive from the over-additive (synergistic) effects. Therefore, the observed effect was called a "borderline case of synergism" or, respectively, "partial synergism". Partial synergism was observed only in the presence of Klebsiella pneumoniae. Additive antimicrobial activity was observed for the combination of the two monosubstances carvacrol plus linalool and thymol plus linalool as well as with the combination of the two essential oils of the carvacrol and linalool chemotypes. An increase of the carvacrol oil concentration from one to two times the MIC resulted in a considerable acceleration of the kill-rate. Thyme oil is composed of several different components that show antimicrobial activity (at least: carvacrol, thymol and linalool). The antimicrobial activity of thyme oil is partly based on additive effects, which might especially enhance the rapidity of the

  20. Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) n.e. Brown.

    Science.gov (United States)

    do Vale, T Gurgel; Furtado, E Couto; Santos, J G; Viana, G S B

    2002-12-01

    Citral, myrcene and limonene (100 and 200 mg/kg body wt., i.p.), constituents of essential oils from Lippia alba chemotypes, decreased not only the number of crossings but also numbers for rearing and grooming, as measured by the open-field test in mice. Although muscle relaxation detected by the rota rod test was seen only at the highest doses of citral (200 mg/kg body wt.) and myrcene (100 and 200 mg/kg body wt.), this effect was observed even at the lowest dose of limonene (50 mg/kg body wt.). Also, citral and myrcene (100 and 200 mg/kg body wt.) increased barbiturate sleeping time as compared to control. Limonene was also effective at the highest dose, and although citral did not increase the onset of sleep, it increased the duration of sleep, which is indicative of a potentiation of sleeping time. Citral (100 and 200 mg/kg body wt.) increased 2.3 and 3.5 times, respectively, the barbiturate sleeping time in mice. Similar effects were observed for myrcene and limonene at the highest dose (200 mg/kg body wt.) which increased the sleeping time around 2.6 times. In the elevated-plus maze, no effect was detected with citral up to 25 mg/kg body wt., while at a high dose it decreased by 46% the number of entries in the open arms. A smaller but significant effect was detected with limonene (5 mg/kg body wt.). While myrcene (10 mg/kg body wt.) decreased only by 22% the number of entries in the open arms, this parameter was decreased by 48% at the highest dose. Our study showed that citral, limonene and myrcene presented sedative as well as motor relaxant effects. Although only at the highest dose, they also produced a potentiation of the pentobarbital-induced sleeping time in mice, which was more intense in the presence of citral. In addition, neither of them showed an anxiolytic effect, but rather a slight anxiogenic type of effect at the higher doses.

  1. Easy identification of leishmania species by mass spectrometry.

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    Oussama Mouri

    2014-06-01

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

  2. Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

    Energy Technology Data Exchange (ETDEWEB)

    Ariza, Antonio; Vickers, Tim J.; Greig, Neil; Fairlamb, Alan H.; Bond, Charles S., E-mail: c.s.bond@dundee.ac.uk [Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH,Scotland (United Kingdom)

    2005-08-01

    The detoxification enzyme glyoxalase I from L. major has been crystallized. Preliminary molecular-replacement calculations indicate the presence of three glyoxalase I dimers in the asymmetric unit. Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

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

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    Maria de Fátima Madeira

    2003-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Leila Martins

    2010-06-01

    amplify a 750 base pair target sequence. In total, 58 subjects (48.7% showed positive PCR amplification and 61 (51.3% were negative. Of the PCR-positive samples, 37 (≅64% were from treated, lesion-free subjects. In conclusion, the PCR technique is efficacious at identifying Leishmania DNA in biopsy and venous blood samples.

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

    Science.gov (United States)

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

    2014-11-01

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

  6. 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.

  7. In Vivo Recognition of Ovalbumin Expressed by Transgenic Leishmania Is Determined by Its Subcellular Localization1

    Science.gov (United States)

    Prickett, Sara; Gray, Peter M.; Colpitts, Sara L.; Scott, Phillip; Kaye, Paul M.; Smith, Deborah F.

    2009-01-01

    The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity. PMID:16585577

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

    Science.gov (United States)

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

    2006-06-15

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

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

    OpenAIRE

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Manoel Sebastião da Costa Lima Junior

    2009-06-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  14. Evaluation of (131)I-pentamidine for scintigraphy of experimentally Leishmania tropica-infected hamsters.

    Science.gov (United States)

    Inceboz, Tonay; Lambrecht, Fatma Yurt; Eren, Mine Şencan; Girginkardeşler, Nogay; Bekiş, Recep; Yilmaz, Osman; Er, Özge; Özbilgin, Ahmet

    2014-06-01

    We aimed to assess the ability of (131)I-Pentamidine scintigraphy to detect the lesions of Leishmania tropica infection. An experimental model of cutaneous leishmaniasis was developed. The presence of cutaneous leishmaniasis was confirmed. Pentamidine was radioiodinated with (131)I. The radiolabeled pentamidine was validated by the requisite quality control tests to check its radiolabeling efficiency, in vitro stability. (131)I-Pentamidine (activity: 18.5 MBq/100 µl) was injected intracardiacally into infected hamsters. Static whole body images of the hamsters were acquired under the gamma camera at 5 and 30 min, 2, 6 and 24 h following the administration. On the scintigrams, anatomically adjusted regions of interest (ROIs) were drawn over the right feet (target) and left feet (not-target) and various organs. Accumulation of (131)I-Pentamidine at sites of infection is expressed as the target to non-target (T/NT) ratio. The results T/NT ratio decreased with time. In concluding the (131)I-Pentamidine has poor sensitivity in detection of L. tropica infection.

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

    Directory of Open Access Journals (Sweden)

    Geraldine De Muylder

    2011-07-01

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

  16. An agent-based model for Leishmania major infection

    Science.gov (United States)

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

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

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

    Science.gov (United States)

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

    1998-01-01

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

  18. 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.

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

    Science.gov (United States)

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

    2006-08-01

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

  20. Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica.

    Science.gov (United States)

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

    2016-04-01

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

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

    OpenAIRE

    Omid Rajabi; Ameneh Sazgarnia; Fatemeh Abbasi; Pouran Layegh

    2015-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  3. Testing of Experimental Compounds for Efficacy Against Leishmania

    Science.gov (United States)

    1986-02-01

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

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

    Science.gov (United States)

    2004-06-01

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

  5. Experimental acquisition, development, and transmission of Leishmania tropica by Phlebotomus duboscqi.

    Science.gov (United States)

    Hanafi, Hanafi A; El-Din, El-Shaimaa M Nour; El-Hossary, Shabaan S I; Kaldas, Rania M; Villinski, Jeffrey T; Furman, Barry D; Fryauff, David J

    2013-01-01

    We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand fly Phlebotomus duboscqi (Neveu-Lemaire). Groups of laboratory-reared female sand flies that fed "naturally" on L. tropica-infected hamsters, or artificially, via membrane feeding device, on a suspension of L. tropica amastigotes, were dissected at progressive time points post-feeding. Acquisition, retention and development of L. tropica through procyclic, nectomonad, and leptomonad stages to the infective metacyclic promastigote stage, and anterior progression of the parasites from abdominal midgut bloodmeal to the thoracic midgut were demonstrated in both groups. Membrane feeding on the concentrated amastigote suspension led to metacyclic promastigote infections in 60% of sand flies, whereas only 3% of P. duboscqi that fed naturally on an infected hamster developed metacyclics. Sand flies from both groups re-fed on naïve hamsters, but despite infections in 25-50% of membrane-fed and 2-3.5% of naturally fed flies, no skin lesions developed in the hamsters. After four months of observation these animals were euthanized and necropsied. Screening of the organs and tissue by polymerase chain reaction (PCR) that targeted the small subunit RNA gene, amplified generic Leishmania DNA from liver, spleen, bone marrow, and blood, but only from hamsters bitten by membrane-infected P. duboscqi. These results are notable in demonstrating the ability of P. duboscqi, originating from Kenya, to acquire, retain, develop, and transmit a Turkish strain of L. tropica originally isolated from a human case of cutaneous leishmaniasis. This marks the first demonstration of complete development and transmission of L. tropica by a member of the Phlebotomus subgenus of sand flies.

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

    Science.gov (United States)

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Bruce Alexander

    2002-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Giselle Aparecida Fagundes-Silva

    2015-09-01

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

  9. Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Isabel M. Vincent

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Alessandro Costagliola

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 皱叶薄荷精油的化学分类特征%Analysis on Chemotype of Volatile Oil of Mentha crispata Schrad. ex Willd

    Institute of Scientific and Technical Information of China (English)

    郭晓恒; 刘涛; 宋登敏; 雨田; 严铸云

    2014-01-01

    This study was aimed to analyze the volatile oil of Mentha crispata Schrad. ex Willd. in order to provide evidence for its chemotype and guidance for its production application. The chemical analysis was detected by headspace GC-MS. The results showed that 64 chemical compounds were detected. It was concluded that the volatile oil of M. crispata Schrad. ex Willd. mainly contained eucalyptol (35.58%), limonene (16.92%) and pinene (15.33%). It was concluded that the analysis on composition characteristics and main compounds of M. crispata Schrad. ex Willd. can provide evidences in its production application and chemotype.%目的:对皱叶薄荷精油进行成分分析,为种源鉴定提供化学分类依据并为生产应用提供指导。方法:采用顶空气相法对皱叶薄荷全草进行化学分析。结果:共得到64个化学成分,且皱叶薄荷主要由桉油素(Eucalyptol ,35.58%)、柠檬烯(Limonene ,16.92%)及蒎烯(Pinene ,15.33%)组成。结论:通过分析皱叶薄荷精油的组成特征及主要成分,可为其生产应用及种源鉴定提供化学分类佐证。

  14. Neuro-protective potential of a vesicular system of a standardized extract of a new chemotype of Withania somnifera Dunal (NMITLI118RT+) against cerebral stroke in rats().

    Science.gov (United States)

    Ahmad, Hafsa; Khandelwal, Kiran; Samuel, Sheeba Saji; Tripathi, Shivangi; Mitra, Kalyan; Sangwan, Rajender Singh; Shukla, Rakesh; Dwivedi, Anil Kumar

    2016-09-01

    Withania somnifera Dunal is an Indian medicinal plant with significant pharmacological properties, such as adaptogenic, anti-inflammatory, anti-oxidant, anti-platelet, anti-hypertensive, hypoglycemic and hypolipidemic effects. Several chemotypes of W. somnifera include NMITLI-101, NMITLI-118 and NMITLI-128. The present work elaborates the optimization and development of a liposomal delivery system for efficient delivery of NMITLI118RT+ [a standardized ethanolic extract of a new chemotype of W. somnifera Dunal (NMITLI-118) roots] against cerebral stroke in rats. Liposomal systems were prepared using thin-film hydration method and characterized on the basis of size, zeta potential, physical stability, FT-IR, DSC-TGA analysis and surface morphological studies by TEM. NMITLI118RT+ and its formulations (NMITLI118RT+LF) were evaluated for biological activity utilizing middle cerebral artery occlusion model in rats. The Z average of the developed liposomal formulation was about 142.6 ± 0.09 nm with a zeta potential of -31.20 ± 1.0 mV. Results of TEM revealed spherical particles in the range of 200 nm. The entrapment efficiency was found to be 94.603 ± 2%. The formulation was found to be physically stable over a 3-week period. Results were suggestive of the fact that both NMITLI118RT+ and its delivery system possess significant neuroprotective activity in cerebral ischemia. The liposomal system largely exhibits better performance over NMITLI118RT+ precisely in the post-treatment group. The present studies could elucidate the successful development of a delivery system for NMITLI118RT+ and demonstrate their beneficial neuro-protective potential in overcoming and reversing the consequences of I/R injury following stroke.

  15. Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival

    Science.gov (United States)

    Gómez, Maria Adelaida; Navas, Adriana; Márquez, Ricardo; Rojas, Laura Jimena; Vargas, Deninson Alejandro; Blanco, Victor Manuel; Koren, Roni; Zilberstein, Dan; Saravia, Nancy Gore

    2014-01-01

    Objectives Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate. Methods Patients with cutaneous leishmaniasis who failed (n = 8) or responded (n = 8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT–PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown. Results Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes. Conclusions These results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular

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

    Science.gov (United States)

    Ahmad, Moaz; Afrin, Farhat; Tuteja, Renu

    2013-11-01

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

  17. Leishmania major methionine sulfoxide reductase A is required for resistance to oxidative stress and efficient replication in macrophages.

    Directory of Open Access Journals (Sweden)

    Fiona M Sansom

    Full Text Available Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H(2O(2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H(2O(2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

  18. The crystal structures of the tryparedoxin-tryparedoxin peroxidase couple unveil the structural determinants of Leishmania detoxification pathway.

    Directory of Open Access Journals (Sweden)

    Annarita Fiorillo

    Full Text Available Leishmaniasis is a neglected disease caused by Leishmania, an intracellular protozoan parasite which possesses a unique thiol metabolism based on trypanothione. Trypanothione is used as a source of electrons by the tryparedoxin/tryparedoxin peroxidase system (TXN/TXNPx to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is not only unique to the parasite but is also essential for its survival; therefore, it constitutes a most attractive drug target. Several forms of TXNPx, with very high sequence identity to one another, have been found in Leishmania strains, one of which has been used as a component of a potential anti-leishmanial polyprotein vaccine. The structures of cytosolic TXN and TXNPx from L. major (LmTXN and LmTXNPx offer a unique opportunity to study peroxide reduction in Leishmania parasites at a molecular level, and may provide new tools for multienzyme inhibition-based drug discovery. Structural analyses bring out key structural features to elucidate LmTXN and LmTXNPx function. LmTXN displays an unusual N-terminal α-helix which allows the formation of a stable domain-swapped dimer. In LmTXNPx, crystallized in reducing condition, both the locally unfolded (LU and fully folded (FF conformations, typical of the oxidized and reduced protein respectively, are populated. The structural analysis presented here points to a high flexibility of the loop that includes the peroxidatic cysteine which facilitates Cys52 to form an inter-chain disulfide bond with the resolving cysteine (Cys173, thereby preventing over-oxidation which would inactivate the enzyme. Analysis of the electrostatic surface potentials of both LmTXN and LmTXNPx unveils the structural elements at the basis of functionally relevant interaction between the two proteins. Finally, the structural analysis of TXNPx allows us to identify the position of the epitopes that make the protein antigenic and therefore potentially suitable

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

    OpenAIRE

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

    2015-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2007-06-01

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

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

    Science.gov (United States)

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

    2008-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Samanta Etel Treiger Borborema

    2013-08-01

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

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

    Science.gov (United States)

    Medeiros, Fernanda Alvarenga Cardoso; Gomes, Luciana Inácia; de Souza, Carolina Senra Alves; Mourão, Maria Vitória; Cota, Gláucia Fernandes; Marques, Letícia Helena dos Santos; Carneiro, Mariângela

    2017-01-01

    A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions. PMID:28163725

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

    曹得萍; 陈建平

    2010-01-01

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

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

    Science.gov (United States)

    Eroglu, Fadime; Koltas, Ismail S; Alabaz, Derya; Uzun, Soner; Karakas, Mehmet

    2015-07-01

    L. infantum was isolated from cutaneous leishmaniasis (CL) skin lesions in patients having no signs and symptoms of visceral leishmaniasis (VL). Similarly, L. tropica had previously been isolated from patients with VL in the absence of cutaneous lesions. It was not certain how visceralization occurred. Smears (207) and bone marrow samples (135) were taken from CL and VL-suspected patients, respectively. Microscopic examination, ITS1-PCR, RFLP and DNA sequencing for all samples were analyzed. The microscopic examination of smears was found to be 61.3% (127/207) in CL-suspected cases and bone marrow samples were found to be positive 8.8% (12/135) in VL-suspected cases. L. tropica 48.6% (72/148), L. infantum 35.8% (53/148), L. major 15.6% (23/148) in CL, and L. infantum 56.3% (18/32), L. donovani 31.2% (10/32), L. tropica 12.5% (4/32) in VL were found with PCR-RFLP. In addition, the DNA sequencing revealed a genetic variation in L. infantum (variants 1-3) and L. tropica (variants 1-5). We assume that the increased disease occurrence may have resulted from geographical expansion of disease, changing patterns of international travel, population migrations, non-immune people into endemic regions of infected people into non-endemic regions. In this study, L. infantum (variant 3) only in CL-patients and L. tropica (variant 2) only in VL-patients were identified. We hypothesize that genetic variation might play a role in the causation of CL and VL in southern Turkey and the genetic variants may differ according to the geographical location among Leishmania strains.

  9. [Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases].

    Science.gov (United States)

    Ertabaklar, Hatice; Ertuğ, Sema; Çalışkan, Serçin Özlem; Bozdoğan, Bülent

    2016-04-01

    The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the

  10. Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome B gene.

    Science.gov (United States)

    Foulet, Françoise; Botterel, Françoise; Buffet, Pierre; Morizot, Gloria; Rivollet, Danièle; Deniau, Michèle; Pratlong, Francine; Costa, Jean-Marc; Bretagne, Stéphane

    2007-07-01

    Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with >or=99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

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

    Directory of Open Access Journals (Sweden)

    Ana Paula FERNANDES

    1997-03-01

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

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

    Science.gov (United States)

    Simon, Stéphane; Veron, Vincent; Carme, Bernard

    2010-02-01

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

  13. Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum.

    Science.gov (United States)

    Mesquita, Juliana Tonini; Pinto, Erika Gracielle; Taniwaki, Noemi Nosomi; Galisteo, Andres Jimenez; Tempone, Andre Gustavo

    2013-12-01

    Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71μg/mL against promastigotes and 6.78μg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.

  14. Molecular identification of Leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of Iran.

    Science.gov (United States)

    Eslami, Gilda; Hajimohammadi, Bahador; Jafari, Abbas Ali; Mirzaei, Farzaneh; Gholamrezai, Mostafa; Anvari, Hossein; Khamesipour, Ali

    2014-12-01

    The most common form of the disease is cutaneous leishmaniasis (CL) which is a public health and social problem in many countries especially Iran. In endemic areas where other diseases with similar clinical symptoms occur, definitive diagnosis of CL is very important. The detection and identification of Leishmania in infected patients is crucial for achieving a correct treatment and prognosis. To our knowledge, this is the first comprehensive study in terms of geographical distribution and molecular identification of Leishmania tropica isolates in central of Iran. This study was performed between 2010 and 2011, during which 218 CL suspected patients referred to Shahid Sadoughi University of Medical Sciences in Yazd, Iran for confirmation were examined. After microscopic analysis, DNA extraction was performed for identification. The molecular target region was ITS1 gene. Results showed that out of 218 isolates, 102 (46.8%) samples were positive for Leishman body using molecular assay. After PCR-RFLP, analysis identified 50 (49.01%) samples as L. major and 52 (50.98%) as L. tropica. Two samples showed a different pattern that were reported as unknown. Among L. tropica, six different isolates were identified in this endemic area. Finally, this study showed heterozygosity among L. tropica isolates in this endemic area such as some other studies from the world. This heterozygosity among the strains may suggest a sexual recombination or genetic exchange between strains.

  15. The gp63 Gene Cluster Is Highly Polymorphic in Natural Leishmania (Viannia) braziliensis Populations, but Functional Sites Are Conserved

    Science.gov (United States)

    Medina, Lilian S.; Souza, Bruno Araújo; Queiroz, Adriano; Guimarães, Luiz Henrique; Lima Machado, Paulo Roberto; M Carvalho, Edgar; Wilson, Mary Edythe; Schriefer, Albert

    2016-01-01

    GP63 or leishmanolysin is the major surface protease of Leishmania spp. involved in parasite virulence and host cell interaction. As such, GP63 is a potential target of eventual vaccines against these protozoa. In the current study we evaluate the polymorphism of gp63 in Leishmania (Viannia) braziliensis isolated from two sets of American tegumentary leishmaniasis (ATL) cases from Corte de Pedra, Brazil, including 35 cases diagnosed between 1994 and 2001 and 6 cases diagnosed between 2008 and 2011. Parasites were obtained from lesions by needle aspiration and cultivation. Genomic DNA was extracted, and 405 bp fragments, including sequences encoding the putative macrophage interacting sites, were amplified from gp63 genes of all isolates. DNA amplicons were cloned into plasmid vectors and ten clones per L. (V.) braziliensis isolate were sequenced. Alignment of cloned sequences showed extensive polymorphism among gp63 genes within, and between parasite isolates. Overall, 45 different polymorphic alleles were detected in all samples, which could be segregated into two clusters. Cluster one included 25, and cluster two included 20 such genotypes. The predicted peptides showed overall conservation below 50%. In marked contrast, the conservation at segments with putative functional domains approached 90% (Fisher’s exact test p<0.0001). These findings show that gp63 is very polymorphic even among parasites from a same endemic focus, but the functional domains interacting with the mammalian host environment are conserved. PMID:27648939

  16. Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation.

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    Angelo Duarte

    Full Text Available Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

  17. The putative Leishmania telomerase RNA (LeishTER undergoes trans-splicing and contains a conserved template sequence.

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    Elton J R Vasconcelos

    Full Text Available Telomerase RNAs (TERs are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER that contains a 5' spliced leader (SL cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs and its role in parasite telomere biology.

  18. Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection.

    Science.gov (United States)

    Luz, Nívea F; Andrade, Bruno B; Feijó, Daniel F; Araújo-Santos, Théo; Carvalho, Graziele Q; Andrade, Daniela; Abánades, Daniel R; Melo, Enaldo V; Silva, Angela M; Brodskyn, Cláudia I; Barral-Netto, Manoel; Barral, Aldina; Soares, Rodrigo P; Almeida, Roque P; Bozza, Marcelo T; Borges, Valéria M

    2012-05-01

    Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

  19. Serological Evidence of Infection by Leishmania (Leishmania) infantum (Synonym: Leishmania (Leishmania) chagasi) in Free-Ranging Wild Mammals in a Nonendemic Region of the State of São Paulo, Brazil.

    Science.gov (United States)

    Paiz, Laís Moraes; Fornazari, Felipe; Menozzi, Benedito Donizete; Oliveira, Gabriela Capriogli; Coiro, Carla Janeiro; Teixeira, Carlos Roberto; da Silva, Valdinei Moraes Campanucci; Donalisio, Maria Rita; Langoni, Helio

    2015-11-01

    Concerns about the interface between wildlife, domestic animals, and humans in the transmission of visceral leishmaniasis (VL) have been growing due to natural or anthropogenic environmental changes. In this context, investigations of the infection in wild mammals are important to assess their exposure to the vector and the parasite. A study of anti-Leishmania (Leishmania) infantum antibodies was carried out using the direct agglutination test (DAT) on 528 free-ranging wild mammals of 38 species from the region of Botucatu, state of São Paulo, Brazil, a municipality that has no records of the vector or of human or canine autochthony. Antibodies were detected, with a cutoff of 1:320, in 9/528 (1.7%; 95% confidence interval [CI] 0.6-2.8%) mammals of the species Callithrix jacchus, Lepus europaeus, Sphiggurus villosus, Nasua nasua, Eira barbara, and Galictis cuja, with high titers (≥1280) for the last three. These three are little-studied species, and previous records of the detection of anti-Leishmania spp. antibodies in Brazil exist only for coatis (N. nasua), whereas worldwide, infection by L. (L.) infantum has been confirmed only in hares (Le. europaeus). On the other hand, opossums and canids, the species most commonly reported to be naturally infected by L. (L.) infantum, were not seropositive. Fifty-eight (58/528; 10.9%) mammals were found to have antibody titers ranging from 20 to 160 and were not included among the seropositive animals due to the adopted cutoff. However, the possibility of infection in these animals should not be discarded, because there is no standard cutoff point for the different wild species. Our findings indicate the need for investigations into the exact role of the seropositive species in the epidemiology of VL and for effective epidemiological surveillance to prevent its expansion, because even in regions where there are no records of canine or human autochthonous cases, there may be parasite circulation among wild mammals.

  20. Differentiation of Leishmania species by FT-IR spectroscopy

    Science.gov (United States)

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

    2015-05-01

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

  1. In vitro activity of amphotericin B cochleates against Leishmania chagasi

    OpenAIRE

    Aretha Molina Sesana; Renata Monti-Rocha; Solange Alves Vinhas; Carlos Gustavo Morais; Reynaldo Dietze; Elenice Moreira Lemos

    2011-01-01

    Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB). In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vi...

  2. [Leishmania tropica in Morocco. IV--Intrafocal enzyme diversity].

    Science.gov (United States)

    Pratlong, F; Rioux, J A; Dereure, J; Mahjour, J; Gallego, M; Guilvard, E; Lanotte, G; Perieres, J; Martini, A; Saddiki, A

    1991-01-01

    Ecoepidemiological analysis of a Moroccan focus of leishmaniasis caused by Leishmania tropica revealed considerable enzymatic diversity. Seven zymodemes belonging to the complex were identified in 149 strains isolated from humans, dogs, and the vector Phlebotomus sergenti. Three distinct subgroups were identifiable, two of which were in turn, composed of three "small variant" zymodemes. The diversity appears to be related to the age of the focus, which may have allowed colonization by zymodemes of different geographic origins. Diversification into "small variants" is apparently the result of recent mutation, possibly associated with genetic exchange.

  3. Aluminium: a natural adjuvant in Leishmania transmission via sand flies?

    Science.gov (United States)

    Maingon, Rhayza; Khela, Amandeep; Sampson, Christopher; Ward, Richard; Walker, Karen; Exley, Christopher

    2008-11-01

    Genetically identical Leishmania chagasi/infantum parasites cause both atypical cutaneous leishmaniasis and visceral leishmaniasis. In this report we have tested the first part of a hypothesis that states that the form of this disease that is manifested depends upon the adjuvant-like activity of aluminium of dietary origin accumulated in the salivary gland of the sand fly vector. In sand flies fed aluminium-supplemented sucrose we have used histochemistry to qualitatively identify aluminium in their salivary glands and graphite furnace atomic absorption spectrometry to quantify the aluminium content of dissected salivary glands. Aluminium may be acting as a natural adjuvant in some forms of leishmaniasis.

  4. The generation of purinome-targeted libraries as a means to diversify ATP-mimetic chemical classes for lead finding.

    Science.gov (United States)

    Felder, Eduard R; Badari, Alessandra; Disingrini, Teresa; Mantegani, Sergio; Orrenius, Christian; Avanzi, Nilla; Isacchi, Antonella; Salom, Barbara

    2012-02-01

    The generation of novel chemotypes in support of our oncology research projects expanded in recent years from a canonical design of kinase-targeted compound libraries to a broader interpretation of purinome-targeted libraries (PTL) addressing the specificity of cancer relevant targets such as kinases and ATPases. Successful screening of structurally diverse ATP-binding targets requires compound libraries covering multiple design elements, which may include phosphate surrogate moieties in ATPase inhibitors or far reaching lipophilic residues stabilizing inactive kinase conformations. Here, we exemplify the design and preparation of drug-like combinatorial libraries and report significantly enhanced screening performance on purinomic targets. We compared overall hit rates of PTL with a simultaneously tested unbiased collection of 200,000 compounds and found consistent superiority of the targeted libraries in all cases. We also analyzed the performance of the largest targeted libraries in comparison with each other and often found striking differences in how a specific target responds to various chemotypes and to whole collections.

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

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    Yang Bin-Bin

    2013-02-01

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

  6. 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.

  7. Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis.

    Science.gov (United States)

    Telleria, Erich L; Sant'Anna, Maurício R V; Ortigão-Farias, João R; Pitaluga, André N; Dillon, Viv M; Bates, Paul A; Traub-Csekö, Yara M; Dillon, Rod J

    2012-04-13

    Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.

  8. Caspar-like Gene Depletion Reduces Leishmania Infection in Sand Fly Host Lutzomyia longipalpis*

    Science.gov (United States)

    Telleria, Erich L.; Sant'Anna, Maurício R. V.; Ortigão-Farias, João R.; Pitaluga, André N.; Dillon, Viv M.; Bates, Paul A.; Traub-Csekö, Yara M.; Dillon, Rod J.

    2012-01-01

    Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on Gram-negative and Gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly. PMID:22375009

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

    Science.gov (United States)

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

    2016-01-01

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

  10. A soro-aglutinação das Leishmanias Agglutination of Leishmanias

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    A. M. da Cunha

    1942-01-01

    Full Text Available The first agglutination experiments (Tables 1 and 2 showed that the serum obtained with any one strain of Leishmania, agglutinates all the others even of another species. This finding reveals the existence of a common antigen. However as the titre of agglutination did not permit a sharp differentiation of species we tried the adsorption method. The first adsorption tests made demonstrated differences in antigenic constitution between a strain of. L. donovani on one hand and strains of L. tropica or L. brasiliensis on the other. Further experiments in which L. chagasi was tested against the other species revealed that the former was antigenically different from the others. These tests were performed by adsorbing an anti-chagasi serum with organisms belonging to the other species or, conversely, adsorbing with L. chagasi sera prepared against the other species (See Tables 9 to 24. On the other hand, the adsorption of a serum prepared against one strain of l. chagasi by another of the same species showed that they had identifical antigenie constitution. These findings suggested the possibility of separating different species of Leishmania by this method. However, tests to separate the other species from one to another gave inconclusive results. (See Tables 27 to 35. It was soon observed that all the strains of L. chagasi were of recent isolation while all the others had been maintained in artificial culture media for a long time. We were led to believe that this condition was responsible for the differences in behaviour encountered. Accordingly, recently isolated strains of L. brasiliensis and L. donovani were tested and shown to be antigenically similar to strains of L. chagasi also recently isolated. The conclusion may be drawn that all strains have the same antigenic constitution when freshly isolated. It has been noted that when a serum which has been prepared against a freshly isolated is adsorbed with an old strain, the amount of agglutinins

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

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    H. Mahmoudzadeh-Niknam , F. Abrishami , M. Doroudian , M. Moradi , M.H. Alimohammadian , P. Parvizi

    2010-12-01

    Full Text Available Background & objectives: Kala-azar is the visceral and most severe form of leishmaniasis thatleads to death if untreated. The causative agents of visceral leishmaniasis (VL are members ofLeishmania (L. donovani complex which includes L. chagasi and L. infantum. Genome sequenceshave raised the question whether L. chagasi and L. infantum are synonymous or different. Thisquestion has important implications for clinical and epidemiological studies, evaluation of vaccinesand drugs, and disease control. LCR1 is an immunogenic molecule discovered from L. chagasiwith potential as a component of a Leishmania subunit vaccine. If this protein has potentials forbeing used in a vaccine or diagnostic testing, there should be little variability in this moleculebetween L. infantum isolates from diverse geographic regions. The aim of this study was to determinewhether lcr1 of an Iranian strain of L. infantum was identical to lcr1 of both L. infantum strainfrom a different geographic region (Spain and that of an L. chagasi isolate from Brazil.Methods: L. infantum isolated from an Iranian kala-azar patient was studied. Lcr1 from this isolatewas PCR amplified, cloned, and studied by restriction digest analysis and sequencing.Results: The sequences of lcr1 of the Iranian L. infantum were completely identical at nucleotidelevel to lcr1 sequences of both the Spanish L. infantum and the Brazilian L. chagasi strains.Conclusion: Complete conservation of the DNA sequence encoding for LCR1 molecule betweengeographically distinct Leishmania species adds credibility to the potential for LCR1 as a componentof a subunit vaccine and diagnostic test for kala-azar.

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

    Science.gov (United States)

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

    2012-07-01

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

  13. The Multiple Forms of Leishmania Major in BALB/C Mice Lung in Iran

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    M Jafari

    2012-06-01

    Full Text Available Cutaneous leishmaniasis is one of the most important parasitic diseases, which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days, the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected.

  14. Metacyclic promastigotes of Leishmania amazonensis selection using gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-07-01

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

  15. Aurapten, a coumarin with growth inhibition against Leishmania major promastigotes

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    Napolitano H.B.

    2004-01-01

    Full Text Available Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compund was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg of shows significant growth inhibition with an LD50 of 30 µM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.

  16. Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

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    Wanderley J.L.M.

    2005-01-01

    Full Text Available Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS exposure and its recognition by a specific receptor (PSR were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

  17. Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

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    Sarah A C Falcão

    2015-03-01

    Full Text Available BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

  18. Glucantime resistant Leishmania promastigotes are sensitive to pentostam

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    Elizabeth Spangler Andrade Moreira

    1992-12-01

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

  19. Natural Leishmania infection of Lutzomyia spp. in Peru.

    Science.gov (United States)

    Perez, J E; Ogusuku, E; Inga, R; Lopez, M; Monje, J; Paz, L; Nieto, E; Arevalo, J; Guerra, H

    1994-01-01

    Natural infection of Lutzomyia spp. with Leishmania was studied with the aid of the polymerase chain reaction (PCR) in Chaute, Lima, Perú, a locality endemic for Andean cutaneous leishmaniasis (uta). The PCR, with primers specific for the L. braziliensis complex, was applied to sandfly pools. Sandflies were sampled from April 1990 to May 1991 with CDC light traps in homes, and from near homes with a Shannon trap using protected human bait. Lu. verrucarum (4 pools) and Lu. peruenis (2 pools) from the anthropophilic collections, and Lu. verrucarum (2 pools) from indoors were found to be infected with Leishmania. The majority of infected sandflies were recorded mainly in April 1991 (4 pools), coinciding with the highest sandfly densities and the maximum number of new cases of uta (7). Non-infected sandflies were found from May to October 1990 and January to March 1991. Thus, these 2 sandfly species play a role in the spread of leishmaniasis among humans and other animals in Chaute.

  20. Proteinases as virulence factors in Leishmania spp. infection in mammals

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    Silva-Almeida Mariana

    2012-08-01

    Full Text Available Abstract Leishmania parasites cause human tegumentary and visceral infections that are commonly referred to as leishmaniasis. Despite the high incidence and prevalence of cases, leishmaniasis has been a neglected disease because it mainly affects developing countries. The data obtained from the analysis of patients’ biological samples and from assays with animal models confirm the involvement of an array of the parasite’s components in its survival inside the mammalian host. These components are classified as virulence factors. In this review, we focus on studies that have explored the role of proteinases as virulence factors that promote parasite survival and immune modulation in the mammalian host. Additionally, the direct involvement of proteinases from the host in lesion evolution is analyzed. The gathered data shows that both parasite and host proteinases are involved in the clinical manifestation of leishmaniasis. It is interesting to note that although the majority of the classes of proteinases are present in Leishmania spp., only cysteine-proteinases, metalloproteinases and, to a lesser scale, serine-proteinases have been adequately studied. Members from these classes have been implicated in tissue invasion, survival in macrophages and immune modulation by parasites. This review reinforces the importance of the parasite proteinases, which are interesting candidates for new chemo or immunotherapies, in the clinical manifestations of leishmaniasis.

  1. Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum.

    Science.gov (United States)

    Brotherton, Marie-Christine; Bourassa, Sylvie; Légaré, Danielle; Poirier, Guy G; Droit, Arnaud; Ouellette, Marc

    2014-08-01

    Amphotericin B (AmB) in its liposomal form is now considered as either first- or second-line treatment against Leishmania infections in different part of the world. Few cases of AmB resistance have been reported and resistance mechanisms toward AmB are still poorly understood. This paper reports a large-scale comparative proteomic study in the context of AmB resistance. Quantitative proteomics using stable isotope labeling of amino acids in cell culture (SILAC) was used to better characterize cytoplasmic and membrane-enriched (ME) proteomes of the in vitro generated Leishmania infantum AmB resistant mutant AmB1000.1. In total, 97 individual proteins were found as differentially expressed between the mutant and its parental sensitive strain (WT). More than half of these proteins were either metabolic enzymes or involved in transcription or translation processes. Key energetic pathways such as glycolysis and TCA cycle were up-regulated in the mutant. Interestingly, many proteins involved in reactive oxygen species (ROS) scavenging and heat-shock proteins were also up-regulated in the resistant mutant. This work provides a basis for further investigations to understand the roles of proteins differentially expressed in relation with AmB resistance.

  2. Genetic diversity of Leishmania infantum field populations from Brazil

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    Marcela Segatto

    2012-02-01

    Full Text Available Leishmania infantum (syn. Leishmania chagasi is the etiological agent of visceral leishmaniasis (VL in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT, random amplification of polymorphic DNA (RAPD and simple sequence repeats-polymerase chain reaction (SSR-PCR, were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

  3. Structure of the SAS-6 cartwheel hub from Leishmania major.

    Science.gov (United States)

    van Breugel, Mark; Wilcken, Rainer; McLaughlin, Stephen H; Rutherford, Trevor J; Johnson, Christopher M

    2014-01-01

    Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001.

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

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    R Salehi

    2009-07-01

    Full Text Available "nBackground: Leishmania is an obligatory intracellular protozoan parasite, which infects human be­ings when infected sand fly vector takes a blood meal.  Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has spe­cial im­portant. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. "nMethods: Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was de­signed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. "nResults: Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltrans­ferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and de­posited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank "nConclusion: We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully.

  5. Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

    Science.gov (United States)

    Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Schneider, Gisbert

    2014-01-07

    Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates.

  6. Systematic analysis of public domain compound potency data identifies selective molecular scaffolds across druggable target families.

    Science.gov (United States)

    Hu, Ye; Wassermann, Anne Mai; Lounkine, Eugen; Bajorath, Jürgen

    2010-01-28

    Molecular scaffolds that yield target family-selective compounds are of high interest in pharmaceutical research. There continues to be considerable debate in the field as to whether chemotypes with a priori selectivity for given target families and/or targets exist and how they might be identified. What do currently available data tell us? We present a systematic and comprehensive selectivity-centric analysis of public domain target-ligand interactions. More than 200 molecular scaffolds are identified in currently available active compounds that are selective for established target families. A subset of these scaffolds is found to produce compounds with high selectivity for individual targets among closely related ones. These scaffolds are currently underrepresented in approved drugs.

  7. The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan Province, Iran.

    Science.gov (United States)

    Spotin, Adel; Rouhani, Soheila; Parvizi, Parviz

    2014-01-01

    Cutaneous leishmaniasis has various phenotypic aspects consisting of polymorphic amastigotes with different genetic ranges. Samples were collected from suspected patients of Khuzestan province. Prepared smears were stained, scaled, and measured using ocular micrometer. The Cyt b, ITS-rDNA, and microsatellite genes of Leishmania were amplified and Leishmania species were identified by molecular analyses. Of 150 examined suspected patients, 102 were identified to Leishmania species (90 L. major, nine L. tropica, and three unidentified). The amastigotes of 90 L. major had regular and different irregular shapes within three clinical lesions with no and/or low genetic diversity. Three haplotypes of Cyt b of L. major were found but no variation was observed using ITS-rDNA gene. Interesting findings were that all nine L. tropica had regular amastigote shapes with more genetic variations, also a patient which had coinfection of L. major, L. tropica, and Crithidia. At least two L. major and L. tropica were identified in suspected patients of the regions. Different irregular amastigotes' shapes of L. major can be explained by various reservoir hosts and vectors. In contrast, more molecular variations in L. tropica could be justified by genetic characters. Unidentified Leishmania could be mixed pathogens or nonpathogens with mammals' Leishmania or Crithidia.

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

    Science.gov (United States)

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

    2015-12-01

    Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

  9. The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

    Science.gov (United States)

    Mahmoudzadeh-Niknam, Hamid; Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-02-01

    Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

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

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    Dhekra Chaara

    2015-12-01

    Full Text Available Leishmania (L. killicki (syn. L. tropica, which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica (from Tunisia, Algeria and Libya and 113 L. tropica specimens (all from Morocco were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE and MultiLocus Microsatellite Typing (MLMT approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica in space and time. Comparison of the L. tropica (exclusively from Morocco and L. killicki (syn. L. tropica population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

  11. Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused bY Leishmania (Leishmania mexicana

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    Erika Ivett Sosa-Bibiano

    2012-06-01

    Full Text Available There is not an experimental model of localized cutaneous leishmaniasis (LCL caused by Leishmania (Leishmania mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L. mexicana. A total of 54 P. yucatanicus (groups of 18 were inoculated with 1x10(6 promastigotes of L. (L. mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18 and in 11.11% (2/18 P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100% biopsies of euthanized P. yucatanicus at three (n = 5 and six (n = 2 months, respectively. These results resembled clinical and histological features caused by L. (L. mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.

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

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    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    , identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some...

  14. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

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    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  15. Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

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    Jörg Schröder

    Full Text Available Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

  16. Anti-leishmanial effect of itraconazole niosome on in vitro susceptibility of Leishmania tropica.

    Science.gov (United States)

    Khazaeli, Payam; Sharifi, Iraj; Talebian, Elham; Heravi, Gioia; Moazeni, Esmaeil; Mostafavi, Mahshid

    2014-07-01

    The novel niosomal system aimed to deliver the active drug entity to the target site. The objective of this study was to prepare and evaluate the effect of itraconazole niosome on the in vitro susceptibility of Leishmania tropica as compared to itraconazole alone or tartar emetic. The overall growth rate of promastigotes treated with various concentrations of itraconazole niosome was significantly lower than that of itraconazole alone (IC₅₀=0.24 μg/ml vs. IC₅₀=0.43 μg/ml, P<0.01). In contrast, the mean multiplication rate of amastigotes inside the macrophages and also the mean number of amastigotes in each macrophage treated with itraconazole niosome (34.9 and 3.0) were significantly lower (P<0.01) than those treated with itraconazole alone (62.0 and 3.8) or tartar emetic (63.9 and 4.2), respectively. These findings indicated that niosomes could be developed as a novel drug delivery for itraconazole in the in vitro model. Further studies are required to evaluate the effect of itraconazole niosome on volunteer human subjects.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Molecular typing of Leishmania infantum isolates from a leishmaniasis outbreak in Madrid, Spain, 2009 to 2012.

    Science.gov (United States)

    Chicharro, C; Llanes-Acevedo, I P; García, E; Nieto, J; Moreno, J; Cruz, I

    2013-07-25

    Leishmaniasis is endemic in south-west Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the south-west of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and -2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a 'new' emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region.

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

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    Nithin eRavooru

    2014-08-01

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

  20. Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

    Science.gov (United States)

    Schröder, Jörg; Noack, Sandra; Marhöfer, Richard J; Mottram, Jeremy C; Coombs, Graham H; Selzer, Paul M

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

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

    Directory of Open Access Journals (Sweden)

    Chau Huynh

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    OpenAIRE

    2010-01-01

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

  4. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

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    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

  5. First case of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Suriname.

    Science.gov (United States)

    Hu, Ricardo V P F; Kent, Alida D; Adams, Emily R; van der Veer, Charlotte; Sabajo, Leslie O A; Mans, Dennis R A; de Vries, Henry J C; Schallig, Henk D F H; Lai A Fat, Rudy F M

    2012-05-01

    The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.

  6. Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

    OpenAIRE

    Kasra Hassani; Elisabeth Antoniak; Armando Jardim; Martin Olivier

    2011-01-01

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 p...

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies...... that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs....

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

    OpenAIRE

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

    2010-01-01

    INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were capt...

  9. New Record of Phlebotomus Sergenti, the Vector of Leishmania Tropica, in the Southern Nile Valley of Egypt

    Science.gov (United States)

    2001-01-01

    SCIENTIFIC NOTE NEW RECORD OF PHLEBOTOMUS SERGENTI, THE VECTOR OF LEISHMANIA TROPICA , IN THE SOUTHERN NILE VALLEY OF EGYPT HANAFI A. HANAFI’ GREGORY M...forms of Leishmania tropica , from southern Egypt . Four female and I male P. sergenti were collected from unlit Centers for Disease Control light traps...sergenti (Parrot) is a widely distrib- uted sand fly species that feeds readily on humans and is a known vector of Leishmania tropica (Ash- ford and

  10. Leishmania infantum AS A CAUSATIVE AGENT OF CUTANEOUS LEISHMANIASIS IN THE STATE OF MATO GROSSO DO SUL, BRAZIL

    Science.gov (United States)

    CASTRO, Ludiele Souza; FRANÇA, Adriana de Oliveira; FERREIRA, Eduardo de Castro; HANS, Günther; HIGA, Minoru German; GONTIJO, Célia Maria Ferreira; PEREIRA, Agnes Antônia Sampaio; DORVAL, Maria Elizabeth Moraes C.

    2016-01-01

    Cutaneous leishmaniasis is caused by different species of theLeishmania genus. Leishmania(Leishmania) infantum, causing cutaneous leishmaniasis, has been described in patients living in areas where visceral leishmaniasis is endemic. In this study, it was possible to characterize this species in seven slides from cutaneous tissue imprints from patients with cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil. PMID:27007566

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

    OpenAIRE

    2004-01-01

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

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

    Science.gov (United States)

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

    1989-02-01

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

  13. Leishmanicidal activity of synthetic chalcones in Leishmania (Viannia) braziliensis.

    Science.gov (United States)

    de Mello, Tatiane F P; Bitencourt, Heriberto R; Pedroso, Raissa B; Aristides, Sandra M A; Lonardoni, Maria V C; Silveira, Thais G V

    2014-01-01

    The treatment of American cutaneous leishmaniasis (ACL) is based on a small group of compounds that were developed decades ago, all of which are highly toxic and have a high rate of treatment failure. The chalcones show leishmanicidal activity, yet few studies have evaluated this activity against Leishmania (Viannia) braziliensis, one of the most important species of Leishmania across Latin America. Four new synthetic chalcones (1-4) were evaluated for inhibitory activity in vitro against promastigotes and intracellular parasites 24h post infection of L. (V.) braziliensis, cytotoxicity for macrophages J774.A1 and red blood cells, and the ability to stimulate nitric oxide production. The results for the inhibitory concentration for 50% of the promastigotes (IC50) (1.38±1.09-6.36±2.04μM), cytotoxic concentration for 50% of the macrophages (CC50) (13.49±3.13-199.43±4.11μM), and selectivity index (SI) (3.76 to 33.94) indicate that all chalcones (1-4) showed an effect on promastigotes of L. (V.) braziliensis; chalcone 2 had the highest SI. The haemolytic assay with chalcones 1 (301.93μM), 2 (534.18μM), 3 (419.46μM) and 4 (381.11μM) showed 0.00%, 2.33%, 0.57% and 1.74% haemolysis, respectively. All chalcones significantly reduced the infection index of macrophages by parasites; for chalcones (1-3) this effect may be dependent on nitric-oxide production by macrophages. The chalcones tested exhibited inhibitory activity for promastigotes and intracellular parasites of L. (V.) braziliensis, with low toxicity for macrophages and red blood cells. The anti-Leishmania activity of chalcones (1-3) may depend on the stimulation of nitric-oxide production in the initial stage of infection. These results show an initially encouraging potential for the use of chalcones (1-4) to treat ACL.

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

    Directory of Open Access Journals (Sweden)

    SA Ali

    2011-09-01

    Full Text Available Background: Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical coun­tries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effec­tive in Leishmania vaccination. Leishmania Soluble Antigen (SLA has also recently been used Leishmania vaccination.Methods: The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of den­dritic cells (DCs loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun.Results: Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administra­tion of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection.Conclusion: The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vac­cine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These find­ings would be helpful in opening new windows in Leishmania vaccine research.

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

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    A. López-Céspedes

    2012-01-01

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

  16. Drought, smallpox, and emergence of Leishmania braziliensis in northeastern Brazil.

    Science.gov (United States)

    Sousa, Anastácio Q; Pearson, Richard

    2009-06-01

    Cutaneous leishmaniasis caused by Leishmania (Vianna) braziliensis is a major health problem in the state of Ceará in northeastern Brazil. We propose that the disease emerged as a consequence of the displacement of persons from Ceará to the Amazon region following the Great Drought and smallpox epidemic of 1877-1879. As the economic and social situation in Ceará deteriorated, approximately 55,000 residents migrated to the Amazon region to find work, many on rubber plantations. Those that returned likely introduced L. (V.) brazilensis into Ceará, where the first cases of cutaneous leishmaniasis were reported early in the 20th century. The absence of an animal reservoir in Ceará, apart from dogs, supports the hypothesis. The spread of HIV/AIDS into the region and the possibility of concurrent cutaneous leishmaniasis raise the possibility of future problems.

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

    Directory of Open Access Journals (Sweden)

    Letícia Oba Galvão

    2000-08-01

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

  18. A Pediatric Case of Concomitant Leishmania and Brucella Infection

    Directory of Open Access Journals (Sweden)

    Perihan Yasemen Canöz

    2014-08-01

    Full Text Available Visceral leishmaniasis (Kala-azar is a disease caused by protozoan parasites of the Leishmania Genus, and Brucellosis is a zoonotic disease infecting human host by infective animals. A sixteen year-old girl presented to our clinic with complaints of fatigue, myalgia, pallor, stomach ache, leg swelling, and diffuse body rash. Physical examination revealed findings of elevated body temperature, splenomegaly, upper and lower extremity edema, and diffuse erythema. Patients’ brucella agglutination test was positive at titers 1: 640. Since the treatment of Brucellosis was unsuccessful, other disease processes were investigated and extracellular and intracellular amastigots were detected in the bone marrow aspirate preparations. Kala-azar dipstick (rk-39 was also positive. We present a 16 year-old girl who was diagnosed with Kala-azar and Brucellosis together infection and successfully treated.

  19. Leishmania: origin, evolution and future since the Precambrian.

    Science.gov (United States)

    Tuon, Felipe Francisco; Neto, Vicente Amato; Amato, Valdir Sabbaga

    2008-11-01

    This brief review discusses the history of leishmaniasis, considering its origin from the Paleoartic, Neoartic or Neotropic. We reassess some of the theories of the likely origin of this protozoan since the beginning of life on Earth, passing through the Mesozoic and continuing to the appearance of humans. The relationship between this parasite or its ancestors, possible vectors and hosts with regard to ecological modifications is discussed. Recent molecular techniques have helped to elucidate some of the evolutionary questions regarding Leishmania, but have also brought doubts about the origin and evolution of this human parasite. PCR has been used for studies in the new discipline of paleoparasitology, helping to elucidate some of the remaining evolutionary questions. Understanding of this global condition is fundamental in determining the best approach to use against the parasite, specifically for the development of an efficient vaccine.

  20. Molecular Cloning, Expression and Characterization of Ribokinase of Leishmania major

    Institute of Scientific and Technical Information of China (English)

    Patrick. O.J. OGBUNUDE; Nadia LAMOUR; Michael P. BARRETT

    2007-01-01

    Ribokinase (EC 2.1.7.15) from Leishmania major was cloned, sequenced and overexpressed in Escherichia coli. The gene expressed an active enzyme that had comparable activity to the same enzyme studied in E. coli. It specifically phosphorylated D-ribose. Under defined conditions, the Km for the substrates D-ribose and ATP were 0.3±0.04 mM and 0.2±0.02 mM, respectively. The turnover numbers of the enzyme for the substrates were 10.8 s-1 and 10.2 s-1, respectively. The enzyme product ribose 5-phosphate inhibited the phosphorylation of D-ribose with an apparent Ki of 0.4 mM, which is close to the Km (0.3 mM) of D-ribose, suggesting that it might play a role in regulating flux through the enzyme.

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

    OpenAIRE

    Aldair Junio Woyames Pinto

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2000-07-01

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

  3. Development of Cutaneous Leishmaniasis after Leishmania Skin Test

    Science.gov (United States)

    Machado, Paulo R.; Carvalho, Augusto M.; Machado, Gustavo U.; Dantas, Marina L.; Arruda, Sérgio

    2011-01-01

    Thirty-year-old female with a previous history of a cutaneous ulcer suspicious of leishmaniasis 20 years ago presented with a new complaint of a depressed papular lesion 8 × 7 mm in the right lower extremity. The lesion was of 10-day duration. Because early cutaneous leishmaniasis (CL) lesions may have a non-ulcerated appearance, a Leishmania skin test (LST) was performed on the forearm with a strong positive result (38 × 32 mm). After 8 days, the lesion in the leg, which was diagnosed as folliculitis, completely healed. However, a typical CL ulcer (26 × 24 mm) developed at the LST site. Histopathology of the new lesion did not identifiy parasites, but the findings were consistent with a diagnosis of CL. Further analysis identified amastigotes by immunohistochemical stain. Mononuclear cells harvested from the patient were stimulated with Leishmania antigen and showed high levels of production of both tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ): 2,943 pg/mL and 2,313 pg/mL, respectively. After 40 days of treatment with antimony and pentoxifylline, the ulcer resolved. The development of CL at the LST site suggests a strong Th1 immune response, and it is an in vivo documentation of the role of the host immune response in the pathology of CL. It teaches us that LST should be cautiously, if at all, used in patients with self-healing CL ulcers. PMID:22162702

  4. Allopurinol Resistance in Leishmania infantum from Dogs with Disease Relapse.

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    Daniel Yasur-Landau

    2016-01-01

    Full Text Available Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of Leishmania strains from dogs to allopurinol has not been described before in clinical studies.Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group showed an average half maximal inhibitory concentration (IC50 of 996 μg/mL. A significantly lower IC50 (P = 0.01 was found for isolates from ten dogs before treatment (NT group, 200 μg/mL, as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL. Axenic amastigotes produced from isolates of the TR group also showed significantly higher (P = 0.002 IC50 compared to the NT group (1678 and 671 μg/mL, respectively. The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (P = 0.002 was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol.This is the first study to demonstrate allopurinol resistance in L. infantum and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.

  5. Leishmania tropica in Stray Dogs in Southeast Iran

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    Mehdi BAMOROVAT

    2015-10-01

    Full Text Available  Background: Cutaneous leishmaniasis (CL caused by Leishmania tropica is endemic in Kerman, southeastern Iran. While dogs have long been implicated as the main domestic reservoirs of L. infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL, they can also carry L. tropica infection. The objective of the present study was to determine molecular identity and to evaluate histopathological changes due to CL in dogs in a well-known focus of anthroponotic CL (ACL in Kerman, southeastern Iran.Methods: This study was carried out in three prospective series from 1994 to 2013 on dogs. Tissue samples were taken from 471 stray dogs. Pathological specimens including skin, spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning and staining for further histopathological examination. PCR amplification of kDNA was performed to identify the causative agent and sequencing. Overall, two out of 471 stray dogs were infected with L. tropica. Hyperplasia of red pulp by the proliferation of histiocytes, lymphocytes, plasma cells and cytoplasm of histiocytes collection of amastigotes was noted.Results: Based on the results of PCR products and sequencing analysis, the parasites isolated from the lesions of two dogs were characterized as L. tropica, corresponding to a band of 830 bp Conclusion: This finding revealed infection with L. tropica in stray dogs in the city and suburbs of Kerman. This information is essential for public health concerns and planning effective future control programs. The role of dogs as potentional reservoir in the epidemiology of ACL needs further investigation. Keywords: Leishmania tropica, Dog, Histopathology, Molecular, Epidemiology, Iran

  6. Antimony quantification in Leishmania by electrothermal atomic absorption spectroscopy.

    Science.gov (United States)

    Roberts, W L; Rainey, P M

    1993-05-15

    Tri- and pentavalent antimony were quantified in Leishmania mexicana pifanoi amastigotes and promastigotes by atomic absorption spectroscopy with electrothermal atomization. Leishmania grown in axenic culture were treated with either potassium antimony tartrate [Sb(III)] or sodium stibogluconate [Sb(V)]. The parasites were collected, digested with nitric acid, and subjected to atomic absorption spectroscopy. The method was linear from 0 to 7 ng of antimony. The interassay coefficients of variation were 9.6 and 5.7% (N = 5) for 0.52 and 3.7-ng samples of leishmanial antimony, respectively. The limit of detection was 95 pg of antimony. The assay was used to characterize Sb(III) and Sb(V) influx and efflux kinetics. Influx rates were determined at antimony concentrations that produced a 50% inhibition of growth (IC50). The influx rates of Sb(V) into amastigotes and promastigotes were 4.8 and 12 pg/million cells/h, respectively, at 200 micrograms antimony/ml. The influx rate of Sb(III) into amastigotes was 41 pg/million cells/h at 20 micrograms antimony/ml. Influx of Sb(III) into promastigotes at 1 microgram antimony/ml was rapid and reached a plateau of 175 pg/million cells in 2 h. Efflux of Sb(III) and Sb(V) from amastigotes and promastigotes exhibited biphasic kinetics. The initial (alpha) half-life of Sb(V) efflux was less than 4 min and that of Sb(III) was 1-2 h. The apparent terminal (beta) half-lives ranged from 7 to 14 h.

  7. Development of cutaneous leishmaniasis after leishmania skin test.

    Science.gov (United States)

    Machado, Paulo R; Carvalho, Augusto M; Machado, Gustavo U; Dantas, Marina L; Arruda, Sérgio

    2011-01-01

    Thirty-year-old female with a previous history of a cutaneous ulcer suspicious of leishmaniasis 20 years ago presented with a new complaint of a depressed papular lesion 8 × 7 mm in the right lower extremity. The lesion was of 10-day duration. Because early cutaneous leishmaniasis (CL) lesions may have a non-ulcerated appearance, a Leishmania skin test (LST) was performed on the forearm with a strong positive result (38 × 32 mm). After 8 days, the lesion in the leg, which was diagnosed as folliculitis, completely healed. However, a typical CL ulcer (26 × 24 mm) developed at the LST site. Histopathology of the new lesion did not identifiy parasites, but the findings were consistent with a diagnosis of CL. Further analysis identified amastigotes by immunohistochemical stain. Mononuclear cells harvested from the patient were stimulated with Leishmania antigen and showed high levels of production of both tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ): 2,943 pg/mL and 2,313 pg/mL, respectively. After 40 days of treatment with antimony and pentoxifylline, the ulcer resolved. The development of CL at the LST site suggests a strong Th1 immune response, and it is an in vivo documentation of the role of the host immune response in the pathology of CL. It teaches us that LST should be cautiously, if at all, used in patients with self-healing CL ulcers.

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

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    Maria Elizabeth Cavalheiros Dorval

    2010-10-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

  11. Molecular modeling, structural analysis and identification of ligand binding sites of trypanothione reductase from Leishmania mexicana

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    Ozal Mutlu

    2013-01-01

    Full Text Available Background & objectives: Trypanothione reductase (TR is a member of FAD-dependent NADPH oxidoreductase protein family and it is a key enzyme which connects the NADPH and the thiol-based redox system. Inhibition studies indicate that TR is an essential enzyme for parasite survival. Therefore, it is an attractive target enzyme for novel drug candidates. There is no structural model for TR of Leishmania mexicana (LmTR in the protein databases. In this work, 3D structure of TR from L. mexicana was identified by template-based in silico homology modeling method, resultant model was validated, structurally analyzed and possible ligand binding pockets were identified. Methods: For computational molecular modeling study, firstly, template was identified by BLAST search against PDB database. Multiple alignments were achieved by ClustalW2. Molecular modeling of LmTR was done and possible drug targeting sites were identified. Refinement of the model was done by performing local energy minimization for backbone, hydrogen and side chains. Model was validated by web-based servers. Results: A reliable 3D model for TR from L. mexicana was modeled by using L. infantum trypanothione reductase (LiTR as a template. RMSD results according to C-alpha, visible atoms and backbone were 0.809 Å, 0.732 Å and 0.728 Å respectively. Ramachandran plot indicates that model shows an acceptable stereochemistry. Conclusion: Modeled structure of LmTR shows high similarity with LiTR based on overall structural features like domains and folding patterns. Predicted structure will provide a source for the further docking studies of various peptide-based inhibitors.

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes

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    Figarella K

    2015-12-01

    Full Text Available The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2 exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    Matthew B Rogers

    2014-01-01

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

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

    Science.gov (United States)

    Smith, Barbara A.; Imamura, Hideo; Sanders, Mandy; Svobodova, Milena; Volf, Petr; Berriman, Matthew; Cotton, James A.; Smith, Deborah F.

    2014-01-01

    Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle. PMID:24453988

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

    Science.gov (United States)

    Ranasinghe, Shalindra; Wickremasinghe, Renu; Hulangamuwa, Sanjeeva; Sirimanna, Ganga; Opathella, Nandimithra; Maingon, Rhaiza D C; Chandrasekharan, Vishvanath

    2015-12-01

    Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

  18. Ocurrence of co-infection by Leishmania (Leishmania chagasi and Trypanosoma (Trypanozoon evansi in a dog in the state of Mato Grosso do Sul, Brazil

    Directory of Open Access Journals (Sweden)

    Elisa San Martin Mouriz Savani

    2005-11-01

    Full Text Available A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L. chagasi, the natural transmission of T. (T. evansi occurs in the area under study.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history...

  20. Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

    Science.gov (United States)

    Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

    2016-06-17

    The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

  1. 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...

  2. Serological survey of Leishmania infantum and Trypanosoma cruzi in dogs from urban areas of Brazil and Colombia

    Science.gov (United States)

    Leishmania infantum and Trypanosoma cruzi are zoonotic parasites that are endemic throughout many parts of Latin America. Infected dogs play an important role in transmission of both parasites to humans. A serological survey of Leishmania and Trypanosoma infection was conducted on 365 dogs from São ...

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

    Science.gov (United States)

    Kumar, Pranav; Lodge, Robert; Raymond, Frédéric; Ritt, Jean-François; Jalaguier, Pascal; Corbeil, Jacques; Ouellette, Marc; Tremblay, Michel J

    2013-08-01

    Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.

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

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    Smandi Sondos

    2012-01-01

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

  5. The effect of temperature on Leishmania (Kinetoplastida: Trypanosomatidae) development in sand flies.

    Science.gov (United States)

    Hlavacova, J; Votypka, J; Volf, P

    2013-09-01

    The spread of leishmaniasis to areas where it was previously considered nonendemic has been recently found in the New and Old Worlds, and climate changes are suspected as a crucial factor responsible for this spread. Ambient temperature is known to significantly affect the metabolism of sand flies and their developmental times, but little is known about the effect of temperature on the Leishmania life cycle in vectors. This study assesses the effect of temperature on the development of two closely related New World Viannia species, Leishmania braziliensis and Leishmania peruviana, in the permissive vector Lutzomyia longipalpis, and on the development of New and Old World Leishmania infantum in its natural vectors Lu. longipalpis and Phlebotomus perniciosus, respectively. The mountain species L. peruviana developed well in sand fly females kept at 20 degrees C, whereas at 26 degrees C, most infections were lost during the defecation ofbloodmeal remains; this suggests an adaptation to the slower metabolism of sand flies living at lower ambient temperature. On the contrary, L. infantum and L. braziliensis developed well at both temperatures tested; heavy late-stage infections were observed in a majority of sand fly females maintained at 20 degrees C as well 26 degrees C. Frequent fully developed infections of L. infantum and L. braziliensis at 20 degrees C suggest a certain risk of the spread of these two Leishmania species to higher latitudes and altitudes.

  6. Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil.

    Science.gov (United States)

    Coelho, Willian Marinho Dourado; Richini-Pereira, Virgínia Bodelão; Langoni, Helio; Bresciani, Katia Denise Saraiva

    2011-03-10

    The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, São Paulo State.

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

    Science.gov (United States)

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

    2002-01-01

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

  9. Identification of the Leishmania major Proteins LmjF07.0430, LmjF07.0440, and LmjF27.2440 as Components of Fatty Acid Synthase II

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    Aner Gurvitz

    2009-01-01

    Full Text Available Leishmania major causes leishmaniasis and is grouped within the Trypanosomatidae family, which also includes the etiologic agent for African sleeping sickness, Trypanosoma brucei. Previous studies on T. brucei showed that acyl carrier protein (ACP of mitochondrial fatty acid synthase type 2 (FASII plays a crucial role in parasite survival. Additionally, 3-oxoacyl-ACP synthase TbKASIII as well as TbHTD2 representing 3-hydroxyacyl-ACP dehydratase were also identified; however, 3-oxoacyl-ACP reductase TbKAR1 has hitherto evaded positive identification. Here, potential Leishmania FASII components LmjF07.0440 and LmjF07.0430 were revealed as 3-hydroxyacyl-ACP dehydratases LmHTD2-1 and LmHTD2-2, respectively, whereas LmjF27.2440 was identified as LmKAR1. These Leishmania proteins were ectopically expressed in Saccharomyces cerevisiae htd2Δ or oar1Δ respiratory deficient cells lacking the corresponding mitochondrial FASII enzymes Htd2p and Oar1p. Yeast mutants producing mitochondrially targeted versions of the parasite proteins resembled the self-complemented cells for respiratory growth. This is the first identification of a FASII-like 3-oxoacyl-ACP reductase from a kinetoplastid parasite.

  10. SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases

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    Pita-Pereira Daniela

    2012-01-01

    Full Text Available Abstract Background Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk. Results A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for Leishmania and Viannia, respectively (p L. (V. braziliensis and L. (V. shawii with a bootstrap value of 100%; while for L. (L. infantum and L. (L. amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6% when compared to species from subgenus Leishmania (average of 48.4%. The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities. Conclusions For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast

  11. Chemo-type of essential oil of Ocimum basilicum L. from DR Congo and relative in vitro antioxidant potential to the polarity of crude extracts

    Institute of Scientific and Technical Information of China (English)

    Dorothe Dinangayi Tshilanda; Philippe Bila Babady; Damase Nguwo Vele Onyamboko; Damien Sha-Tshibey Tshibangu; Koto-te-Nyiwa Ngbolua; Philippe Vuka Tsalu; Pius Tshimankinda Mpiana

    2016-01-01

    Objective: To carry out a phyto-chemical characterization of essential oil from Ocimum basilicum L. (O. basilicum) harvested in DR Congo and to assess the antioxidant potential of crude extracts with respect to the polarity for comparison reason. Methods: The phyto-chemical characterization of essential oil produced by hydro-distillation was performed by coupled gas chromatography-mass spectrometer analysis and the antioxidant potential evaluation by in vitro 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity method. Results: A previously weighed amount of fresh leaves of O. basilicum produced 0.65% of essential oil that led to the identification of a set of 84.44% out of 99.98% as major com-pounds (> 1.5%). The chemo-type of this essential oil was linalool-methyl chavicol. Chemical components of oil were characterized by oxygenated aromatic hydrocarbons (46.00%) and oxygenated monoterpenes (26.75%). With respect to the amount of compo-nents, methyl chavicol also known as estragole (35.72%) constituted the very large quantity afterward linalool (21.25%) and then epi-a-cadinol (8.02%), a-bergamotene (6.56%), eugenol (4.60%), 1,8-cineole (4.04%), germacrene D (2.06%), thymol (1.64%), and (E)-citral (1.55%), respectively. Essential oil exhibited antioxidant potential and IC50=(1.180 ± 0.015) mg/mL. Non-polar crude extracts yields were low compared to the one of polar extracts. Only methanol and ethyl acetate had considerably manifested antioxidant potential with IC50 values equal to (0.025 ± 0.013) mg/mL and (0.085 ± 0.012) mg/mL, respectively. As concerns to IC50 values, essential oil was less active than methanol and ethyl acetate extracts. The methanol crude extract exhibited the highest activity. Non-polar extracts showed insignificant radical scavenging ability that did not allow assessing IC50 values. These results highlighted the occurrence of antioxidant potential compounds in polar media. Conclusions: Essential oil and crude extracts of O

  12. Unusual domain architecture of aminoacyl tRNA synthetases and their paralogs from Leishmania major

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    Gowri V S

    2012-11-01

    Full Text Available Abstract Background Leishmania major, a protozoan parasite, is the causative agent of cutaneous leishmaniasis. Due to the development of resistance against the currently available anti-leishmanial drugs, there is a growing need for specific inhibitors and novel drug targets. In this regards, aminoacyl tRNA synthetases, the linchpins of protein synthesis, have received recent attention among the kinetoplastid research community. This is the first comprehensive survey of the aminoacyl tRNA synthetases, their paralogs and other associated proteins from L. major. Results A total of 26 aminoacyl tRNA synthetases were identified using various computational and bioinformatics tools. Phylogenetic analysis and domain architectures of the L. major aminoacyl tRNA synthetases suggest a probable archaeal/eukaryotic origin. Presence of additional domains or N- or C-terminal extensions in 11 aminoacyl tRNA synthetases from L. major suggests possibilities such as additional tRNA binding or oligomerization or editing activity. Five freestanding editing domains were identified in L. major. Domain assignment revealed a novel asparagine tRNA synthetase paralog, asparagine synthetase A which has been so far reported from prokaryotes and archaea. Conclusions A comprehensive bioinformatic analysis revealed 26 aminoacyl tRNA synthetases and five freestanding editing domains in L. major. Identification of two EMAP (endothelial monocyte-activating polypeptide II-like proteins similar to human EMAP II-like proteins suggests their participation in multisynthetase complex formation. While the phylogeny of tRNA synthetases suggests a probable archaeal/eukaryotic origin, phylogeny of asparagine synthetase A strongly suggests a bacterial origin. The unique features identified in this work provide rationale for designing inhibitors against parasite aminoacyl tRNA synthetases and their paralogs.

  13. Diverse modes of binding in structures of Leishmania majorN-myristoyltransferase with selective inhibitors

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    James A. Brannigan

    2014-07-01

    Full Text Available The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

  14. Disseminated Leishmaniasis Caused by Leishmania Tropica in a Puppy from Karaj, Central Iran

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    M Mohebali

    2011-06-01

    Full Text Available A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was exam­ined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large num­ber of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT. DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.

  15. Geographical Distribution of Leishmania Species of Human Cutaneous Leishmaniasis in Fars Province, Southern Iran

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    M Akhoundi

    2013-03-01

    Full Text Available Background: The goal of this study was to know the identity of Leishmania species responsible of cutaneous leishmaniasis (CL in Fars Province, southern Iran.Methods: Five counties of Shiraz, Firouz Abad, Ghir-Karzin, Farashband and Larestan were pros­pected. Forty-four patients exhibiting cutaneous lesions were selected. Samples collected on skin lesions were examined both microscopically (after Giemsa staining and molecularly (after PCR-RFLP.Results: On the 44 examined patients, 39 exhibit Leishmania sp. by microscopical examination, all confirmed by PCR. For five patients with negative microscopical examination, PCR was positive for three of them. Among these 42 positive samples, 3 (7% were infected by L. tropica and 39 (93% by L. major.Conclusions: Leishmania major is the most prevalent species in prospected area and L. tropica occurs in Shiraz and Ghir-Karzin counties.

  16. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis.

    Science.gov (United States)

    Kato, Hirotomo; Gomez, Eduardo A; Martini-Robles, Luiggi; Muzzio, Jenny; Velez, Lenin; Calvopiña, Manuel; Romero-Alvarez, Daniel; Mimori, Tatsuyuki; Uezato, Hiroshi; Hashiguchi, Yoshihisa

    2016-07-01

    A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.

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

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    A.J.A. Barbosa

    1991-03-01

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

  18. Molecular diagnosis of Leishmania mexicana in a cutaneous leishmaniasis case in Sinaloa, Mexico.

    Science.gov (United States)

    Ochoa-Diaz, Yssete O; Lopez-Moreno, Carmina Y; Rendon-Maldonado, Jose G; Lopez-Moreno, Hector S

    2012-01-01

    Leishmaniasis has been considered endemic in Sinaloa, Mexico, since 1994. Despite that Leishmania mexicana is the main etiological agent of cutaneous leishmaniasis (CL) in other regions of Mexico, the species causing CL in patients from Sinaloa state has not been previously established, although Leishmania braziliensis has been found in the neighboring southern state, Nayarit. L. braziliensis is also associated with mucocutaneous leishmaniasis, which is a more complicated clinical variant. Due to the implications on individual and public health, the objective of this report was to identify the Leishmania species present in Sinaloa, Mexico. Using the first internal transcribed spacer (ITS-1) polymerase chain reaction-restriction fragment length polymorphism, we identified L. mexicana in a CL patient from Sinaloa and confirmed the extended distribution of this parasite in Mexico.

  19. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis

    Science.gov (United States)

    Kato, Hirotomo; Gomez, Eduardo A.; Martini-Robles, Luiggi; Muzzio, Jenny; Velez, Lenin; Calvopiña, Manuel; Romero-Alvarez, Daniel; Mimori, Tatsuyuki; Uezato, Hiroshi; Hashiguchi, Yoshihisa

    2016-01-01

    A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas. PMID:27410039

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

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    Adrien Weingärtner

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

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

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    MORA Ana Mariela

    1999-01-01

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

  2. Manifestations of paediatric Leishmania infantum infections in Malta.

    Science.gov (United States)

    Pace, David; Williams, Thomas N; Grochowska, Alicja; Betts, Alexandra; Attard-Montalto, Simon; Boffa, Michael J; Vella, Cecil

    2011-01-01

    Leishmania infantum is endemic in the Maltese archipelago, a group of islands in the Mediterranean which are visited frequently by tourists from Northern European countries. The burden of leishmaniasis is highest in children who may present with cutaneous or visceral manifestations. We describe systematically the manifestations, diagnosis and management of leishmaniasis in children Malta, from 2004 to 2008. Eleven children were diagnosed with leishmaniasis; 8 children (15-44 months of age) had visceral disease and three (aged 9-13 years) suffered cutaneous infections. Prolonged high grade fever, pallor, hepatosplenomegaly, and pancytopenia were common presenting features of visceralisation. Diagnosis was based on the visualisation of amastigotes from bone marrow aspirates. Pentavalent antimonials were associated with treatment failure in two children, whilst liposomal amphotericin B was curative in all. Children with cutaneous leishmaniasis had dry crusted ulcero-nodular lesions on exposed areas which responded to intra-lesional instillation of sodium stibogluconate or to cryotherapy. Leishmaniasis should be included in the differential diagnosis of fever and hepatosplenomegaly or chronic cutaneous lesions in children who travel to Malta.

  3. In vitro activity of amphotericin B cochleates against Leishmania chagasi

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    Aretha Molina Sesana

    2011-03-01

    Full Text Available Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB. In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vitro activity of CAMB against L. chagasi was found to be similar to that of the reference drug AmB deoxycholate, with ED50s of 0.017 μg/mL and 0.021 μg/mL, respectively. Considering that L. chagasi affects organs amenable to cochleate-mediated delivery of AmB, we hypothesize that CAMB will be an effective lipid system for the treatment of visceral leishmaniasis.

  4. In vitro activity of amphotericin B cochleates against Leishmania chagasi.

    Science.gov (United States)

    Sesana, Aretha Molina; Monti-Rocha, Renata; Vinhas, Solange Alves; Morais, Carlos Gustavo; Dietze, Reynaldo; Lemos, Elenice Moreira

    2011-03-01

    Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB). In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vitro activity of CAMB against L. chagasi was found to be similar to that of the reference drug AmB deoxycholate, with ED50s of 0.017 μg/mL and 0.021 μg/mL, respectively. Considering that L. chagasi affects organs amenable to cochleate-mediated delivery of AmB, we hypothesize that CAMB will be an effective lipid system for the treatment of visceral leishmaniasis.

  5. Studies on Stibanate unresponsive isolates of Leishmania donovani

    Indian Academy of Sciences (India)

    Anindita Bhattacharyya; Mandira Mukherjee; Swadesh Duttagupta

    2002-09-01

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

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

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    Omid Rajabi

    2015-09-01

    Full Text Available Objective(s:Cutaneous Leishmaniasis is a common and endemic disease in Khorasan province in North-East of Iran. The pentavalant antimony (Sb V is the mainstay of treatment that has many side effects and resistance to the drug has been reported. The microbicidal effect of ozone was proven in different microorganisms. Since there is no study in this respect and to achieve a low cost and effective treatment, we decided to evaluate the efficacy of ozone against promastigotes of Leishmania major,in vitro. Materials and Methods: Ozonated olive oil was prepared after production of ozone by bubbling ozone-oxygen gas produced by ozone generator through olive oil until it solidified. Promastigotes of L. major were cultivated in two phasic media. After calculation of the number of promastigotes, they were incubated with ozonated olive oil (0, 0.626, 0.938, 1.25, 2.5, 5, 10 mcg/ml at 28 °c for 24 hr. Parasites survival percentage was evaluated using MTS and microscopic assay, and then compared with Glucantime and non-ozonated olive oil. Results:According to the results, there were significant differences in parasites survival percentage between ozonated olive oil and non-ozonated olive oil, at similar concentrations (P

  7. Efficacy of a diarylheptanoid derivative against Leishmania amazonensis

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    Alves Luciana Vignólio

    2003-01-01

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

  8. Multilocus sequence analysis for Leishmania braziliensis outbreak investigation.

    Directory of Open Access Journals (Sweden)

    Mariel A Marlow

    2014-02-01

    Full Text Available With the emergence of leishmaniasis in new regions around the world, molecular epidemiological methods with adequate discriminatory power, reproducibility, high throughput and inter-laboratory comparability are needed for outbreak investigation of this complex parasitic disease. As multilocus sequence analysis (MLSA has been projected as the future gold standard technique for Leishmania species characterization, we propose a MLSA panel of six housekeeping gene loci (6pgd, mpi, icd, hsp70, mdhmt, mdhnc for investigating intraspecific genetic variation of L. (Viannia braziliensis strains and compare the resulting genetic clusters with several epidemiological factors relevant to outbreak investigation. The recent outbreak of cutaneous leishmaniasis caused by L. (V. braziliensis in the southern Brazilian state of Santa Catarina is used to demonstrate the applicability of this technique. Sequenced fragments from six genetic markers from 86 L. (V. braziliensis strains from twelve Brazilian states, including 33 strains from Santa Catarina, were used to determine clonal complexes, genetic structure, and phylogenic networks. Associations between genetic clusters and networks with epidemiological characteristics of patients were investigated. MLSA revealed epidemiological patterns among L. (V. braziliensis strains, even identifying strains from imported cases among the Santa Catarina strains that presented extensive homogeneity. Evidence presented here has demonstrated MLSA possesses adequate discriminatory power for outbreak investigation, as well as other potential uses in the molecular epidemiology of leishmaniasis.

  9. Human genetic susceptibility and infection with Leishmania peruviana

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, M.A.; Davis, C.R.; Collins, A. [and others

    1995-11-01

    Racial differences, familial clustering, and murine studies are suggestive of host genetic control of Leishmania infections. Complex segregation analysis has been carried out by use of the programs POINTER and COMDS and data from a total population survey, comprising 636 nuclear families, from an L. perurviana endemic area. The data support genetic components controlling susceptibility to clinical leishmaniasis, influencing severity of disease and resistance to disease among healthy individuals. A multifactorial model is favored over a sporadic model. Two-locus models provided the best fit to the data, the optimal model being a recessive gene (frequency .57) plus a modifier locus. Individuals infected at an early age and with recurrent lesions are genetically more susceptible than those infected with a single episode of disease at a later age. Among people with no lesions, those with a positive skin-test response are genetically less susceptible than those with a negative response. The possibility of the involvement of more than one gene together with environmental effects has implications for the design of future linkage studies. 31 refs., 7 tabs.

  10. Coxiella burnetii and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses

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    Jess A Millar

    2015-08-01

    Full Text Available Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up- and 311 genes down-regulated in C. burnetii infected cells, whereas 698 genes (534 genes up- and 164 genes down-regulated were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (~7% are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs that were expressed in THP-1 cells and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections. Intriguingly, our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches.

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

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    S Molaie

    2013-09-01

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

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

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    Mimori Tatsuyuki

    2006-09-01

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

  13. Novel approach to in vitro drug susceptibility assessment of clinical strains of Leishmania spp.

    Science.gov (United States)

    Fernández, Olga; Diaz-Toro, Yira; Valderrama, Liliana; Ovalle, Clemencia; Valderrama, Mabel; Castillo, Harry; Perez, Mauricio; Saravia, Nancy Gore

    2012-07-01

    Resistance to antimonial drugs has been documented in Leishmania isolates transmitted in South America, Europe, and Asia. The frequency and distribution of resistance to these and other antileishmanial drugs are unknown. Technical constraints have limited the assessment of drug susceptibility of clinical strains of Leishmania. Susceptibility of experimentally selected lines and 130 clinical strains of Leishmania panamensis, L. braziliensis, and L. guyanensis to meglumine antimoniate and miltefosine was determined on the basis of parasite burden and percentage of infected U-937 human macrophages. Reductions of infection at single predefined concentrations of meglumine antimoniate and miltefosine and 50% effective doses (ED(50)s) were measured and correlated. The effects of 34°C and 37°C incubation temperatures and different parasite-to-host cell ratios on drug susceptibility were evaluated at 5, 10, and 20 parasites/cell. Reduction of the intracellular burden of Leishmania amastigotes in U-937 cells exposed to the predefined concentrations of meglumine antimoniate or miltefosine discriminated sensitive and experimentally derived resistant Leishmania populations and was significantly correlated with ED(50) values of clinical strains (for meglumine antimoniate, ρ = -0.926 and P < 0.001; for miltefosine, ρ = -0.906 and P < 0.001). Incubation at 37°C significantly inhibited parasite growth compared to that at 34°C in the absence of antileishmanial drugs and resulted in a significantly lower ED(50) in the presence of drugs. Susceptibility assessment was not altered by the parasite-to-cell ratio over the range evaluated. In conclusion, measurement of the reduction of parasite burden at a single predetermined drug concentration under standardized conditions provides an efficient and reliable strategy for susceptibility evaluation and monitoring of clinical strains of Leishmania.

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

    Directory of Open Access Journals (Sweden)

    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.

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

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    Norris-Mullins Brianna

    2014-01-01

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

  16. Activation of human T lymphocytes by Leishmania lipophosphoglycan

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  17. Riesgo de transmisión de Leishmania (Kinetoplastida: Trypanosomatidae en Mérida Venezuela

    Directory of Open Access Journals (Sweden)

    Elsa Nieves

    2014-09-01

    Full Text Available La leishmaniasis es una enfermedad causada por la infección de un parásito protozoario del género Leishmania, transmitido por la picada de insectos hematófagos conocidos como flebotominos. El estudio tiene como objetivo determinar la presencia de flebotominos en los Distritos Sanitarios del estado Mérida y diseñar un mapa de riesgo de transmisión entomológico. Se utilizaron cuatro métodos de captura de flebotominos, los ejemplares se identificaron y se les determinó la infección natural por Leishmania. Se estimó la riqueza de especies, y se realizó un proceso analítico Jerárquico. Los resultados muestran la presencia de diversas especies de flebotominos en los Distritos Sanitarios del estado Mérida, siendo las especies de mayor frecuencia L. youngi, L. gomezi, L. ovallesi y L. walkeri. Se detectó 2,1% de infección natural con Leishmania, la cual se encontró en las 4 especies más frecuentes. Se presenta un mapa de riesgo de transmisión entomológico para el estado Mérida. El conocimiento de la situación actual de los vectores de Leishmania en el estado Mérida y el riesgo de transmisión son relevantes a la hora de considerar la prevención y posible surgimiento de nuevos brotes de leishmaniasis. Abstract (english The leishmaniasis is a disease caused by infection with a protozoan parasite of the genus Leishmania, transmitted by the bite of blood-sucking insects known as sandflies. The study aims to determine the presence of sandflies in Merida state health districts and design a map of entomological risk of transmission. Four methods capture sandflies were used, the specimens were identified and natural Leishmania infection was determined. The richness species was estimated and analityc Hierarchie procesess was performed. The results show the presence of various species of sandflies in Merida state health districts, L. youngi, L. gomezi, L. ovallesi and L. walkeri were most abundant species. The 2.1% of natural infection

  18. Innate Immune Activation and Subversion of Mammalian Functions by Leishmania Lipophosphoglycan

    Directory of Open Access Journals (Sweden)

    Luis H. Franco

    2012-01-01

    Full Text Available Leishmania promastigotes express several prominent glycoconjugates, either secreted or anchored to the parasite surface. Of these lipophosphoglycan (LPG is the most abundant, and along with other phosphoglycan-bearing molecules, plays important roles in parasite infectivity and pathogenesis in both the sand fly and the mammalian host. Besides its contribution for parasite survival in the sand fly vector, LPG is important for modulation the host immune responses to favor the establishment of mammalian infection. This review will summarize the current knowledge regarding the role of LPG in Leishmania infectivity, focusing on the interaction of LPG and innate immune cells and in the subversion of mammalian functions by this molecule.

  19. 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

  20. Vectorial competence of Phlebotomus papatasi (Diptera: Psychodidae) to transmit two old world Leishmania species: Leishmania major and L. Tropica.

    Science.gov (United States)

    Darwish, A B; Tewfick, M K; Doha, S A; Abo-Ghalia, A H; Soliman, B A

    2011-12-01

    The vectorial competence of Phlebotomus papatasi for two old world Leishmania species, L. major & L. tropica was investigated. Phlebotomus papatasi originally collected from Suez Governorate, were membrane fed on homogenized hamster's lesion infected with L. major, MHOM/EG/06/RTC-63, and L. tropica, MGER/EG/06/RTC-74 identified from patients with suspected CL in Northern Sinai, Egypt. Fed flies were dissected at different time intervals and examined microscopically to determine the infection rate and parasite intensity. The feeding rate of P. papatasi on L. major (58.69%) was found higher than on L. tropica (45.99%). Infection rate with L. major (60.19%) was significantly higher than that with L. tropica (39.73%). Transmission by bites in case of P. papatasi/L. tropica failed. A characteristic L. major lesion was developed on the foot pads region 120 days post infective bites on healthy hamster. It is therefore concluded that P. papatasi is a much more effective vector for L. major than for L. tropica.

  1. Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides.

    Science.gov (United States)

    Keller, Andrea-Anneliese; Breitling, Reinhard; Hemmerich, Peter; Kappe, Katarina; Braun, Maria; Wittig, Berith; Schaefer, Buerk; Lorkowski, Stefan; Reissmann, Siegmund

    2014-02-01

    Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

  2. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1.

    Directory of Open Access Journals (Sweden)

    Goutam Mandal

    2015-02-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Directory of Open Access Journals (Sweden)

    André Luiz Rodrigues Roque

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

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    Manoel Barral-Netto

    1986-03-01

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

  8. Leishmania Surveillance and Diagnostic Capability in Support of the Joint Biological Agent Identification and Diagnostic System (JBAIDS) and Leishmania Vector Surveillance

    Science.gov (United States)

    2013-02-07

    biosurveillance uses and pre-clinical test phase will be conducted under separate protocols. Proposed follow-on activities are directed at near-term...commercialization of biosurveillance kits and ultimately transition and FDA clearance using the Next Generation Diagnostic System (NGDS). Project... biosurveillance uses and pre-clinical test phase will be conducted under a separate protocol. 3. Validate performance of the Leishmania epidemiology

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

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    Kucknoor Ashwini S

    2005-02-01

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

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

    Science.gov (United States)

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

    2007-09-01

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

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

    Science.gov (United States)

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

    2007-01-01

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

  12. Comparison of molecular markers for strain typing of Leishmania infantum.

    Science.gov (United States)

    Botilde, Yanick; Laurent, Thierry; Quispe Tintaya, Wilber; Chicharro, Carmen; Cañavate, Carmen; Cruz, Israel; Kuhls, Katrin; Schönian, Gabriele; Dujardin, Jean-Claude

    2006-11-01

    The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles (kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV- patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.

  13. Functional analysis of Leishmania cyclopropane fatty acid synthetase.

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    Samuel O Oyola

    Full Text Available The single gene encoding cyclopropane fatty acid synthetase (CFAS is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular and host (intracellular stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.

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

    Science.gov (United States)

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

    2014-12-01

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

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

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    Chalé-balboa W.G.

    2009-09-01

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

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

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    Nathálya dos Santos Martins

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

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    Regina Coeli Cunha Dórea

    2003-11-01

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

  19. Insecticide Treated Camouflage Sceening Reduces Sand Fly Numbers in Leishmania-Endemic Regions in Kenya

    Science.gov (United States)

    Current U.S. military operations in deserts face persistent threats from sand flies that transmit human Leishmania. In this study we investigated the efficacy of artificial barriers treated with residual insecticide to potentially reduce the risk of human infection from leishmaniasis by reducing the...

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  1. Leishmania tropica: the effect of darkness and light on biological activities in vitro.

    Science.gov (United States)

    Allahverdiyev, Adil M; Koc, Rabia Cakir; Ates, Sezen Canim; Bagirova, Malahat; Elcicek, Serhat; Oztel, Olga Nehir

    2011-08-01

    Leishmania parasites can be exposed to effects of light in their vectors and hosts, at various periods. However, there is no information about the effects of light on Leishmania parasites. The aim of this study is to investigate the effects of light on various cell parameters of Leishmania tropica, in vitro. All experiments were conducted on L. tropica promastigotes and amastigote-macrophage cultures, using flow cytometric analysis, MTT and phenol-sulfuric acid assay, DAPI and Giemsa. The results showed that the morphology of parasites has changed; the cell cycle has been affected and this caused parasites to remain at G0/G1 phase. Furthermore the proliferation, infectivity, glucose consumption and mitochondrial dehydrogenase activities of parasites were decreased. Thus, for the first time, in this study, the effects of light on biological activities of Leishmania parasites were shown. These new information about parasites' biology, would be very important to investigate the effects of light on the parasites in infected vectors and hosts.

  2. Molecular characterization of Leishmania species isolated from cutaneous leishmaniasis in Yemen.

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    Mohammed A K Mahdy

    Full Text Available BACKGROUND: Cutaneous leishmaniasis (CL is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited. METHODOLOGY/PRINCIPAL FINDINGS: This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1 gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types. CONCLUSIONS/SIGNIFICANCE: The predominance of the anthroponotic species (i.e. L. tropica indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person-to-person transmission as the main dynamic of transmission of CL.

  3. Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice.

    OpenAIRE

    Buffet, P. A.; Sulahian, A.; Garin, Y J; Nassar, N.; Derouin, F

    1995-01-01

    We developed a microtitration method to determine the parasite burdens in homogenized organs of mice infected with Leishmania infantum. This method proved more sensitive than direct enumeration of amastigotes in stained organs, was appropriate for describing the kinetics of infection, and can be considered for physiopathological or pharmaceutical experimental studies.

  4. Molecular Diagnosis and Identification of Leishmania Species in Jordan from Saved Dry Samples

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    Nawal Hijjawi

    2016-01-01

    Full Text Available Diagnosis of the endemic cutaneous leishmaniasis (CL in Jordan relies on patient clinical presentation and microscopic identification. Studies toward improved identification of the causative Leishmania species, especially in regions where multiple species exist, and the introduction of these techniques into medical diagnosis is paramount. This study looked at the current epidemiology of CL in Jordan. Clinically diagnosed 41 patients with CL were tested for the presence of Leishmania parasite using both Giemsa staining from skin scraps on glass slides and ITS1-PCR from samples blotted onto storage cards (NucleoCards®. Microscopically, 28 out of the 41 (68.3% collected samples were positive for amastigotes, whereas the molecular ITS1-PCR amplification successfully identified 30 of the 41 samples (73.2%. Furthermore, PCR-RFLP analysis allowed species identification which is impossible microscopically. Of the 30 PCR positive samples, 28 were Leishmania major positive and the other two samples were Leishmania tropica. This indicates that L. major is the most prevalent species in Jordan and the two L. tropica cases originated from Syria indicating possible future L. tropica outbreaks. Diagnosis of CL based on clinical presentation only may falsely increase its prevalence. Although PCR is more sensitive, it is still not available in our medical laboratories in Jordan.

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

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    A.J.A. Barbosa

    1991-03-01

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

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

    Directory of Open Access Journals (Sweden)

    F. T. Silveira

    1984-10-01

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

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

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    Sergio Sifontes-Rodríguez

    2015-04-01

    Full Text Available Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC and 50% inhibitory concentration (IC50 values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl-furan (furvina and 2-bromo-5-(2-methyl-2-nitrovinyl-furan (UC245 also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r (a furvina-containing antifungal ointment for the treatment of CL.

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

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    Vanessa Diniz Atayde

    2015-11-01

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

  9. Role of the ABC transporter PRP1 (ABCC7) in pentamidine resistance in Leishmania amastigotes.

    Science.gov (United States)

    Coelho, Adriano C; Messier, Nadine; Ouellette, Marc; Cotrim, Paulo C

    2007-08-01

    Pentamidine is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance mechanism are not well understood. In this work, we show that the intracellular ABC protein PRP1 (pentamidine resistance protein 1) (ABCC7) can confer resistance to pentamidine in Leishmania sp. parasites in the intracellular stage.

  10. Occurrence of Leishmania (Leishmania chagasi in a domestic cat (Felis catus in Andradina, São Paulo, Brazil: case report Ocorrência de Leishmania (Leishmania chagasi em gato doméstico (Felis catus em Andradina, São Paulo, Brasil: relato de caso

    Directory of Open Access Journals (Sweden)

    Willian Marinho Dourado Coelho

    2010-12-01

    Full Text Available This work describes natural infection by Leishmania in a domestic cat where amastigote forms of the parasite were observed in the popliteal lymph node imprint. Positive and negative serological reactions were observed by enzyme-linked immunosorbent assay (ELISA and indirect immunofluorescence assay (IFA, respectively. Polymerase chain reaction (PCR revealed that the nucleotide sequence of the sample was identical to Leishmania (L. chagasi. This is the first report of the disease in felines of the city of Andradina, SP, an area considered endemic for canine and human visceral leishmaniasis.Neste trabalho, é relatada a infecção natural por Leishmania em um gato doméstico no qual, formas amastigotas do parasito foram observadas em imprint de linfonodo poplíteo. Reações sorológicas positivas e negativas foram observadas pelo teste de imunoadsorção enzimática (ELISA e reação de imunofluorescência indireta (RIFI, respectivamente. A reação em cadeia da polimerase (PCR revelou que a sequência de nucleotídeos foi idêntica à Leishmania (L. chagasi. Este é o primeiro relato da doença em felino da cidade de Andradina, Estado de São Paulo, Brasil, área considerada endêmica para leishmaniose visceral canina e humana.

  11. 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 ...

  12. Prevalence of antibodies to Leishmania infantum and Toxoplasma gondii in horses from the north of Portugal

    Science.gov (United States)

    Background Leishmania infantum and Toxoplasma gondii are protozoa with zoonotic and economic importance. Prevalences of antibodies to these agents were assessed in 173 horses from the north of Portugal. Findings Antibodies to L. infantum were detected by the direct agglutination test (DAT); seven (...

  13. Cutaneous leishmaniasis (Leishmania tropica) in a German tourist after travel to Greece.

    Science.gov (United States)

    Berens-Riha, Nicole; Fleischmann, Erna; Pratlong, Francine; Bretzel, Gisela; von Sonnenburg, Frank; Löscher, Thomas

    2009-01-01

    We report on a German tourist returning from vacations in Southern Greece with cutaneous leishmaniasis (CL) presenting as multiple erythematosquamous lesions caused by Leishmania tropica (zymodeme MON-57). In spite of its endemicity, only few data are available on the incidence and current distribution of CL in Greece, which may allow for an assessment of the risk for travelers.

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

    Science.gov (United States)

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

    2017-01-01

    ABSTRACT The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission. PMID:28096483

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

    DEFF Research Database (Denmark)

    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...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    OBJECTIVE: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission...

  17. The pathology of cutaneous leishmaniasis due to Leishmania major in Sudan

    DEFF Research Database (Denmark)

    Gaafar, A; el Kadaro, A Y; Theander, T G

    1995-01-01

    The pathology of cutaneous leishmaniasis in Sudan, where the disease is caused by Leishmania major, was studied by light and electron microscopy. Lesions were classified into four distinct groups based on the ratio of different cell types, especially lymphocytes, macrophages, and plasma cells...

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

    Energy Technology Data Exchange (ETDEWEB)

    Lourenco, Cecilia de Oliveira

    2000-07-01

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

  19. A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.

    Science.gov (United States)

    Santrich, C; Moore, L; Sherwin, T; Bastin, P; Brokaw, C; Gull, K; LeBowitz, J H

    1997-12-01

    We demonstrate a functional role for the paraflagellar rod (PFR) in motility of Leishmania mexicana. The PFR is a complex cytoskeletal structure running parallel to the axoneme in the flagella of kinetoplastid protozoa. The PFR is composed of a latticework of protein filaments whose major constituents are two related proteins (PFR-1 and PFR-2 in Leishmania). The molecular details of their assembly into PFR filaments are unknown as is the biological function of the PFR. As an approach to understanding the structure and function of the PFR in Leishmania, we made L. mexicana null mutants of PFR-2. PFR-2 minus parasites grow and divide normally in culture and still express the PFR-1 protein. They lack most of the PFR structure demonstrating that the PFR-2 protein is an essential constituent of the PFR. Detailed ultrastructural analysis of the PFR-2 null mutant reveals the presence of a residual inner substructure of the PFR which contains PFR-1 protein, indicating that PFR-1 can polymerize in the absence of PFR-2. The PFR-2 null mutant displays pronounced changes in flagellar beat waveform and forward swimming velocity, compared to wild type parasites consistent with decreased internal elastic bending resistance in PFR-lacking flagella, and indicating a functional role for the PFR in the motility of Leishmania.

  20. Can Sergentomyia (Diptera, Psychodidae) play a role in the transmission of mammal-infecting Leishmania?

    Science.gov (United States)

    Maia, Carla; Depaquit, Jérôme

    2016-01-01

    Leishmaniases are parasitic diseases caused by protozoa of the genus Leishmania. The parasites, which infect various wild and domestic mammals, including humans, are transmitted by the bite of phlebotomine sand flies belonging to the Phlebotomus genus in the Old World and to several genera (including Lutzomyia, Psychodopygus and Nyssomyia) in the New World. In this paper, we consider the genus Sergentomyia as divided into seven subgenera, mainly based on spermathecal morphology: Sergentomyia, Sintonius, Parrotomyia, Rondanomyia, Capensomyia, Vattieromyia and Trouilletomyia. We also include the groups Grassomyia and Demeillonius but exclude the genera Spelaeomyia and Parvidens. The possible role of Sergentomyia in the circulation of mammalian leishmaniases in the Old World has been considered as Leishmania DNA and/or parasites have been identified in several species. However, several criteria must be fulfilled to incriminate an arthropod as a biological vector of leishmaniasis, namely: it must be attracted to and willing to feed on humans and any reservoir host, and be present in the same environment; several unambiguously identified wild female flies not containing blood meals have to be found infected (through isolation and/or typing of parasites) with the same strain of Leishmania as occurs in humans or any reservoir host; the presence of infective forms of Leishmania on naturally infected females and/or on colonized sand flies infected experimentally should be observed; and finally, the vector has to be able to transmit parasites as a result of blood-feeding on a susceptible mammal. PMID:27921993