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Sample records for leishmania braziliensis isolation

  1. Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis.

    PubMed

    Rojas, Agustina; García-Lugo, Pablo; Crisante, Gladys; Añez-Rojas, Néstor; Añez, Néstor

    2008-02-01

    GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.

  2. GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

    PubMed Central

    FERREIRA, Lasaro Teixeira; GOMES, Aparecida Helena de Souza; PEREIRA-CHIOCCOLA, Vera Lucia

    2015-01-01

    Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods: Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus. Results: Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci. PMID:26200968

  3. Extreme inbreeding in Leishmania braziliensis

    PubMed Central

    Rougeron, Virginie; De Meeûs, Thierry; Hide, Mallorie; Waleckx, Etienne; Bermudez, Herman; Arevalo, Jorge; Llanos-Cuentas, Alejandro; Dujardin, Jean-Claude; De Doncker, Simone; Le Ray, Dominique; Ayala, Francisco J.; Bañuls, Anne-Laure

    2009-01-01

    Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 ± 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy−Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites. PMID:19497885

  4. Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

    PubMed Central

    Alves-Ferreira, Eliza V. C.; Toledo, Juliano S.; De Oliveira, Arthur H. C.; Ferreira, Tiago R.; Ruy, Patricia C.; Pinzan, Camila F.; Santos, Ramon F.; Boaventura, Viviane; Rojo, David; López-Gonzálvez, Ángelez; Rosa, Jose C.; Barbas, Coral; Barral-Netto, Manoel; Barral, Aldina; Cruz, Angela K.

    2015-01-01

    Background Leishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection. Methodology/Principal Findings We observed no major genomic divergences among the clinical isolates by molecular karyotype and genomic sequencing. RT-PCR revealed that the isolates lacked Leishmania RNA virus (LRV). However, the isolates exhibited distinct in vivo pathogenesis in BALB/c mice; the LbrC isolates were more virulent than the LbrM isolates. Metabolomic analysis revealed significantly increased levels of 14 metabolites in LbrC parasites and 31 metabolites in LbrM parasites that were mainly related to inflammation and chemotaxis. A proteome comparative analysis revealed the overexpression of LbrPGF2S (prostaglandin f2-alpha synthase) and HSP70 in both LbrC isolates. Overexpression of LbrPGF2S in LbrC and LbrM promastigotes led to an increase in infected macrophages and the number of amastigotes per cell at 24–48 h post-infection (p.i.). Conclusions/Significance Despite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that

  5. [Leishmania braziliensis Vianna, 1911 in French Guiana].

    PubMed

    Garin, J P; Piens, M A; Pratlong, F; Rioux, J A

    1989-01-01

    A new case of cutaneous Leishmaniasis to L. braziliensis Vianna, 1911, contracted in French Guyana is reported. The parasite, isolated in culture, is identified by enzymatic typing (13 zymoden). The identified zymodem is zymodem MON-43. It is the same of the WHO reference strain L. braziliensis s. st.

  6. In vitro characterization of Leishmania (Viannia) braziliensis isolates from patients with different responses to Glucantime(®) treatment from Northwest Paraná, Brazil.

    PubMed

    Fernandes, Andrea Claudia Bekner Silva; Pedroso, Raíssa Bocchi; de Mello, Tatiane França Perles; Donatti, Lucélia; Venazzi, Eneide Aparecida Sabaini; Demarchi, Izabel Galhardo; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Silveira, Thaís Gomes Verzignassi

    2016-08-01

    Leishmaniasis is a group of diseases that presents various clinical manifestations. Many studies have shown that the parasite plays an important role in the clinical manifestations and prognosis of this disease. The cutaneous and mucosal forms of American tegumentary leishmaniasis (ATL) are associated with Leishmania (Viannia) braziliensis, which exhibits intraspecific genetic polymorphisms and various clinical manifestations. The present study focused on four different L. braziliensis strains that were isolated from patients with distinct Glucantime(®) treatment responses. The isolates were described based on their molecular, biological, and infective characteristics. Growth patterns in culture medium and different grow phases were analyzed, MID-Logarithimic (Mid-LOG), Logarithimic (LOG) and Stationary (STAT) phases. Complement resistance was evaluated using guinea pig serum. Infection to murine peritoneal macrophages, cytokine and nitric oxide were analyzed. Ultrastructural features were determined by transmission electron microscopy, and molecular characteristics were determined based on random amplified polymorphic DNA (RAPD). All of the L. braziliensis isolates showed typical growth and similar complement sensitivity patterns. Markedly lower infectivity indexes were observed for all strains in the LOG phase, with different cytokine profiles. The ultrastructure analysis revealed distinct differences between the MID-LOG, LOG, and STAT phases. The RAPD results showed a divergence between the isolates of the L. braziliensis. The in vitro characterization of L. braziliensis isolates from humans with different treatment responses using various parameters enabled us to observe differences among the isolates. Molecular and in vivo characterizations are currently under study to improve understanding of the parasite-host interaction that can imply in the clinical manifestation differences. PMID:27181585

  7. BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

    PubMed Central

    Costa, Diego L.; Carregaro, Vanessa; Lima-Júnior, Djalma S.; Silva, Neide M.; Milanezi, Cristiane M.; Cardoso, Cristina R.; Giudice, Ângela; de Jesus, Amélia R.; Carvalho, Edgar M.; Almeida, Roque P.; Silva, João S.

    2011-01-01

    Background Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult. Methodology/Principal Findings Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S). Conclusion/Significance We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens

  8. Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

    PubMed Central

    Hurrell, Benjamin P.; Celes, Fabiana S.; Curvelo, Rebecca P.; Prates, Deboraci B.; Barral, Aldina; Borges, Valeria M.; Tacchini-Cottier, Fabienne; de Oliveira, Camila I.

    2015-01-01

    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. PMID:25756874

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

    PubMed Central

    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

  10. Cutaneous leishmaniasis in western Venezuela caused by infection with Leishmania venezuelensis and L. braziliensis variants.

    PubMed

    Bonfante-Garrido, R; Meléndez, E; Barroeta, S; de Alejos, M A; Momen, H; Cupolillo, E; McMahon-Pratt, D; Grimaldi, G

    1992-01-01

    Between 1975 and 1987, epidemiological studies were carried out in several rural and urban communities in the central part of western Venezuela, especially in the state of Lara. 115 positive cultures were obtained from human cases and identified by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay; 53 were Leishmania venezuelensis and 62 were L. braziliensis. Most of these stocks were also characterized by isoenzyme electrophoresis, which confirmed the identification of the L. venezuelensis isolates. The enzyme electrophoretic profiles of the L. braziliensis isolates, however, revealed two populations with distinct electromorphs, one related to the World Health Organization L. braziliensis reference strain while the other population appeared to be a hybrid between L. braziliensis and L. guyanensis. L. braziliensis variants showed the widest geographical distribution, and were found in 7 states: Districto Federal (Caracas); Lara (Barquisimeto, Crespo, Iribarren, Jimenez, Morán, Palavecino, Torres, Urdaneta); Nueva Esparta (Margarita); Portuguesa (Las Cruces, Rio Amarillo); Trujillo (Cuicas); Yaracuy (Agua Fria, Cambural, Guaremal); and Zulia (Zipa-Yare). L. venezuelensis was found in the following endemic regions: Lara (Barquisimeto, Iribarren, Jimenez, Morán); Merida (Zéa); and Yaracuy (Campos Elias), showing that this parasite has a much wider geographical distribution than was initially recognized and that both these species can occur simultaneously within the same endemic region. Five isolates of L. braziliensis were made from infected donkeys (Equus asinus) in Urdaneta, Lara State, suggesting a possible domestic reservoir of L. braziliensis. PMID:1440772

  11. Cutaneous leishmaniasis in western Venezuela caused by infection with Leishmania venezuelensis and L. braziliensis variants.

    PubMed

    Bonfante-Garrido, R; Meléndez, E; Barroeta, S; de Alejos, M A; Momen, H; Cupolillo, E; McMahon-Pratt, D; Grimaldi, G

    1992-01-01

    Between 1975 and 1987, epidemiological studies were carried out in several rural and urban communities in the central part of western Venezuela, especially in the state of Lara. 115 positive cultures were obtained from human cases and identified by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay; 53 were Leishmania venezuelensis and 62 were L. braziliensis. Most of these stocks were also characterized by isoenzyme electrophoresis, which confirmed the identification of the L. venezuelensis isolates. The enzyme electrophoretic profiles of the L. braziliensis isolates, however, revealed two populations with distinct electromorphs, one related to the World Health Organization L. braziliensis reference strain while the other population appeared to be a hybrid between L. braziliensis and L. guyanensis. L. braziliensis variants showed the widest geographical distribution, and were found in 7 states: Districto Federal (Caracas); Lara (Barquisimeto, Crespo, Iribarren, Jimenez, Morán, Palavecino, Torres, Urdaneta); Nueva Esparta (Margarita); Portuguesa (Las Cruces, Rio Amarillo); Trujillo (Cuicas); Yaracuy (Agua Fria, Cambural, Guaremal); and Zulia (Zipa-Yare). L. venezuelensis was found in the following endemic regions: Lara (Barquisimeto, Iribarren, Jimenez, Morán); Merida (Zéa); and Yaracuy (Campos Elias), showing that this parasite has a much wider geographical distribution than was initially recognized and that both these species can occur simultaneously within the same endemic region. Five isolates of L. braziliensis were made from infected donkeys (Equus asinus) in Urdaneta, Lara State, suggesting a possible domestic reservoir of L. braziliensis.

  12. Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis.

    PubMed Central

    Kutner, S; Pellerin, P; Breniere, S F; Desjeux, P; Dedet, J P

    1991-01-01

    We examined the expression and the antigenicity of the major surface polypeptides of Leishmania braziliensis braziliensis and Leishmania donovani chagasi, parasites which commonly coexist in the same endemic areas of Bolivia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles from surface-iodinated promastigotes showed the presence of a unique iodinatable polypeptide of 72 kDa on the L. b. braziliensis surface and of two major components of 65 and 50 kDa exposed at the surface of L. d. chagasi. Comparison of the peptide digestion profiles of the major iodinated polypeptides of both strains showed no similarity between the maps of the 72- and the 65-kDa polypeptides of L. b. braziliensis and L. d. chagasi, respectively. Immunoprecipitation of surface-labeled L. b. braziliensis Nonidet P-40 extracts with 35 serum specimens obtained from Bolivian patients with cutaneous and mucocutaneous leishmaniasis showed that all serum specimens recognized predominantly the 72-kDa antigen and high-molecular-mass proteins in some cases. The recognition patterns were independent of the geographical origin of the patient, the type of lesion, and the serum antibody titer. Serum specimens from children with visceral leishmaniasis did not precipitate the L. b. braziliensis 72-kDa antigen. Hamster hyperimmune serum against L. b. braziliensis also recognized the 72-kDa surface antigen. However, this recognition was inhibited in the presence of the homologous nonlabeled antigen but not in the presence of heterologous (L. d. chagasi and Trypanosoma cruzi) antigens. The specific recognition of 72-kDa surface antigen in both natural and experimental L. b. braziliensis infections suggests that this antigen could be a good candidate for use in the differential immunodiagnosis and prognosis of the disease. Images PMID:2037677

  13. In Vitro Sensitivity of Paired Leishmania (Viannia) braziliensis Samples Isolated before Meglumine Antimoniate Treatment and after Treatment Failure or Reactivation of Cutaneous Leishmaniasis

    PubMed Central

    Baptista, Cibele; Miranda, Luciana de Freitas Campos; Madeira, Maria de Fátima; Leon, Leonor Laura Pinto; Conceição-Silva, Fátima; Schubach, Armando de Oliveira

    2015-01-01

    This study evaluated the in vitro sensitivity of paired Leishmania braziliensis samples isolated from the same patient before pentavalent antimonial treatment (Sample A) and after treatment failure or cutaneous leishmaniasis reactivation (Sample B) in patients undergoing intralesional administration or injections (5 mgSbV/kg/d) of meglumine antimoniate. Fourteen samples from 7 patients were studied. After 24 h of drug exposure, 50% lethal dose (LD50) values for promastigotes ranged from 0.37 mg/mL to 5.86 mg/mL for samples obtained before treatment (A) and 0.89 mg/mL to 7.80 mg/mL for samples obtained after treatment (B). After 48 h, LD50 values ranged from 0.37 mg/mL to 5.75 mg/mL and 0.70 mg/mL to 7.68 mg/mL for A and B samples, respectively. After 48 h, LD50 values for amastigotes ranged from 11.7 to 44.3 μg/mL for A samples and 13.7 to 52.7 μg/mL for B samples. Of 7 patients, 1 discontinued treatment and 6 were cured after retreatment with amphotericin B (4 cases) or meglumine antimoniate (2 cases). Overall the B samples had higher LD50 values than A samples; however the difference was not significant. These results do not support the hypothesis that low-dose and intralesional treatments induce selection of resistant parasites in vitro and suggest that other factors may influence therapeutic outcome in patients with poor response to initial treatment. PMID:25802480

  14. Low plasma membrane expression of the miltefosine transport complex renders Leishmania braziliensis refractory to the drug.

    PubMed

    Sánchez-Cañete, María P; Carvalho, Luís; Pérez-Victoria, F Javier; Gamarro, Francisco; Castanys, Santiago

    2009-04-01

    Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its beta subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the beta subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent. PMID:19188379

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

    PubMed

    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

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

    PubMed Central

    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

  17. 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▿

    PubMed Central

    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

  18. Drought, Smallpox, and Emergence of Leishmania braziliensis in Northeastern Brazil

    PubMed Central

    Pearson, Richard

    2009-01-01

    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, ≈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. PMID:19523291

  19. Is Leishmania (Viannia) braziliensis preferentially restricted to the cutaneous lesions of naturally infected dogs?

    PubMed

    Madeira, Maria de Fátima; Schubach, Armando de O; Schubach, Tânia M P; Serra, Cathia M B; Pereira, Sandro A; Figueiredo, Fabiano B; Confort, Eliame Mouta; Quintella, Leonardo P; Marzochi, Mauro C A

    2005-08-01

    Nineteen dogs naturally infected with Leishmania (Viannia) braziliensis were studied in order to determine the presence of the parasite outside cutaneous lesions. Eleven (57.9%) animals showed single cutaneous or mucosal lesions and eight (42.1%) presented two or three lesions. Twenty-eight active lesions were biopsied. Isolation in culture and characterization by enzyme electrophoresis were possible in 100% of cases and amastigote forms were visualized upon histopathological examination in three samples (n=25, 12%). Isolation of the parasite in culture from peripheral blood and intact skin fragments obtained from the scapular region was negative in all animals, as was the histopathological analysis of skin from this region. Serological reactivity determined by an immunofluorescent antibody test and/or enzyme-linked immunosorbent assay was demonstrated in 15 animals. The results obtained suggest that L. braziliensis preferentially remains at the site of lesion, in contrast to the systemic distribution of parasites observed in dogs infected with L. (Leishmania) chagasi. A better understanding of this aspect may help direct diagnostic and control strategies applicable to areas characterized by the simultaneous occurrence of the cutaneous and visceral forms of leishmaniasis, as is the case for the Municipality of Rio de Janeiro, Brazil.

  20. Is Leishmania (Viannia) braziliensis preferentially restricted to the cutaneous lesions of naturally infected dogs?

    PubMed

    Madeira, Maria de Fátima; Schubach, Armando de O; Schubach, Tânia M P; Serra, Cathia M B; Pereira, Sandro A; Figueiredo, Fabiano B; Confort, Eliame Mouta; Quintella, Leonardo P; Marzochi, Mauro C A

    2005-08-01

    Nineteen dogs naturally infected with Leishmania (Viannia) braziliensis were studied in order to determine the presence of the parasite outside cutaneous lesions. Eleven (57.9%) animals showed single cutaneous or mucosal lesions and eight (42.1%) presented two or three lesions. Twenty-eight active lesions were biopsied. Isolation in culture and characterization by enzyme electrophoresis were possible in 100% of cases and amastigote forms were visualized upon histopathological examination in three samples (n=25, 12%). Isolation of the parasite in culture from peripheral blood and intact skin fragments obtained from the scapular region was negative in all animals, as was the histopathological analysis of skin from this region. Serological reactivity determined by an immunofluorescent antibody test and/or enzyme-linked immunosorbent assay was demonstrated in 15 animals. The results obtained suggest that L. braziliensis preferentially remains at the site of lesion, in contrast to the systemic distribution of parasites observed in dogs infected with L. (Leishmania) chagasi. A better understanding of this aspect may help direct diagnostic and control strategies applicable to areas characterized by the simultaneous occurrence of the cutaneous and visceral forms of leishmaniasis, as is the case for the Municipality of Rio de Janeiro, Brazil. PMID:15986254

  1. Structural and functional studies of Leishmania braziliensis Hsp90.

    PubMed

    Silva, K P; Seraphim, T V; Borges, J C

    2013-01-01

    The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing "client" proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k(cat)=0.320min(-1)) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC(50) of 0.7μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis. PMID:22910377

  2. In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

    PubMed

    Pinto, Juliana Guerra; Fontana, Letícia Correa; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

    2016-07-01

    Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 μg/ml up to 7.8 μg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

  3. American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis resistant to meglumine antimoniate, but with good response to pentamidine: a case report.

    PubMed

    Pimentel, Maria Inês Fernandes; Baptista, Cibele; Rubin, Evelyn Figueiredo; Vasconcellos, Erica de Camargo Ferreira e; Lyra, Marcelo Rosandiski; Salgueiro, Mariza de Matos; Saheki, Maurício Naoto; Rosalino, Cláudia Maria Valete; Madeira, Maria de Fátima; Silva, Aline Fagundes da; Confort, Eliame Mouta; Schubach, Armando de Oliveira

    2011-01-01

    This is a case report of a Brazilian soldier with cutaneous leishmaniasis. The lesion relapsed following two systemic treatments with meglumine antimoniate. The patient was treated with amphotericin B, which was interrupted due to poor tolerance. Following isolation of Leishmania sp., six intralesional infiltrations of meglumine antimoniate resulted in no response. Leishmania sp promastigotes were again isolated. The patient was submitted to intramuscular 4 mg/kg pentamidine. Parasites from the first and second biopsies were identified as Leishmania (Viannia) braziliensis; those isolated from the first biopsy were more sensitive to meglumine antimoniate in vitro than those isolated from the second biopsy. No relapse was observed.

  4. American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis resistant to meglumine antimoniate, but with good response to pentamidine: a case report.

    PubMed

    Pimentel, Maria Inês Fernandes; Baptista, Cibele; Rubin, Evelyn Figueiredo; Vasconcellos, Erica de Camargo Ferreira e; Lyra, Marcelo Rosandiski; Salgueiro, Mariza de Matos; Saheki, Maurício Naoto; Rosalino, Cláudia Maria Valete; Madeira, Maria de Fátima; Silva, Aline Fagundes da; Confort, Eliame Mouta; Schubach, Armando de Oliveira

    2011-01-01

    This is a case report of a Brazilian soldier with cutaneous leishmaniasis. The lesion relapsed following two systemic treatments with meglumine antimoniate. The patient was treated with amphotericin B, which was interrupted due to poor tolerance. Following isolation of Leishmania sp., six intralesional infiltrations of meglumine antimoniate resulted in no response. Leishmania sp promastigotes were again isolated. The patient was submitted to intramuscular 4 mg/kg pentamidine. Parasites from the first and second biopsies were identified as Leishmania (Viannia) braziliensis; those isolated from the first biopsy were more sensitive to meglumine antimoniate in vitro than those isolated from the second biopsy. No relapse was observed. PMID:21552747

  5. A vaccine combining two Leishmania braziliensis proteins offers heterologous protection against Leishmania infantum infection.

    PubMed

    Duarte, Mariana C; Lage, Daniela P; Martins, Vívian T; Costa, Lourena E; Lage, Letícia M R; Carvalho, Ana Maria R S; Ludolf, Fernanda; Santos, Thaís T O; Roatt, Bruno M; Menezes-Souza, Daniel; Fernandes, Ana Paula; Tavares, Carlos A P; Coelho, Eduardo A F

    2016-08-01

    In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4(+) and CD8(+) T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts.

  6. A vaccine combining two Leishmania braziliensis proteins offers heterologous protection against Leishmania infantum infection.

    PubMed

    Duarte, Mariana C; Lage, Daniela P; Martins, Vívian T; Costa, Lourena E; Lage, Letícia M R; Carvalho, Ana Maria R S; Ludolf, Fernanda; Santos, Thaís T O; Roatt, Bruno M; Menezes-Souza, Daniel; Fernandes, Ana Paula; Tavares, Carlos A P; Coelho, Eduardo A F

    2016-08-01

    In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4(+) and CD8(+) T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts. PMID:27387277

  7. Pterocarpanquinone LQB-118 induces apoptosis in Leishmania (Viannia) braziliensis and controls lesions in infected hamsters.

    PubMed

    Costa, Luciana; Pinheiro, Roberta O; Dutra, Patrícia M L; Santos, Rosiane F; Cunha-Júnior, Edézio F; Torres-Santos, Eduardo C; da Silva, Alcides J M; Costa, Paulo R R; Da-Silva, Silvia A G

    2014-01-01

    Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the

  8. Pterocarpanquinone LQB-118 Induces Apoptosis in Leishmania (Viannia) braziliensis and Controls Lesions in Infected Hamsters

    PubMed Central

    Costa, Luciana; Pinheiro, Roberta O.; Dutra, Patrícia M. L.; Santos, Rosiane F.; Cunha-Júnior, Edézio F.; Torres-Santos, Eduardo C.; da Silva, Alcides J. M.; Costa, Paulo R. R.; Da-Silva, Silvia A. G.

    2014-01-01

    Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the

  9. Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico.

    PubMed

    Sanchez-Tejeda, G; Rodríguez, N; Parra, C I; Hernandez-Montes, O; Barker, D C; Monroy-Ostria, A

    2001-01-01

    An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.

  10. Ultradeformable Archaeosomes for Needle Free Nanovaccination with Leishmania braziliensis Antigens.

    PubMed

    Higa, Leticia H; Arnal, Laura; Vermeulen, Mónica; Perez, Ana Paula; Schilrreff, Priscila; Mundiña-Weilenmann, Cecilia; Yantorno, Osvaldo; Vela, María Elena; Morilla, María José; Romero, Eder Lilia

    2016-01-01

    Total antigens from Leishmania braziliensis promastigotes, solubilized with sodium cholate (dsLp), were formulated within ultradeformable nanovesicles (dsLp-ultradeformable archaeosomes, (dsLp-UDA), and dsLp-ultradeformable liposomes (dsLp-UDL)) and topically administered to Balb/c mice. Ultradeformable nanovesicles can penetrate the intact stratum corneum up to the viable epidermis, with no aid of classical permeation enhancers that can damage the barrier function of the skin. Briefly, 100 nm unilamellar dsLp-UDA (soybean phosphatidylcholine: Halorubrum tebenquichense total polar lipids (TPL): sodium cholate, 3:3:1 w:w) of -31.45 mV Z potential, containing 4.84 ± 0.53% w/w protein/lipid dsLp, 235 KPa Young modulus were prepared. In vitro, dsLp-UDA was extensively taken up by J774A1 and bone marrow derive cells, and the only that induced an immediate secretion of IL-6, IL-12p40 and TNF-α, followed by IL-1β, by J774A1 cells. Such extensive uptake is a key feature of UDA ascribed to the highly negatively charged archaeolipids of the TPL, which are recognized by a receptor specialized in uptake and not involved in downstream signaling. Despite dsLp alone was also immunostimulatory on J774A1 cells, applied twice a week on consecutive days along 7 weeks on Balb/c mice, it raised no measurable response unless associated to UDL or UDA. The highest systemic response, IgGa2 mediated, 1 log lower than im dsLp Al2O3, was elicited by dsLp-UDA. Such findings suggest that in vivo, UDL and UDA acted as penetration enhancers for dsLp, but only dsLp-UDA, owed to its pronounced uptake by APC, succeeded as topical adjuvants. The actual TPL composition, fully made of sn2,3 ether linked saturated archaeolipids, gives the UDA bilayer resistance against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers. Together, these properties make UDA a promising platform for topical drug targeted delivery and vaccination, that may be of help for countries with

  11. Ultradeformable Archaeosomes for Needle Free Nanovaccination with Leishmania braziliensis Antigens

    PubMed Central

    Higa, Leticia H.; Arnal, Laura; Vermeulen, Mónica; Perez, Ana Paula; Schilrreff, Priscila; Mundiña-Weilenmann, Cecilia; Yantorno, Osvaldo; Vela, María Elena; Morilla, María José; Romero, Eder Lilia

    2016-01-01

    Total antigens from Leishmania braziliensis promastigotes, solubilized with sodium cholate (dsLp), were formulated within ultradeformable nanovesicles (dsLp-ultradeformable archaeosomes, (dsLp-UDA), and dsLp-ultradeformable liposomes (dsLp-UDL)) and topically administered to Balb/c mice. Ultradeformable nanovesicles can penetrate the intact stratum corneum up to the viable epidermis, with no aid of classical permeation enhancers that can damage the barrier function of the skin. Briefly, 100 nm unilamellar dsLp-UDA (soybean phosphatidylcholine: Halorubrum tebenquichense total polar lipids (TPL): sodium cholate, 3:3:1 w:w) of -31.45 mV Z potential, containing 4.84 ± 0.53% w/w protein/lipid dsLp, 235 KPa Young modulus were prepared. In vitro, dsLp-UDA was extensively taken up by J774A1 and bone marrow derive cells, and the only that induced an immediate secretion of IL-6, IL-12p40 and TNF-α, followed by IL-1β, by J774A1 cells. Such extensive uptake is a key feature of UDA ascribed to the highly negatively charged archaeolipids of the TPL, which are recognized by a receptor specialized in uptake and not involved in downstream signaling. Despite dsLp alone was also immunostimulatory on J774A1 cells, applied twice a week on consecutive days along 7 weeks on Balb/c mice, it raised no measurable response unless associated to UDL or UDA. The highest systemic response, IgGa2 mediated, 1 log lower than im dsLp Al2O3, was elicited by dsLp-UDA. Such findings suggest that in vivo, UDL and UDA acted as penetration enhancers for dsLp, but only dsLp-UDA, owed to its pronounced uptake by APC, succeeded as topical adjuvants. The actual TPL composition, fully made of sn2,3 ether linked saturated archaeolipids, gives the UDA bilayer resistance against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers. Together, these properties make UDA a promising platform for topical drug targeted delivery and vaccination, that may be of help for countries with

  12. Ultradeformable Archaeosomes for Needle Free Nanovaccination with Leishmania braziliensis Antigens.

    PubMed

    Higa, Leticia H; Arnal, Laura; Vermeulen, Mónica; Perez, Ana Paula; Schilrreff, Priscila; Mundiña-Weilenmann, Cecilia; Yantorno, Osvaldo; Vela, María Elena; Morilla, María José; Romero, Eder Lilia

    2016-01-01

    Total antigens from Leishmania braziliensis promastigotes, solubilized with sodium cholate (dsLp), were formulated within ultradeformable nanovesicles (dsLp-ultradeformable archaeosomes, (dsLp-UDA), and dsLp-ultradeformable liposomes (dsLp-UDL)) and topically administered to Balb/c mice. Ultradeformable nanovesicles can penetrate the intact stratum corneum up to the viable epidermis, with no aid of classical permeation enhancers that can damage the barrier function of the skin. Briefly, 100 nm unilamellar dsLp-UDA (soybean phosphatidylcholine: Halorubrum tebenquichense total polar lipids (TPL): sodium cholate, 3:3:1 w:w) of -31.45 mV Z potential, containing 4.84 ± 0.53% w/w protein/lipid dsLp, 235 KPa Young modulus were prepared. In vitro, dsLp-UDA was extensively taken up by J774A1 and bone marrow derive cells, and the only that induced an immediate secretion of IL-6, IL-12p40 and TNF-α, followed by IL-1β, by J774A1 cells. Such extensive uptake is a key feature of UDA ascribed to the highly negatively charged archaeolipids of the TPL, which are recognized by a receptor specialized in uptake and not involved in downstream signaling. Despite dsLp alone was also immunostimulatory on J774A1 cells, applied twice a week on consecutive days along 7 weeks on Balb/c mice, it raised no measurable response unless associated to UDL or UDA. The highest systemic response, IgGa2 mediated, 1 log lower than im dsLp Al2O3, was elicited by dsLp-UDA. Such findings suggest that in vivo, UDL and UDA acted as penetration enhancers for dsLp, but only dsLp-UDA, owed to its pronounced uptake by APC, succeeded as topical adjuvants. The actual TPL composition, fully made of sn2,3 ether linked saturated archaeolipids, gives the UDA bilayer resistance against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers. Together, these properties make UDA a promising platform for topical drug targeted delivery and vaccination, that may be of help for countries with

  13. Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

    PubMed

    Parmentier, Laurent; Cusini, Alexia; Müller, Norbert; Zangger, Haroun; Hartley, Mary-Anne; Desponds, Chantal; Castiglioni, Patrik; Dubach, Patrick; Ronet, Catherine; Beverley, Stephen M; Fasel, Nicolas

    2016-04-01

    Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B.

  14. Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

    PubMed

    Parmentier, Laurent; Cusini, Alexia; Müller, Norbert; Zangger, Haroun; Hartley, Mary-Anne; Desponds, Chantal; Castiglioni, Patrik; Dubach, Patrick; Ronet, Catherine; Beverley, Stephen M; Fasel, Nicolas

    2016-04-01

    Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B. PMID:26834198

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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

  18. Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects.

    PubMed

    Carvalho, F S; Wenceslau, A A; Albuquerque, G R; Munhoz, A D; Gross, E; Carneiro, P L S; Oliveira, H C; Rocha, J M; Santos, I A; Rezende, R P

    2015-01-01

    Leishmaniasis is an endemic disease present in 98 countries. In Brazil, the northeast region accounts for approximately half of the cases in humans, and has experienced an increased number of positive cases in dogs. In this study, we investigated the epidemiology of canine leishmaniasis in the city of Ilhéus, Bahia, using serological and molecular techniques and evaluated the possible environmental risk factors and associated clinical signs. Blood samples were collected from 560 dogs in urban and peri-urban areas in Ilhéus, northeastern Brazil. Genomic DNA was extracted from the selected animals and subjected to molecular analysis using Leishmania species-specific primers and diagnosis of Trypanosoma cruzi. A total of 54.72% of dogs were positive for Leishmania braziliensis, and animals positive for both Leishmania infantum and T. cruzi were not identified. Hematologic variables were not statistically associated with cases of L. braziliensis. However, the positive animal group showed lower red blood cell and platelet counts and higher levels of urea and serum creatinine. Few dogs presented clinical signs compatible with the presence of Leishmania. Age of more than 2 years and specific hair colors were associated with positive results for L. braziliensis. The geoclimatic characteristics of the region may improve parasite survival, reproduction, and vectors. This may explain the higher rate of dogs identified as positive in this study.

  19. Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects.

    PubMed

    Carvalho, F S; Wenceslau, A A; Albuquerque, G R; Munhoz, A D; Gross, E; Carneiro, P L S; Oliveira, H C; Rocha, J M; Santos, I A; Rezende, R P

    2015-01-01

    Leishmaniasis is an endemic disease present in 98 countries. In Brazil, the northeast region accounts for approximately half of the cases in humans, and has experienced an increased number of positive cases in dogs. In this study, we investigated the epidemiology of canine leishmaniasis in the city of Ilhéus, Bahia, using serological and molecular techniques and evaluated the possible environmental risk factors and associated clinical signs. Blood samples were collected from 560 dogs in urban and peri-urban areas in Ilhéus, northeastern Brazil. Genomic DNA was extracted from the selected animals and subjected to molecular analysis using Leishmania species-specific primers and diagnosis of Trypanosoma cruzi. A total of 54.72% of dogs were positive for Leishmania braziliensis, and animals positive for both Leishmania infantum and T. cruzi were not identified. Hematologic variables were not statistically associated with cases of L. braziliensis. However, the positive animal group showed lower red blood cell and platelet counts and higher levels of urea and serum creatinine. Few dogs presented clinical signs compatible with the presence of Leishmania. Age of more than 2 years and specific hair colors were associated with positive results for L. braziliensis. The geoclimatic characteristics of the region may improve parasite survival, reproduction, and vectors. This may explain the higher rate of dogs identified as positive in this study. PMID:26505353

  20. Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

    PubMed

    Adaui, Vanessa; Lye, Lon-Fye; Akopyants, Natalia S; Zimic, Mirko; Llanos-Cuentas, Alejandro; Garcia, Lineth; Maes, Ilse; De Doncker, Simonne; Dobson, Deborah E; Arevalo, Jorge; Dujardin, Jean-Claude; Beverley, Stephen M

    2016-01-01

    Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

  1. A Cutaneous Ulcer Resulting from Mycobacterium ulcerans—Leishmania braziliensis Coinfection in South America

    PubMed Central

    Mougin, Benjamin; Avenel-Audran, Martine; Hasseine, Lilia; Martin, Ludovic; Cottin, Jane; Pomares, Christelle; Delaunay, Pascal; Marty, Pierre; Ravel, Christophe; Chabasse, Dominique; Abgueguen, Pierre

    2011-01-01

    Buruli ulcer is a tropical skin disease caused by Mycobacterium ulcerans. Its mode of transmission is not yet clearly understood. We report here a cutaneous ulcer in a European traveler in South America resulting from a coinfection detected specifically for Mycobacterium ulcerans and Leishmania braziliensis DNA with real-time polymerase chain reaction. This observation of a unique cutaneous ulcer raises the issue about possible modes of transmission of those two pathogens by the same vector. PMID:22049045

  2. Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform.

    PubMed

    Rezende-Soares, F A; Carvalho-Campos, C; Marques, M J; Porcino, G N; Giarola, N L L; Costa, B L S; Taunay-Rodrigues, A; Faria-Pinto, P; Souza, M A; Diniz, V A; Corte-Real, S; Juliano, M A; Juliano, L; Vasconcelos, E G

    2010-04-01

    An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response. PMID:19961654

  3. Can Equids Be a Reservoir of Leishmania braziliensis in Endemic Areas?

    PubMed Central

    Truppel, Jessé Henrique; Otomura, Flavio; Teodoro, Ueslei; Massafera, Rubens; da Costa-Ribeiro, Magda Clara Vieira; Catarino, Carolina Motter; Dalagrana, Luana; Costa Ferreira, Maria Eugênia Moreira; Thomaz-Soccol, Vanete

    2014-01-01

    In this study, we detected Leishmania (Viannia) braziliensis infection in equids living in endemic regions of cutaneous leishmaniasis. To determine the role of these animals in the Leishmania cycle, we used two approaches: serological and molecular methods. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay (ELISA). Blood samples were collected and tested by polymerase chain reaction (PCR), and the positive products were sequenced. The results showed that 11.0% (25/227) of the equids were seropositive for Leishmania sp, and 16.3% (37/227) were PCR positive. Antibodies were detected in 20 horses, 3 donkeys, and 2 mules, and the parasite DNA was detected in 30 horses, 5 donkeys, and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. braziliensis. Our results show that equids are infected with L. braziliensis, which could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the cutaneous leishmaniasis cycle. PMID:24721908

  4. Can equids be a reservoir of Leishmania braziliensis in endemic areas?

    PubMed

    Truppel, Jessé Henrique; Otomura, Flavio; Teodoro, Ueslei; Massafera, Rubens; da Costa-Ribeiro, Magda Clara Vieira; Catarino, Carolina Motter; Dalagrana, Luana; Costa Ferreira, Maria Eugênia Moreira; Thomaz-Soccol, Vanete

    2014-01-01

    In this study, we detected Leishmania (Viannia) braziliensis infection in equids living in endemic regions of cutaneous leishmaniasis. To determine the role of these animals in the Leishmania cycle, we used two approaches: serological and molecular methods. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay (ELISA). Blood samples were collected and tested by polymerase chain reaction (PCR), and the positive products were sequenced. The results showed that 11.0% (25/227) of the equids were seropositive for Leishmania sp, and 16.3% (37/227) were PCR positive. Antibodies were detected in 20 horses, 3 donkeys, and 2 mules, and the parasite DNA was detected in 30 horses, 5 donkeys, and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. braziliensis. Our results show that equids are infected with L. braziliensis, which could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the cutaneous leishmaniasis cycle.

  5. Co-Infection of Leishmania (Viannia) braziliensis and HIV: report of a case of mucosal leishmaniasis in Cochabamba, Bolivia.

    PubMed

    Torrico, Faustino; Parrado, Rudy; Castro, Rosario; Marquez, Carla Jimena; Torrico, Mary Cruz; Solano, Marco; Reithinger, Richard; García, Ana Lineth

    2009-10-01

    We describe the first case of Leishmania/HIV co-infection reported in Bolivia. Initially hospitalized with a diagnosis of pneumonia and bronchitis, the patient had numerous cutaneous and mucosal lesions caused by Leishmania (Viannia) braziliensis. The patient was also diagnosed as severely immunocompromised because of HIV infection.

  6. Ultrastructural and morphological changes in Leishmania (Viannia) braziliensis treated with synthetic chalcones.

    PubMed

    de Mello, Tatiane F P; Cardoso, Bruna M; Bitencourt, Heriberto R; Donatti, Lucélia; Aristides, Sandra M A; Lonardoni, Maria V C; Silveira, Thais G V

    2016-01-01

    Cutaneous leishmaniasis has an estimated incidence of 1.5 million new cases per year and the treatment options available are old, expensive, toxic, and difficult to administer. Chalcones have shown good activity against several species of Leishmania. However few studies have discussed the mechanisms of action and drug target of this group of compounds in Leishmania. The synthetic chalcones that were evaluated in the present study were previously shown to exhibit activity against Leishmania (Viannia) braziliensis. The objective of the present study was to identify ultrastructural and morphological changes in L. (V.) braziliensis after treatment with three synthetic chalcones (1-3). Promastigotes were treated with chalcones 1-3 and evaluated by transmission and scanning electron microscopy. Cellular and nuclear morphology of the parasites, changes in membrane permeability, and DNA fragmentation in agarose electrophoresis gel were also investigated after exposure to synthetic chalcones. All three synthetic chalcones (1-3) induced ultrastructural alterations in mitochondria, intense vacuolization, two nuclei with rounding of parasites, and cellular and nuclear shrinkage. Chalcones 1-3 also induced no changes in membrane permeability, and presence of nucleosome-sized DNA fragments. Synthetic chalcones 1-3 induced ultrastructural and morphological changes, suggesting that chalcones 1-3 induce apoptosis-like cell death. Further studies should be conducted to elucidate other aspects of the action of these chalcones against Leishmania spp. and their use for the treatment of cutaneous leishmaniasis.

  7. Characterization of regulatory T cell (Treg) function in patients infected with Leishmania braziliensis.

    PubMed

    Costa, Diego L; Guimarães, Luiz H; Cardoso, Thiago M; Queiroz, Adriano; Lago, Ednaldo; Roselino, Ana M; Bacellar, Olívia; Carvalho, Edgar M; Silva, João S

    2013-12-01

    Th1 immune responses are crucial for eliminating Leishmania parasites. However, despite strong Th1 responses, cutaneous leishmaniasis (CL) patients infected with Leishmania braziliensis develop the disease, while milder Th1 responses are found in sub-clinical (SC) infections. Therefore, CL patients may experience impaired regulatory T cell (Treg) function, causing excessive Th1 responses and tissue damage. To address this hypothesis, we characterized the function of circulating Tregs in L. braziliensis infected CL patients and compared them to Tregs from uninfected controls (UC) and SC subjects. The frequency of circulating Tregs was similar in CL patients, UC and SC subjects. Moreover, CL patients Tregs suppressed lymphocyte proliferation and PBMC pro-inflammatory cytokine production more efficiently than UC Tregs, and also produced higher levels of IL-10 than UC and SC Tregs. Furthermore, PBMC and mononuclear cells from lesions of CL patients responded normally to Treg-induced suppression. Therefore, the lesion development in CL patients infected with L. braziliensis is not associated with impairment in Treg function or failure of cells to respond to immunomodulation. Rather, the increased Treg activation in CL patients may impair parasite elimination, resulting in establishment of chronic infection. Thus, immunological strategies that interfere with this response may improve leishmaniasis treatment.

  8. Intermediate Monocytes Contribute to Pathologic Immune Response in Leishmania braziliensis Infections

    PubMed Central

    Passos, Sara; Carvalho, Lucas P.; Costa, Rúbia S.; Campos, Taís M.; Novais, Fernanda O.; Magalhães, Andréa; Machado, Paulo R. L.; Beiting, Daniel; Mosser, David; Carvalho, Edgar M.; Scott, Phillip

    2015-01-01

    Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16+ (both intermediate [CD14+CD16+] and nonclassical [CD14dimCD16+]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16+ monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response. PMID:25139016

  9. Genomic profiling of human Leishmania braziliensis lesions identifies transcriptional modules associated with cutaneous immunopathology.

    PubMed

    Novais, Fernanda O; Carvalho, Lucas P; Passos, Sara; Roos, David S; Carvalho, Edgar M; Scott, Phillip; Beiting, Daniel P

    2015-01-01

    The host immune response has a critical role not only in protection from human leishmaniasis but also in promoting disease severity. Although candidate gene approaches in mouse models of leishmaniasis have been extremely informative, a global understanding of the immune pathways active in lesions from human patients is lacking. To address this issue, genome-wide transcriptional profiling of Leishmania braziliensis-infected cutaneous lesions and normal skin controls was carried out. A signature of the L. braziliensis skin lesion was defined, which includes over 2,000 differentially regulated genes. Pathway-level analysis of this transcriptional response revealed key biological pathways present in cutaneous lesions, generating a testable 'metapathway' model of immunopathology and providing new insights for treatment of human leishmaniasis.

  10. Products of Leishmania braziliensis glucose catabolism: release of D-lactate and, under anaerobic conditions, glycerol

    SciTech Connect

    Darling, T.N.; Davis, D.G.; London, R.E.; Blum, J.J.

    1987-10-01

    Leishmania braziliensis panamensis promastigotes were incubated with glucose as the sole carbon source. About one-fifth of the glucose consumed under aerobic conditions was oxidized to CO/sub 2/. Nuclear magnetic resonance studies with (1-/sup 13/C)glucose showed that the other products released were succinate, acetate, alanine, pyruvate, and lactate. Under anaerobic conditions, lactate output increased, glycerol became a major product, and, surprisingly, glucose consumption decreased. Enzymatic assays showed that the lactate formed was D(-)-lactate. The release of alanine during incubation with glucose as the sole carbon source suggested that appreciable proteolysis occurred, consistent with our observation that a large amount of ammonia was released under these conditions. The discoveries that D-lactate is a product of L. braziliensis glucose catabolism, that glycerol is produced under anaerobic conditions, and that the cells exhibit a reverse Pasteur effect open the way for detailed studies of the pathways of glucose metabolism and their regulation in this organism.

  11. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

    PubMed

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C; Wilson, Mary E; Carvalho, Edgar M; Bacellar, Olívia

    2016-05-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects.

  12. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis

    PubMed Central

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C.; Wilson, Mary E.; Carvalho, Edgar M.; Bacellar, Olívia

    2016-01-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects. PMID:27167379

  13. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

    PubMed

    Skraba, Cissiara Manetti; Pedroso, Raíssa Bocchi; Fiorini, Adriana; Rosado, Fábio Rogério; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Teixeira, Jorge Juarez Vieira; Silveira, Thaís Gomes Verzignassi

    2014-04-01

    This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients. PMID:24485589

  14. In vitro leishmanicidal activities of sesquiterpene lactones from Tithonia diversifolia against Leishmania braziliensis promastigotes and amastigotes.

    PubMed

    de Toledo, Juliano S; Ambrósio, Sergio R; Borges, Carly H G; Manfrim, Viviane; Cerri, Daniel G; Cruz, Angela K; Da Costa, Fernando B

    2014-01-01

    Natural compounds represent a rich and promising source of novel, biologically active chemical entities for treating leishmaniasis. Sesquiterpene lactones are a recognized class of terpenoids with a wide spectrum of biological activities, including activity against Leishmania spp. In this work, a sesquiterpene lactone-rich preparation-a leaf rinse extract (LRE) from Tithonia diversifolia-was tested against promastigote forms of L. braziliensis. The results revealed that the LRE is a rich source of potent leishmanicidal compounds, with an LD50 value 1.5 ± 0.50 µg·mL-1. Therefore, eight sesquiterpene lactones from the LRE were initially investigated against promastigote forms of L. braziliensis. One of them did not present any significant leishmanicidal effect (LD50 > 50 µg·mL-1). Another had a cytotoxic effect against macrophages (4.5 µg·mL-1). The five leishmanicidal compounds with the highest level of selectivity were further evaluated against intracellular parasites (amastigotes) using peritoneal macrophages. Tirotundin 3-O-methyl ether, tagitinin F, and a guaianolide reduced the internalization of parasites after 48 h, in comparison with the negative control. This is the first report on sesquiterpene lactones that have potent leishmanicidal effects on both developmental stages of L. braziliensis. PMID:24830711

  15. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

    PubMed

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

  16. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

    PubMed Central

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P.

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections. PMID:22792440

  17. Host-biting rate and susceptibility of some suspected vectors to Leishmania braziliensis

    PubMed Central

    2014-01-01

    Background American tegumentary leishmaniasis is a serious Brazilian public health problem. This diseases is attributed to seven species of Leishmania, however, the majority of cases are associated with Leishmania braziliensis. Some phlebotomine species have been implicated in the transmission of this parasite, nonetheless only Psychodopygus wellcomei has had its vectorial competence demonstrated. Thus this study sought to assess some parameters related to the vectorial capacity of anthropophilic species of sand fly occurring in São Paulo state: Pintomyia fischeri, Migonemyia migonei Nyssomyia intermedia, Nyssomyia whitmani, Expapillata firmatoi and Psychodopygus ayrozai, under laboratory conditions. These parameters were the duration of the gonotrophic cycle, proportion of females which feed on hamster, the rate of infection by L. braziliensis and the duration of the extrinsic incubation period. Methods The sandflies were collected in three regions of the São Paulo state: Greater São Paulo and the Mogi Guaçu and Iporanga municipalities. To assess the proportion of engorged females the insects were fed on hamsters to estimate the duration of the gonotrophic cycle. To estimate the susceptibility to infection of each species, their females were fed on hamsters infected with Leishmania braziliensis and dissected to ascertain the localization of the flagellates and estimate the extrinsic incubation period. Results Low hamster attractiveness to Ps. ayrozai was observed. A high proportion of engorged females was observed when the hamster had its whole body exposed. The gonotrophic cycle ranged between three and eight days. Mg. migonei, Pi. fischeri, Ny. neivai, Ny. intermedia, Ny. whitmani and Ex.firmatoi presented susceptibility to infection by L. braziliensis. The highest infection rate (34.4%) was observed for Ny. whitmani and the lowest for Ny. intermedia (6.6%). Mg. migonei presented late-stage infection forms on the fifth day after feeding, but in the other

  18. Protective and pathological functions of CD8+ T cells in Leishmania braziliensis infection.

    PubMed

    Cardoso, Thiago Marconi; Machado, Álvaro; Costa, Diego Luiz; Carvalho, Lucas P; Queiroz, Adriano; Machado, Paulo; Scott, Phillip; Carvalho, Edgar M; Bacellar, Olívia

    2015-03-01

    Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8(+) T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8(+) T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8(+) IFN-γ(+) cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8(+) T cells induced more apoptosis of infected monocytes than did SC CD8(+) T cells. Granzyme B production in CD8(+) T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8(+) T cells are more cytotoxic and may be involved in pathology.

  19. Protective and Pathological Functions of CD8+ T Cells in Leishmania braziliensis Infection

    PubMed Central

    Cardoso, Thiago Marconi; Machado, Álvaro; Costa, Diego Luiz; Carvalho, Lucas P.; Queiroz, Adriano; Machado, Paulo; Scott, Phillip; Carvalho, Edgar M.

    2014-01-01

    Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8+ T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4+ IFN-γ+ and CD8+ IFN-γ+ T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8+ T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8+ IFN-γ+ cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8+ T cells induced more apoptosis of infected monocytes than did SC CD8+ T cells. Granzyme B production in CD8+ T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8+ T cells are more cytotoxic and may be involved in pathology. PMID:25534940

  20. Differential Midgut Attachment of Leishmania (Viannia) braziliensis in the Sand Flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia

    PubMed Central

    Soares, Rodrigo P.; Margonari, Carina; Secundino, Nágila C.; Macêdo, Maria E.; da Costa, Simone M.; Rangel, Elizabeth F.; Pimenta, Paulo F.; Turco, Salvatore J.

    2010-01-01

    The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG). An upregulation of β-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia) braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V.) braziliensis with its natural vectors Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. Using two in vitro binding techniques, phosphoglycans (PGs) derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was ∼11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL) in Brazil is discussed. PMID:20011070

  1. A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome

    PubMed Central

    Coelho, Adriano C.; Oliveira, Jordana C.; Espada, Caroline R.; Reimão, Juliana Q.; Trinconi, Cristiana T.; Uliana, Silvia R. B.

    2016-01-01

    Background Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine. Methodology/Principal Findings A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine. Conclusions/Significance Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration. PMID:27144739

  2. The Role of Nitric Oxide and Reactive Oxygen Species in the Killing of Leishmania braziliensis by Monocytes from Patients with Cutaneous Leishmaniasis

    PubMed Central

    Carneiro, Pedro Paulo; Conceição, Jacilara; Macedo, Michael; Magalhães, Viviane; Carvalho, Edgar M.; Bacellar, Olivia

    2016-01-01

    Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, presents an exaggerated Th1 response that is associated with ulcer development. Macrophages are the primary cells infected by Leishmania parasites and both reactive oxygen species (ROS) and nitric oxide (NO) are important in the control of Leishmania by these cells. The mechanism involved in the killing of L. braziliensis is not well established. In this study, we evaluate the role of ROS and NO in the control of L. braziliensis infection by monocytes from CL patients. After in vitro infection with L. braziliensis, the oxidative burst by monocytes from CL patients was higher when compared to monocytes from healthy subjects (HS). Inhibition of the ROS pathway caused a significant decrease in the oxidative burst in L. braziliensis infected monocytes from both groups. In addition, we evaluated the intracellular expression of ROS and NO in L. braziliensis-infected monocytes. Monocytes from CL patients presented high expression of ROS after infection with L. braziliensis. The expression of NO was higher in monocytes from CL patients as compared to expression in monocytes from HS. A strong positive correlation between NO production and lesion size of CL patients was observed. The inhibition of ROS production in leishmania-infected monocytes from CL patients allowed the growth of viable promastigotes in culture supernatants. Thus, we demonstrate that while production of ROS is involved in L. braziliensis killing, NO alone is not sufficient to control infection and may contribute to the tissue damage observed in human CL. PMID:26840253

  3. The Role of Nitric Oxide and Reactive Oxygen Species in the Killing of Leishmania braziliensis by Monocytes from Patients with Cutaneous Leishmaniasis.

    PubMed

    Carneiro, Pedro Paulo; Conceição, Jacilara; Macedo, Michael; Magalhães, Viviane; Carvalho, Edgar M; Bacellar, Olivia

    2016-01-01

    Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, presents an exaggerated Th1 response that is associated with ulcer development. Macrophages are the primary cells infected by Leishmania parasites and both reactive oxygen species (ROS) and nitric oxide (NO) are important in the control of Leishmania by these cells. The mechanism involved in the killing of L. braziliensis is not well established. In this study, we evaluate the role of ROS and NO in the control of L. braziliensis infection by monocytes from CL patients. After in vitro infection with L. braziliensis, the oxidative burst by monocytes from CL patients was higher when compared to monocytes from healthy subjects (HS). Inhibition of the ROS pathway caused a significant decrease in the oxidative burst in L. braziliensis infected monocytes from both groups. In addition, we evaluated the intracellular expression of ROS and NO in L. braziliensis-infected monocytes. Monocytes from CL patients presented high expression of ROS after infection with L. braziliensis. The expression of NO was higher in monocytes from CL patients as compared to expression in monocytes from HS. A strong positive correlation between NO production and lesion size of CL patients was observed. The inhibition of ROS production in leishmania-infected monocytes from CL patients allowed the growth of viable promastigotes in culture supernatants. Thus, we demonstrate that while production of ROS is involved in L. braziliensis killing, NO alone is not sufficient to control infection and may contribute to the tissue damage observed in human CL.

  4. Lutzomyia longipalpis Saliva or Salivary Protein LJM19 Protects against Leishmania braziliensis and the Saliva of Its Vector, Lutzomyia intermedia

    PubMed Central

    Tavares, Natalia M.; Silva, Robson A.; Costa, Dirceu J.; Pitombo, Maiana A.; Fukutani, Kiyoshi F.; Miranda, José C.; Valenzuela, Jesus G.; Barral, Aldina; de Oliveira, Camila I.; Barral-Netto, Manoel; Brodskyn, Claudia

    2011-01-01

    Background Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. Methodology/Principal Findings Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-β. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression. Conclusions/Significance Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    França, F; Lago, E L; Marsden, P D

    1996-01-01

    This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis (L(V)b) among the rural population of a cocoa-producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plants species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale, Anacardiaceae), used by 65% of the population, folha-fogo (Clidemia hirta,Melastomataceae) 39%, alfavaca-grossa (Plectranthus amboinicus, Lamiaceae) 33%, mastruz (Chenopodium ambrosioides, Chenopodiaceae) 31%, erva-de-santa-maria (Solanum americanum, Solanaceae) (25%) and transagem (Plantago major, Plantaginaceae) 2%. PMID:8701041

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

    PubMed

    França, F; Lago, E L; Marsden, P D

    1996-01-01

    This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis (L(V)b) among the rural population of a cocoa-producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plants species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale, Anacardiaceae), used by 65% of the population, folha-fogo (Clidemia hirta,Melastomataceae) 39%, alfavaca-grossa (Plectranthus amboinicus, Lamiaceae) 33%, mastruz (Chenopodium ambrosioides, Chenopodiaceae) 31%, erva-de-santa-maria (Solanum americanum, Solanaceae) (25%) and transagem (Plantago major, Plantaginaceae) 2%.

  9. Functional Transcriptomics of Wild-Caught Lutzomyia intermedia Salivary Glands: Identification of a Protective Salivary Protein against Leishmania braziliensis Infection

    PubMed Central

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

    2013-01-01

    Background Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. Methods and Findings A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11—coding for a 4.5-kDa protein—induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. Conclusions We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis. PMID:23717705

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

    PubMed

    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.

  11. CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes

    PubMed Central

    Zylbersztejn, Ana Madeira Brito; de Morais, Carlos Gustavo Vieira; Lima, Ana Karina Castro; Souza, Joyce Eliza de Oliveira; Lopes, Angela Hampshire; Da-Silva, Sílvia Amaral Gonçalves; Silva-Neto, Mário Alberto Cardoso; Dutra, Patrícia Maria Lourenço

    2015-01-01

    CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis. PMID:26120579

  12. CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

    PubMed

    Zylbersztejn, Ana Madeira Brito; de Morais, Carlos Gustavo Vieira; Lima, Ana Karina Castro; Souza, Joyce Eliza de Oliveira; Lopes, Angela Hampshire; Da-Silva, Sílvia Amaral Gonçalves; Silva-Neto, Mário Alberto Cardoso; Dutra, Patrícia Maria Lourenço

    2015-01-01

    CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.

  13. Immunoproteomic and bioinformatic approaches to identify secreted Leishmania amazonensis, L. braziliensis, and L. infantum proteins with specific reactivity using canine serum.

    PubMed

    Lima, B S S; Fialho, L C; Pires, S F; Tafuri, W L; Andrade, H M

    2016-06-15

    Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection. PMID:27198787

  14. Disposition of antimony in rhesus monkeys infected with Leishmania braziliensis and treated with meglumine antimoniate.

    PubMed

    Friedrich, Karen; Vieira, Flávia A; Porrozzi, Renato; Marchevsky, Renato S; Miekeley, Norbert; Grimaldi, Gabriel; Paumgartten, Francisco J R

    2012-01-01

    Antimony (Sb) disposition and toxicity was evaluated in Leishmania braziliensis-infected monkeys (Macaca mulatta) treated with a 21-d course of low (LOW) or standard (STD) meglumine antimoniate (MA) dosage regimens (5 or 20 mg Sb(V)/kg body weight/d im). Antimony levels in biological matrices were determined by inductively coupled plasma mass spectrometry (ICPMS), while on-line ion chromatography coupled to ICPMS was used to separate and quantify Sb species in plasma. Nadir Sb levels rose steadily from 19.6 ± 4 and 65.1 ± 17.4 ng/g, 24 h after the first injection, up to 27.4 ± 5.8 and 95.7 ± 6.6 ng/g, 24 h after the 21st dose in LOW and SDT groups, respectively. Subsequently, Sb plasma levels gradually declined with a terminal elimination phase half-life of 35.8 d. Antimony speciation in plasma on posttreatment days 1-9 indicated that as total Sb levels declined, proportion of Sb(V) remained nearly constant (11-20%), while proportion of Sb(III) rose from 5% (d 1) to 50% (d 9). Plasma [Sb]/erythrocyte [Sb] ratio was >1 until 12 h after dosing and reversed thereafter. Tissue Sb concentrations (posttreatment days 55 and 95) were as follows: >1000 ng/g in thyroid, nails, liver, gall bladder and spleen; >200 and <1000 ng/g in lymph nodes, kidneys, adrenals, bones, skeletal muscles, heart and skin; and <200 ng/g in various brain structures, thymus, stomach, colon, pancreas. and teeth. Results from this study are therefore consistent with view that Sb(V) is reduced to Sb(III), the active form, within cells from where it is slowly eliminated. Localization of Sb active forms in the thyroid gland and liver and the pathophysiological consequences of marked Sb accumulation in these tissues warrant further studies.

  15. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

    PubMed

    Kato, Hirotomo; Cáceres, Abraham G; Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.

  16. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

    PubMed

    Kato, Hirotomo; Cáceres, Abraham G; Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area. PMID:26735142

  17. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru

    PubMed Central

    Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area. PMID:26735142

  18. American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis: assessment of parasite genetic variability at intra- and inter-patient levels

    PubMed Central

    2013-01-01

    Background The genetic variability of Leishmania (Viannia) braziliensis was assessed at intra and interpatient levels of individuals with different clinical manifestations of American tegumentary leishmaniasis (ATL). Methods Fifty-two samples, of which 13 originated from cutaneous lesions and 39 from mucosal lesions, provided by 35 patients, were examined by low-stringency single-specific-primer PCR (LSSP-PCR) and phenetic analysis. Genetic variability of L. (V.) braziliensis, in kinetoplast DNA (kDNA) signatures, was compared both from different patients and from different lesions of the same patient. Phenetic analysis was performed to evaluate the degree of heterogeneity of the kDNA minicircles. In order to evaluate inter and intrapatient L. (V.) braziliensis genetic variability, the percentage of shared bands and analysis of the coefficients of similarity were analyzed. Results Different genetic profiles, representing kDNA signatures of the parasite, were obtained by LSSP-PCR analysis of each sample. Phenetic analysis grouped genetic profiles of different levels of differentiation from more similar to most divergent. The percentage of shared bands at the inter and intrapatient levels was 77% and 89%, respectively. Comparison of the average inter and intrapatient coefficients of similarity and their standard deviations were statistically significant (p < 0.001). Conclusion Genetic variability at the intrapatient level was less pronounced than that between different patients. A conceptual model was proposed to better understand the complexity at both levels. PMID:23786878

  19. Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

    PubMed

    Moreira, Douglas de Souza; Pescher, Pascale; Laurent, Christine; Lenormand, Pascal; Späth, Gerald F; Murta, Silvane M F

    2015-09-01

    Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.

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

    PubMed Central

    Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M. R.

    2015-01-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  1. Upregulation of Cysteine Synthase and Cystathionine β-Synthase Contributes to Leishmania braziliensis Survival under Oxidative Stress

    PubMed Central

    Téllez, Jair; Romanha, Alvaro José; Steindel, Mario

    2015-01-01

    Cysteine metabolism is considered essential for the crucial maintenance of a reducing environment in trypanosomatids due to its importance as a precursor of trypanothione biosynthesis. Expression, activity, functional rescue, and overexpression of cysteine synthase (CS) and cystathionine β-synthase (CβS) were evaluated in Leishmania braziliensis promastigotes and intracellular amastigotes under in vitro stress conditions induced by hydrogen peroxide (H2O2), S-nitroso-N-acetylpenicillamine, or antimonial compounds. Our results demonstrate a stage-specific increase in the levels of protein expression and activity of L. braziliensis CS (LbrCS) and L. braziliensis CβS (LbrCβS), resulting in an increment of total thiol levels in response to both oxidative and nitrosative stress. The rescue of the CS activity in Trypanosoma rangeli, a trypanosome that does not perform cysteine biosynthesis de novo, resulted in increased rates of survival of epimastigotes expressing the LbrCS under stress conditions compared to those of wild-type parasites. We also found that the ability of L. braziliensis promastigotes and amastigotes overexpressing LbrCS and LbrCβS to resist oxidative stress was significantly enhanced compared to that of nontransfected cells, resulting in a phenotype far more resistant to treatment with the pentavalent form of Sb in vitro. In conclusion, the upregulation of protein expression and increment of the levels of LbrCS and LbrCβS activity alter parasite resistance to antimonials and may influence the efficacy of antimony treatment of New World leishmaniasis. PMID:26033728

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

    PubMed

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

    2016-01-01

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

  3. Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis

    PubMed Central

    Santos, Diego M.; Petersen, Antonio L. O. A.; Celes, Fabiana S.; Borges, Valeria M.; Veras, Patricia S. T.; de Oliveira, Camila I.

    2014-01-01

    Background Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces L. (L.) amazonensis infection in vitro. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with L. (V.) braziliensis. Methodology/Principal findings Exposure of axenic L. (V.) braziliensis promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to L. (V.) braziliensis-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O−2) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated in vivo, employing BALB/c mice infected with L. braziliensis. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability. Conclusion/Significance 17-AAG effectively inhibited the growth of L. braziliensis, both in vitro and in vivo. Given the chronicity of L. (V.) braziliensis infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis. PMID:25340794

  4. Transcriptome Patterns from Primary Cutaneous Leishmania braziliensis Infections Associate with Eventual Development of Mucosal Disease in Humans

    PubMed Central

    Maretti-Mira, Ana Claudia; Bittner, Jaime; Oliveira-Neto, Manoel Paes; Liu, Minghsun; Kang, Dezhi; Li, Huiying; Pirmez, Claude; Craft, Noah

    2012-01-01

    Introduction Localized Cutaneous Leishmaniasis (LCL) and Mucosal Leishmaniasis (ML) are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not. Methods Using RNA-seq, we analyzed both the human and Leishmania transcriptomes in primary cutaneous lesions. Results Limited number of reads mapping to Leishmania transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome. Conclusions We were able to simultaneously sequence both human and Leishmania mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of L. braziliensis infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity. PMID:23029578

  5. Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescens D-mannose sensitive fimbriae.

    PubMed

    Moraes, Caroline S; Seabra, Sergio H; Albuquerque-Cunha, José Maurício; Castro, Daniele P; Genta, Fernando A; de Souza, Wanderley; Brazil, Reginaldo P; Garcia, Eloi S; Azambuja, Patrícia

    2009-06-01

    In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 degrees C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 degrees C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

    PubMed

    Porcino, Gabriane Nascimento; Carvalho-Campos, Cristiane; Maia, Ana Carolina Ribeiro Gomes; Detoni, Michelle Lima; Faria-Pinto, Priscila; Coimbra, Elaine Soares; Marques, Marcos José; Juliano, Maria Aparecida; Juliano, Luiz; Diniz, Vanessa Álvaro; Corte-Real, Suzana; Vasconcelos, Eveline Gomes

    2012-10-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. PMID:22921497

  8. Salivary gland homogenates from wild-caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice.

    PubMed

    Francesquini, Fernanda C; Silveira, Fernando T; Passero, Luiz Felipe D; Tomokane, Thaise Y; Carvalho, Ana Kely; Corbett, Carlos Eduardo P; Laurenti, Márcia D

    2014-12-01

    During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu. flaviscutellata and Lu. (P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used.

  9. Evaluation of photodynamic antimicrobial therapy (PACT) against promastigotes form of the Leishmania (Viannia) braziliensis: in vitro study

    NASA Astrophysics Data System (ADS)

    Barbosa, Artur F. S.; Sangiorgi, Bruno B.; Galdino, Suely L.; Pitta, Ivan R.; Barral Netto, Manoel; Correia, Neandder A.; Pinheiro, Antônio L. B.

    2012-03-01

    Leishmaniasis is a complex disease that affects more than 12 million people in 88 countries worldwide. Leishmania (Viannia) braziliensis is the most common species in the Americas and the most important causative agent of cutaneous and mucocutaneous leishmaniasis in Brazil. The therapeutic arsenal routinely employed to treat patients with leishmaniasis is limited and unsatisfactory. For cutaneous leishmaniasis, pentavalent antimonials are the first line therapeutic scheme recommended by the WHO. These compounds are highly toxic, poorly tolerated and their effectiveness highly variable. In this work, a technique with, so far, an unknown disadvantage is discussed. The aim of this study was to verify the effectiveness of PACT in vitro, as a new technique for the treatment of Leishmaniasis. For this, semiconductor laser (λ = 660nm, 40mW, 4.2J/cm2, CW) associated to phenothiazine's derivatives (5 and 10 μg/ml, TBO, Methylene Blue or Phenothiazine) on the promastigotes form of Leishmania braziliensis in a single session was used. Viability of the parasites was assessed in quadruplicates of each group. The samples were removed and analyzed in a hemocytometer 72h after PACT. We found an important decrease in the number of viable parasites on all treated groups in comparison to their controls. The results of present study showed significant percentage of lethality (above 95%) of the protocol. The 99.23% of lethality was achieved with 10 μg/ml of TBO. No lethality was seen on groups treated neither with laser nor with each compounds separately. The results are promising and indicative that the use of PACT may be a powerful treatment of leishmaniasis when compared to already available ones.

  10. Functional analysis of iron superoxide dismutase-A in wild-type and antimony-resistant Leishmania braziliensis and Leishmania infantum lines.

    PubMed

    Tessarollo, N G; Andrade, J M; Moreira, D S; Murta, S M F

    2015-04-01

    In this work, we characterized the gene encoding iron superoxide dismutase-A (FeSOD-A) in wild-type (WTS) and antimony-resistant (SbR) L. (Viannia) braziliensis and L. (Leishmania) infantum lines, which were selected in vitro. FeSOD-A transcript and protein expression were similar in all tested lines; however, specific enzyme activity analysis revealed higher superoxide dismutase activity in SbIII-resistant LbSbR and LiSbR lines than in the corresponding WTS lines. These parasites were also more tolerant to oxidative stress generated by the herbicide paraquat. Functional analysis showed that in comparison to non-transfected lines, wild-type LbWTS and LiWTS clones overexpressing the FeSOD-A enzyme are 1.6- and 1.7-fold more resistant to SbIII, respectively. Our results suggest that FeSOD-A is involved in the antimony resistance phenotype in L. (V.) braziliensis and L. (L.) infantum.

  11. Native Rodent Species Are Unlikely Sources of Infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru

    PubMed Central

    Shender, Lisa A.; De Los Santos, Maxy; Montgomery, Joel M.; Conrad, Patricia A.; Ghersi, Bruno M.; Razuri, Hugo; Lescano, Andres G.; Mazet, Jonna A. K.

    2014-01-01

    An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region. PMID:25062033

  12. Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis.

    PubMed

    Duarte, Mariana C; Pimenta, Daniel C; Menezes-Souza, Daniel; Magalhães, Rubens D M; Diniz, João L C P; Costa, Lourena E; Chávez-Fumagalli, Miguel A; Lage, Paula S; Bartholomeu, Daniela C; Alves, Maria Julia M; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos A P; Gonçalves, Denise U; Rocha, Manoel O C; Coelho, Eduardo A F

    2015-11-01

    The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.

  13. Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

    PubMed Central

    Duarte, Mariana C.; Pimenta, Daniel C.; Menezes-Souza, Daniel; Magalhães, Rubens D. M.; Diniz, João L. C. P.; Costa, Lourena E.; Chávez-Fumagalli, Miguel A.; Lage, Paula S.; Bartholomeu, Daniela C.; Alves, Maria Julia M.; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos A. P.; Gonçalves, Denise U.; Rocha, Manoel O. C.

    2015-01-01

    The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL. PMID:26376929

  14. Infectiousness of Sylvatic and Synanthropic Small Rodents Implicates a Multi-host Reservoir of Leishmania (Viannia) braziliensis

    PubMed Central

    F. Brito, Maria E.; Carvalho, Francisco G.; Carvalho, Ana Waléria S.; Soares, Fábia; Carvalho, Silvia M.; Costa, Pietra L.; Zampieri, Ricardo; Floeter-Winter, Lucile M.; Shaw, Jeffrey J.; Brandão-Filho, Sinval P.

    2015-01-01

    Background The possibility that a multi-host wildlife reservoir is responsible for maintaining transmission of Leishmania (Viannia) braziliensis causing human cutaneous and mucocutaneous leishmaniasis is tested by comparative analysis of infection progression and infectiousness to sandflies in rodent host species previously shown to have high natural infection prevalences in both sylvatic or/and peridomestic habitats in close proximity to humans in northeast Brazil. Methods The clinical and parasitological outcomes, and infectiousness to sandflies, were observed in 54 colonized animals of three species (18 Necromys lasiurus, 18 Nectomys squamipes and 18 Rattus rattus) experimentally infected with high (5.5×106/ml) or low (2.8×105/ml) dose L. (V.) braziliensis (MBOL/BR/2000/CPqAM95) inoculum. Clinical signs of infection were monitored daily. Whole animal xenodiagnoses were performed 6 months post inoculation using Lutzomyia longipalpis originating from flies caught in Passira, Pernambuco, after this parasite evaluation was performed at necropsy. Heterogeneities in Leishmania parasite loads were measured by quantitative PCR in ear skin, liver and spleen tissues. Results All three rodent species proved to establish infection characterized by short-term self-resolving skin lesions, located on ears and tail but not on footpads (one site of inoculation), and variable parasite loads detected in all three tissues with maximum burdens of 8.1×103 (skin), 2.8×103 (spleen), and 8.9×102 (liver). All three host species, 18/18 N. lasiurus, 10/18 N. squamipes and 6/18 R. rattus, also proved infectious to sandflies in cross-sectional study. R. rattus supported significantly lower tissue parasite loads compared to those in N. lasiurus and N. squamipes, and N. lasiurus appeared to be more infectious, on average, than either N. squamipes or R. rattus. Conclusions A multi-host reservoir of cutaneous leishmaniasis is indicated in this region of Brazil, though with apparent

  15. Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

    PubMed

    Batista, Fernanda A H; Almeida, Glessler S; Seraphim, Thiago V; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2015-01-01

    The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. PMID:25369258

  16. Eco-epidemiological survey of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in Ribeira Valley River, Paraná State, Brazil.

    PubMed

    de Castro, Edilene Alcântara; Luz, Ennio; Telles, Flávio Queiroz; Pandey, Ashok; Biseto, Alceu; Dinaiski, Marlene; Sbalqueiro, Ives; Soccol, Vanete Thomaz

    2005-02-01

    Leishmaniasis is endemic since last century in Adrianópolis Municipality, Ribeira Valley and is a serious public health. A study carried out during 1993-2003 on epidemiological surveys conducted in rural communities showed 339 new cases of cutaneous leishmaniasis (CL) detected from four municipalities (Adrianópolis, Cerro Azul, Doutor Ulysses and Rio Branco do Sul). A larger prevalence of cutaneous lesions was observed in rural workers (36%), women with domestic activities (18%), and younger students (31%). Multiple lesions were noticed in 53% of patients, but only one case of mucocutaneous leishmaniasis was reported. Twenty stocks were isolated from patients with characteristics lesions and were identified as Leishmania (Viannia) braziliensis using multi-locus enzyme electrophoresis (MLEE) and Random Amplified DNA (RAPD). In Phlebotominae survey, five species were obtained. Lutzomyia intermedia sl. represented 97.5% in peridomiciliar area and 100% in domicile. A canine serological survey made (Indirect Immunofluorescence Antibody Test, IFAT and Enzyme Linked Immunosorbent Assay, ELISA) in six rural county of Adrianópolis Municipality during 1998-1999 showed that 15.1% (24/159) of dogs were sera reactive. No lesions were observed in dogs and no parasite was isolated from lymph node aspirates and biopsies. In wild reservoirs study, only seven animals (Cricetidae, Desmodus sp. and edentates) were captured, but no parasites were found in culture from deep organs. The paper presents results of our 10 years study on cutaneous leishmaniasis survey in the Ribeira River Valley, East Region of Paraná State, Brazil. Environment changes in this region are also discussed. PMID:15652328

  17. Comparative Efficacies of Two Antimony Regimens To Treat Leishmania braziliensis-Induced Cutaneous Leishmaniasis in Rhesus Macaques (Macaca mulatta)▿

    PubMed Central

    Grimaldi, G.; Porrozzi, R.; Friedrich, K.; Teva, A.; Marchevsky, R. S.; Vieira, F.; Miekeley, N.; Paumgartten, F. J. R.

    2010-01-01

    This study compared the efficacies of two N-methylglucomine antimoniate (MA) dose regimens for treating macaques with Leishmania braziliensis-induced chronic skin disease. Whereas all animals treated with the full dose (20 mg MA/kg/day) were cured, 50% of the monkeys receiving a low-dose regimen (5 mg MA/kg/day) relapsed. The antimony concentrations in macaque plasma and tissue samples were greater in the full-dose group than in that receiving a subtherapeutic MA regimen. Our data also suggest the presence of drug-induced hepatic pathology. PMID:19822700

  18. Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry.

    PubMed

    Dores-Silva, P R; Silva, E R; Gomes, F E R; Silva, K P; Barbosa, L R S; Borges, J C

    2012-04-15

    The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether, LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. PMID:22387434

  19. In vitro study of the photodynamic antimicrobial therapy (PACT) against promastigotes form of the leishmania (viannia) braziliensis: in vitro study

    NASA Astrophysics Data System (ADS)

    Barbosa, Artur F. S.; Sangiorgi, Bruno B.; Galdino, Suely L.; Pitta, Ivan R.; Barral-Netto, Manoel; Pinheiro, Antônio L. B.

    2013-03-01

    Leishmaniasis, a protozoan parasitic disease that remains a major worldwide health problem with high endemicity in developing countries. Treatment of cutaneous Leishmaniasis (CL) should be decided by the clinical lesions, etiological species and its potential to develop into mucosal Leishmaniasis. High cost, systemic toxicity, and diminished efficacy due to development of parasite resistance are the serious drawbacks of current treatment options. Thus, identifying new, effective, and safer anti-leishmanial drug(s) is of paramount importance. The aim of this study was to verify the effectiveness of PACT in vitro, as a new technique for the treatment of Leishmaniasis. For this, semiconductor laser (λ = 660nm, 40mW, 8.4J/cm2, CW) associated to phenothiazine's derivatives (5 and 10 μg/ml, TBO, Methylene Blue or Phenothiazine) on the promastigotes form of Leishmania braziliensis in a single session was used. Viability of the parasites was assessed in quadruplicates of each group. The samples were removed and analyzed in a hemocytometer 72h after PACT. We found an important decrease in the number of viable parasites on all treated groups in comparison to their controls. The results of present study showed significant percentage of lethality (above 92%) of the protocol. The 98.33% of lethality was achieved with 10 μg/ml of FTZ. No lethality was seen on groups treated neither with laser nor with each compounds separately. The results are promising and indicative that the use of PACT may be a powerful treatment of Leishmaniasis when compared to already available ones.

  20. Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

    PubMed

    Batista, Fernanda A H; Seraphim, Thiago V; Santos, Clelton A; Gonzaga, Marisvanda R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2016-06-15

    Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved. PMID:27103305

  1. Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

    PubMed

    Batista, Fernanda A H; Seraphim, Thiago V; Santos, Clelton A; Gonzaga, Marisvanda R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2016-06-15

    Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

  2. Epitope Mapping of the HSP83.1 Protein of Leishmania braziliensis Discloses Novel Targets for Immunodiagnosis of Tegumentary and Visceral Clinical Forms of Leishmaniasis

    PubMed Central

    Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Reis-Cunha, João Luís; Nagem, Ronaldo Alves Pinto; Carneiro, Cláudia Martins; Coelho, Eduardo Antônio Ferraz; Galvão, Lúcia Maria da Cunha; Fujiwara, Ricardo Toshio

    2014-01-01

    Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution. PMID:24807053

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

    PubMed

    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.

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

    PubMed

    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. PMID:16520002

  5. Antihelminthic Therapy and Antimony in Cutaneous Leishmaniasis: A Randomized, Double-Blind, Placebo-Controlled Trial in Patients Co-Infected with Helminths and Leishmania braziliensis

    PubMed Central

    Newlove, Tracey; Guimarães, Luiz H.; Morgan, Daniel J.; Alcântara, Leda; Glesby, Marshall J.; Carvalho, Edgar M.; Machado, Paulo R.

    2011-01-01

    Helminth infections influence the clinical response to certain diseases and are associated with delayed healing time of patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. We conducted a randomized, double-blind, placebo-controlled clinical trial to examine the role of early versus deferred treatment of intestinal helminth infection on the clinical course of patients with CL treated with pentavalent antimony. (Clinicaltrials.gov number NCT00469495). A total of 90 patients were enrolled, 51.1% (N = 23) of control patients had persistent lesions at Day 90, compared with 62.2% (N = 28) in the treatment group (difference 11.1%, 95% confidence interval = −9.1–30.0%). There was no statistically significant difference in overall time to cure between groups, although there was a tendency for shorter cure times in the control group. This study shows that early introduction of antihelminthic therapy does not improve clinical outcome in patients co-infected with helminths and L. braziliensis. PMID:21460008

  6. Combination of In Silico Methods in the Search for Potential CD4(+) and CD8(+) T Cell Epitopes in the Proteome of Leishmania braziliensis.

    PubMed

    E Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals. PMID:27621732

  7. Combination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis

    PubMed Central

    e Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals.

  8. Combination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis

    PubMed Central

    e Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals. PMID:27621732

  9. Combination of In Silico Methods in the Search for Potential CD4(+) and CD8(+) T Cell Epitopes in the Proteome of Leishmania braziliensis.

    PubMed

    E Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals.

  10. Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

    PubMed Central

    Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio

    2015-01-01

    Background The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis. Methodology/Principal Findings We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis. Conclusions/Significance The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions. PMID:25569432

  11. Linear B-cell epitope mapping of MAPK3 and MAPK4 from Leishmania braziliensis: implications for the serodiagnosis of human and canine leishmaniasis.

    PubMed

    Menezes-Souza, Daniel; de Oliveira Mendes, Tiago Antônio; de Araújo Leão, Ana Carolina; de Souza Gomes, Matheus; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella Castanheira

    2015-02-01

    The correct and early identification of humans and dogs infected with Leishmania are key steps in the control of leishmaniasis. Additionally, a method with high sensitivity and specificity at low cost that allows the screening of a large number of samples would be extremely valuable. In this study, we analyzed the potential of mitogen-activated protein kinase 3 (MAPK3) and mitogen-activated protein kinase 4 (MAPK4) proteins from Leishmania braziliensis to serve as antigen candidates for the serodiagnosis of human visceral and tegumentary leishmaniasis, as well as canine visceral disease. Moreover, we mapped linear B-cell epitopes in these proteins and selected those epitopes with sequences that were divergent in the corresponding orthologs in Homo sapiens, in Canis familiaris, and in Trypanosoma cruzi. We compared the performance of these peptides with the recombinant protein using ELISA. Both MAPK3 and MAPK4 recombinant proteins showed better specificity in the immunodiagnosis of human and canine leishmaniasis than soluble parasite antigens and the EIE-leishmaniose-visceral-canina-bio-manguinhos (EIE-LVC) kit. Furthermore, the performance of this serodiagnosis assay was improved using synthetic peptides corresponding to B-cell epitopes derived from both proteins.

  12. Identification and Biological Characterization of Leishmania (Viannia) guyanensis Isolated from a Patient with Tegumentary Leishmaniasis in Goiás, a Nonendemic Area for This Species in Brazil

    PubMed Central

    Pires, Alause da Silva; Borges, Arissa Felipe; Cappellazzo Coelho, Adriano; Dorta, Miriam Leandro; Lino Junior, Ruy de Souza; Pereira, Ledice Inacia de Araújo; Pinto, Sebastião Alves; Pelli de Oliveira, Milton Adriano; de Matos, Grazzielle Guimarães; Abrahamsohn, Ises A.; Uliana, Silvia Reni B.; Lima, Glória Maria Collet de Araújo; Ribeiro-Dias, Fátima

    2015-01-01

    The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil. PMID:26583102

  13. Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

    PubMed

    Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Nagem, Ronaldo Alves Pinto; Santos, Thaís Teodoro de Oliveira; Silva, Ana Luíza Teixeira; Santoro, Marcelo Matos; de Carvalho, Silvio Fernando Guimarães; Coelho, Eduardo Antônio Ferraz; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio

    2014-01-01

    The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL

  14. A galactosyl(alpha 1-3)mannose epitope on phospholipids of Leishmania mexicana and L. braziliensis is recognized by trypanosomatid-infected human sera.

    PubMed Central

    Avila, J L; Rojas, M

    1990-01-01

    An immunoglobulin M antibody reactive with galactosyl(alpha 1-3)mannose [Gal(alpha 1-3)Man] residues present on phospholipids extracted from Leishmania mexicana and L. braziliensis was found to be present in high titer in the serum of every normal individual studied. Periodate oxidation, acid hydrolysis, or acetylation suppressed immunoreactivity, suggesting that an oligosaccharide chain was responsible for antibody binding. Interaction occurs only with alpha-Gal terminal residues, since treatment of purified glycophospholipids with alpha-galactosidase but not with beta-galactosidase abolished it. Antibody bound to galactosyl(alpha 1-3)galactose-linked synthetic antigens but did not bind to the same residues present in rabbit, rat, and guinea pig erythrocytes or in murine laminin. Antigen-antibody binding was strongly blocked with Gal(alpha 1-3)Man and Gal(beta 1-4)Man. These results plus inhibition studies with several oligosaccharides suggest that they are indeed different from antibodies against the galactosyl(alpha 1-3)galactose residue. Anti-Gal(alpha 1-3)Man antibody values were significantly elevated in 89% of patients with diffuse cutaneous leishmaniasis, 84% of patients with localized cutaneous leishmaniasis, 69% of patients with mucocutaneous leishmaniasis, and 44 and 62% of patients with Trypanosoma cruzi or T. rangeli infection, respectively, but not in patients with 15 other different infectious and inflammatory diseases. Anti-Gal(alpha 1-3)Man antibody readily absorbed to American Leishmania and Trypanosoma culture forms, suggesting a surface membrane localization of reactive epitope. Gal(alpha 1-3)Man-bearing glycophospholipid was easily extracted from American Leishmania promastigotes and T. cruzi trypomastigotes as well as from American Trypanosoma culture forms. The possibility that this antibody arises against parasitic glycophospholipid-linked Gal(alpha 1-3)Man terminal residues is proposed. PMID:1696285

  15. Comparative evaluation of lesion development, tissue damage, and cytokine expression in golden hamsters (Mesocricetus auratus) infected by inocula with different Leishmania (Viannia) braziliensis concentrations.

    PubMed

    Ribeiro-Romão, Raquel P; Moreira, Otacílio C; Osorio, Elvia Yaneth; Cysne-Finkelstein, Lea; Gomes-Silva, Adriano; Valverde, Joanna G; Pirmez, Claude; Da-Cruz, Alda Maria; Pinto, Eduardo Fonseca

    2014-12-01

    The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.

  16. In vitro and in vivo leishmanicidal activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis.

    PubMed

    de Lima, Silvio César Gomes; Teixeira, Maria Jania; Lopes, José Evaldo Gonçalves; de Morais, Selene Maia; Torres, Alba Fabiola; Braga, Milena Aguiar; Rodrigues, Raphael Oliveira; Santiago, Gilvandete Maria Pinheiro; Martins, Alice Costa; Nagao-Dias, Aparecida Tiemi

    2014-01-01

    The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 10(6) promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 10(7) L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 10(6) L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium. PMID:24829921

  17. In Vitro and In Vivo Leishmanicidal Activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis

    PubMed Central

    de Lima, Silvio César Gomes; Teixeira, Maria Jania; Lopes Júnior, José Evaldo Gonçalves; de Morais, Selene Maia; Torres, Alba Fabiola; Braga, Milena Aguiar; Rodrigues, Raphael Oliveira; Santiago, Gilvandete Maria Pinheiro; Martins, Alice Costa; Nagao-Dias, Aparecida Tiemi

    2014-01-01

    The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 106  promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 107  L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 106  L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium. PMID:24829921

  18. Topical and Intradermal Efficacy of Photodynamic Therapy with Methylene Blue and Light-Emitting Diode in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis

    PubMed Central

    Sbeghen, Mônica Raquel; Voltarelli, Evandra Maria; Campois, Tácito Graminha; Kimura, Elza; Aristides, Sandra Mara Alessi; Hernandes, Luzmarina; Caetano, Wilker; Hioka, Noboru; Lonardoni, Maria Valdrinez Campana; Silveira, Thaís Gomes Verzignassi

    2015-01-01

    Introduction: The topical and intradermal photodynamic therapy (PDT) effect of methylene blue (MB) using light-emitting diode (LED) as light source (MB/LED-PDT) in the treatment of lesions of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis in hamsters were investigated. Methods: Hamsters were infected in the footpad with 4×107 promastigotes of L. braziliensis and divided in 4 groups: Control group was not treated, AmB group was treated with amphotericin B, MB-Id group received intradermal MB at the edge of the lesion and MB-Tp group received MB topic. After treatment with MB, the animals were illuminated using red LEDs at the 655 nm wavelength for 1 hour. The MB/LED-PDT was carried out three times a week for 12 weeks. Results: Animals of MB-Tp group presented lesion healing with significant diminution in extent of the lesion, and reduced parasite burden compared to control group; however, no significant difference was seen compared to the AmB group. MB-Tp group also showed reconstitution of the epithelium, the formation of collagen fibers, organization in the epidermis, a little disorganization and inflammation in the dermis. MB-Id was ineffective in all parameters evaluated, and it was comparable to the control group results. Conclusion: These data show that PDT with the use of MB-Tp and LED may be an alternative for the treatment of ACL. However, additional studies are being conducted to assess the potential of MB/LED-PDT, alone or in combination with conventional therapy, for the treatment of ACL. PMID:26464777

  19. Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.

    PubMed

    Kima, Peter E; Dunn, Waltraud

    2005-04-01

    We have developed a simple scheme for the isolation of parasitophorous vacuoles (PVs) that harbor Leishmania parasites. This scheme exploits the observation that PVs display endoplasmic reticulum molecules, including the transmembrane protein calnexin. The presence of calnexin at the surface of the PVs distinguishes them from late endosomal vesicles of comparable density. As a result, PVs can be isolated by calnexin affinity selection from an enriched PV fraction obtained by sucrose density fractionation.

  20. Low Resolution Structural Studies Indicate that the Activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis Has an Elongated Shape Which Allows Its Interaction with Both N- and M-Domains of Hsp90

    PubMed Central

    Seraphim, Thiago V.; Alves, Marina M.; Silva, Indjara M.; Gomes, Francisco E. R.; Silva, Kelly P.; Murta, Silvane M. F.; Barbosa, Leandro R. S.; Borges, Júlio C.

    2013-01-01

    The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins’ interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90. PMID:23826147

  1. Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90.

    PubMed

    Seraphim, Thiago V; Alves, Marina M; Silva, Indjara M; Gomes, Francisco E R; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2013-01-01

    The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins' interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90. PMID:23826147

  2. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed Central

    Wirth, D F; Pratt, D M

    1982-01-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. Images PMID:6960359

  3. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed

    Wirth, D F; Pratt, D M

    1982-11-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. PMID:6960359

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

    PubMed

    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.

  5. First Isolation of Leishmania from Northern Thailand: Case Report, Identification as Leishmania martiniquensis and Phylogenetic Position within the Leishmania enriettii Complex

    PubMed Central

    Pothirat, Thatawan; Tantiworawit, Adisak; Chaiwarith, Romanee; Jariyapan, Narissara; Wannasan, Anchalee; Siriyasatien, Padet; Supparatpinyo, Khuanchai; Bates, Michelle D.; Kwakye-Nuako, Godwin; Bates, Paul A.

    2014-01-01

    Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as “L. siamensis” also appear to be L. martiniquensis. PMID:25474647

  6. 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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    López-Céspedes, A.; Longoni, S. S.; Sauri-Arceo, C. H.; Sánchez-Moreno, M.; Rodríguez-Vivas, R. I.; Escobedo-Ortegón, F. J.; Barrera-Pérez, M. A.; Bolio-González, M. E.; Marín, C.

    2012-01-01

    Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico). A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high. PMID:22927792

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

    PubMed

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

    2006-01-01

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

  10. Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

    PubMed Central

    Mahdy, Mohammed A. K.; Al-Mekhlafi, Hesham M.; Al-Mekhlafi, Abdulsalam M.; Lim, Yvonne A. L.; Bin Shuaib, Naemah O. M.; Azazy, Ahmed A.; Mahmud, Rohela

    2010-01-01

    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. PMID:20862227

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

    PubMed Central

    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

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

    PubMed

    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; Arruda, Carla Cardozo Pinto de

    2014-12-01

    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.

  13. [Leishmania infantum MON-1 isolated from a golden jackal (Canis aureus) in Grande Kabylie (Algeria)].

    PubMed

    Bessad, A; Mouloua, K; Kherrachi, I; Benbetka, S; Benikhlef, R; Mezai, G; Harrat, Z

    2012-02-01

    In the north of Algeria, Leishmania infantum is responsible for two clinical forms of leishmaniasis: visceral and cutaneous leishmaniasis, for which dogs are the only proven reservoir host. In this study, the authors report, for the first time, the isolation of L. infantum from a golden jackal (Canis aureus) trapped in the Illoulen ou Malou region (Grande Kabylie). Two isolates were thus obtained from bone marrow and spleen and were identified by starch gel isoenzyme electrophoresis as L. infantum MON-1, the widespread zymodeme in the north of the country. Leishmania parasites have also been detected by polymerase chain reaction (PCR) in the biopsy of the spleen. The golden jackal, a prevalent wild canid in Northern Africa, could play a predominant role in the sylvatic foci of leishmaniasis and in the dissemination of the parasite in this region. PMID:21874583

  14. [Leishmania infantum MON-1 isolated from a golden jackal (Canis aureus) in Grande Kabylie (Algeria)].

    PubMed

    Bessad, A; Mouloua, K; Kherrachi, I; Benbetka, S; Benikhlef, R; Mezai, G; Harrat, Z

    2012-02-01

    In the north of Algeria, Leishmania infantum is responsible for two clinical forms of leishmaniasis: visceral and cutaneous leishmaniasis, for which dogs are the only proven reservoir host. In this study, the authors report, for the first time, the isolation of L. infantum from a golden jackal (Canis aureus) trapped in the Illoulen ou Malou region (Grande Kabylie). Two isolates were thus obtained from bone marrow and spleen and were identified by starch gel isoenzyme electrophoresis as L. infantum MON-1, the widespread zymodeme in the north of the country. Leishmania parasites have also been detected by polymerase chain reaction (PCR) in the biopsy of the spleen. The golden jackal, a prevalent wild canid in Northern Africa, could play a predominant role in the sylvatic foci of leishmaniasis and in the dissemination of the parasite in this region.

  15. Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania.

    PubMed Central

    Ryan, K A; Garraway, L A; Descoteaux, A; Turco, S J; Beverley, S M

    1993-01-01

    Trypanosomatid parasites of the genus Leishmania cause a spectrum of widespread tropical diseases. In the vertebrate host they reside within the macrophage phagolysosome; however, the mechanisms employed in this remarkable survival strategy are not well understood. Recent advances in the molecular genetics of these parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant lipophosphoglycan, an abundant glycosyl-phosphatidylinositol-anchored polysaccharide. LPG1, the gene product identified by complementation of the R2D2 mutant, appears to be a glycosyltransferase responsible for the addition of galactofuranosyl residues to the nascent lipophosphoglycan chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism. Images Fig. 5 PMID:8378337

  16. Epidemiologic, genetic, and clinical associations among phenotypically distinct populations of Leishmania (Viannia) in Colombia.

    PubMed

    Saravia, N G; Segura, I; Holguin, A F; Santrich, C; Valderrama, L; Ocampo, C

    1998-07-01

    Phenotypic characterization of 511 strains of Leishmania, subgenus Viannia, isolated from Colombian patients was conducted based on electrophoretic polymorphisms of 13 isoenzymes. Ninety-one Colombian strains of L. braziliensis were the most heterogeneous, constituting seven zymodemes while 397 L. panamensis and 22 L. guyanensis strains yielded five and three zymodemes, respectively. Phosphogluconate dehydrogenase, nucleoside hydrolase, and superoxide dismutase were the most polymorphic enzymes in this collection of strains, and together with glucose-6-phosphate dehydrogenase, allowed the discrimination of the three aforementioned species. Hierarchical cluster analysis of the zymodemes using Jaccard's coefficient of similarities revealed two clusters, one constituted by L. braziliensis zymodemes, and another by three subgroups consisting of zymodemes of L. panamensis closely related to the species reference strain, another consisting of L. guyanensis zymodemes, and a third group distinguished by new electromorphs of proline iminopeptidase and aspartate aminotransferase that reacted with the L. panamensis-specific monoclonal antibody B-11. Multiple zymodemes of L. panamensis and L. guyanensis were found to be sympatrically transmitted in foci along the Pacific coast. Leishmania braziliensis variants were ubiquitous throughout the territory of Colombia; L. panamensis was prevalent in the western region and L. guyanensis was prevalent in the Orinoco and Amazon river basins in the eastern half of the country. Distinct zymodemes of L. panamensis predominated in the northern and southern regions of the Pacific coast. Nine zymodemes of all three species were isolated from mucosal lesions. Zymodeme 1.1 of L. braziliensis had the highest frequency of mucosal involvement (10% of the cases), and disease caused by this zymodeme had the longest mean time of evolution (31 months; P = 0.002). In addition to being useful in describing epidemiologic relationships, the intraspecific

  17. Heterogeneity of Wild Leishmania major Isolates in Experimental Murine Pathogenicity and Specific Immune Response

    PubMed Central

    Kébaïer, C.; Louzir, H.; Chenik, M.; Salah, A. Ben; Dellagi, K.

    2001-01-01

    Virulence variability was investigated by analyzing the experimental pathogenicity of 19 Leishmania major strains in susceptible BALB/c mice. Twelve strains were isolated from Tunisian patients with zoonotic cutaneous leishmaniasis; seven strains were isolated in Syria (n = 1), Saudi Arabia (n = 2), Jordan (n = 2), or Israel (n = 2). BALB/c mice were injected in the hind footpad with 2 × 106 amastigotes of the various isolates, and lesion progression was recorded weekly for 9 weeks. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) production of lymph node mononuclear cells activated in vitro with parasite antigens were evaluated 5 weeks after infection. We show that disease progression induced by different L. major isolates was largely heterogeneous although reproducible results were obtained when using the same isolate. Interestingly, isolates from the Middle East induced a more severe disease than did the majority of Tunisian isolates. Strains with the highest virulence tend to generate more IL-4 and less IFN-γ in vitro at week 5 postinfection as well as higher levels of early IL-4 mRNA in the lymph node draining the inoculation site at 16 h postinfection. These results suggest that L. major isolates from the field may differ in virulence, which influences the course of the disease induced in mice and the type of immune response elicited by the infected host. PMID:11447167

  18. Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

    PubMed

    Ghatee, Mohammad Amin; Sharifi, Iraj; Kuhls, Katrin; Kanannejad, Zahra; Harandi, Majid Fasihi; de Almeida, Marcos E; Hatam, Gholamreza; Mirhendi, Hossein

    2014-09-01

    Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica. PMID:24932536

  19. Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

    PubMed

    Miranda, Aracelis; Samudio, Franklyn; González, Kadir; Saldaña, Azael; Brandão, Adeilton; Calzada, Jose E

    2016-08-01

    A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

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

    PubMed

    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).

  1. The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite

    PubMed Central

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

    2014-01-01

    Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana complex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development. PMID:25336895

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

    PubMed

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

    2014-01-01

    Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the "Mexicana complex", reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development. PMID:25336895

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

    PubMed

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

    2013-02-27

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

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Pedral-Sampaio, Geraldo; Alves, Jessé S; Schriefer, Albert; Magalhães, Andréa; Meyer, Roberto; Glesby, Marshall J; Carvalho, Edgar M; Carvalho, Lucas P

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  10. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis

    PubMed Central

    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

  11. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis.

    PubMed

    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. PMID:27410039

  12. Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

    PubMed

    Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

    2006-08-01

    Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

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

    PubMed

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

    2016-03-01

    Chemical investigation of the stem of Thalictrum foliolosum resulted in the isolation of two new bisbenzylisoquinoline alkaloids (1 and 2) along with known protoberberine group of isoquinoline alkaloids thalifendine (3) and berberine (4). The structures of the new compounds were established by detailed 2D NMR spectral analysis with their configurations determined from their optical rotation values and confirmed using circular dichroism. Inhibitory activities of these four compounds against DNA topoisomerase IB of Leishmania donovani were evaluated. Compound 2 exhibited almost complete inhibition of the enzyme activity at 50 μM concentration and it was found to be effective in killing both wild type as well as SAG resistant promastigotes of the parasite. PMID:26625837

  14. Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum.

    PubMed

    Gómez Pérez, Verónica; García-Hernandez, Raquel; Corpas-López, Victoriano; Tomás, Ana M; Martín-Sanchez, Joaquina; Castanys, Santiago; Gamarro, Francisco

    2016-08-01

    Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime(®), 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to Sb(III) for promastigotes and >3-fold to Sb(III) and 3-fold to Sb(V) for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates. PMID:27317865

  15. Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum.

    PubMed

    Gómez Pérez, Verónica; García-Hernandez, Raquel; Corpas-López, Victoriano; Tomás, Ana M; Martín-Sanchez, Joaquina; Castanys, Santiago; Gamarro, Francisco

    2016-08-01

    Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime(®), 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to Sb(III) for promastigotes and >3-fold to Sb(III) and 3-fold to Sb(V) for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.

  16. Glycoconjugates isolated from Trypanosoma cruzi but not from Leishmania species membranes trigger nitric oxide synthesis as well as microbicidal activity in IFN-gamma-primed macrophages.

    PubMed

    Camargo, M M; Andrade, A C; Almeida, I C; Travassos, L R; Gazzinelli, R T

    1997-12-15

    In the present study, we investigated the role of glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) from Trypanosoma cruzi trypomastigotes in triggering the synthesis of nitric oxide as well as the microbicidal activity in murine macrophages. Our results show that GPI-mucins isolated from trypomastigote membranes are potent inducers of nitric oxide synthesis by IFN-gamma-primed macrophages, even at concentrations as low as 10 ng/ml. Our data also indicate the important role of glycosylphosphatidylinositol anchors from GPI-mucins as the second signal responsible for induction of nitric oxide synthesis by macrophages. To further investigate the role of these parasite molecules in inducing parasiticidal function, we cultured macrophages in the presence or absence of trypomastigote GPI-mucins and/or IFN-gamma and then infected these cells with either Leishmania spp. or T. cruzi. IFN-gamma was sufficient to induce microbial activity in macrophages infected with T. cruzi trypomastigotes. In contrast, killing of different species of Leishmania was further enhanced when macrophages exposed to IFN-gamma were also costimulated with trypomastigote-derived GPI-mucins. Our results also indicate that different glycolipids obtained from Leishmania major or Leishmania donovani (i.e., lipophosphoglycans or glycoinositolphospholipids) were unable to potentiate nitric oxide synthesis and/or microbicidal activity displayed by IFN-gamma-primed macrophages.

  17. Investigation of Double-Band Electrophoretic Pattern of ITS-rDNA Region in Iranian Isolates of Leishmania tropica

    PubMed Central

    Ghatee, MA; Sharifi, I; Mirhendi, H; Kanannejad, Z; Hatam, G

    2013-01-01

    Background Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs. Methods Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products. Results Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis. Conclusion Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles. PMID:23914240

  18. Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil.

    PubMed

    Rocha, Leonardo S; Falqueto, Aloísio; dos Santos, Claudiney B; Ferreira, Adelson L; da Graça, Grazielle C; Grimaldi, Gabriel; Cupolillo, Elisa

    2010-07-01

    In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors.

  19. Molecular diagnosis of Leishmania mexicana in a cutaneous leishmaniasis case in Sinaloa, Mexico.

    PubMed

    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.

  20. Inter- and Intraspecific Variations of Leishmania Strains Isolated from Patients with Cutaneous and Visceral Leishmaniases in Fars Province, South of Iran

    PubMed Central

    Sarkari, Bahador; Ahmadpour, Niloofar Bavarsad; Motazedian, Mohammad Hossein; Mirjalali, Hamed; Akhoundi, Mohammad; Mohebali, Mehdi; Hajjaran, Homa

    2016-01-01

    Background: Cutaneous and visceral leishmaniases are present in Fars Province in the south of Iran. The current study aimed to evaluate the inter- and intragenic diversities of Leishmania species isolated from patients with leishmaniasis in Fars Province, using PCR-based analyses and DNA sequencing of the N-acetylglucosamine-1-phosphate transferase (nagt) gene. Methods: Clinical samples were taken from the skin lesions of 120 individuals with clinical suspicion of cutaneous leishmaniasis (CL) referred to the major health centers of Shiraz. Along with microscopic examination, a part of each sample was used for in vitro cultivation. DNA was extracted from the cultured parasites and the nagt gene was PCR-amplified. For RFLP analysis, the PCR product of the nagt gene was digested with the Acc1 restriction enzyme. Moreover, the PCR products of 23 isolates were sequenced and analyzed, using MEGA5. Results: From the 120 patients with clinical suspicion of CL, 110 (91.7%) cases were found to be positive by direct microscopy while 77 (64.1%) of the cultures were positive. Digestion of the PCR product with the Acc1 restriction enzyme detected L. major in 57 out of the 77 (74.1%) and L. tropica, in 20 out of the 77 (25.9%) cases with CL. Phylogenetic analysis grouped the Leishmania isolates into 3 main clades, representing L. major, L. infantum, and L. tropica, encompassing 2, 2, and 2 haplotypes, respectively. Within the clades, the L. tropica intraspecies divergence was more pronounced in L. major. Conclusion: The findings of this study demonstrated that the causative agent of CL in Fars Province was mainly L. majorz and that there was considerable heterogeneity between the Leishmania species and also within the L. major isolates. PMID:27217605

  1. Detection of Leishmania in Unaffected Mucosal Tissues of Patients with Cutaneous Leishmaniasis Caused by Leishmania (Viannia) Species

    PubMed Central

    Figueroa, Roger Adrian; Lozano, Leyder Elena; Romero, Ibeth Cristina; Cardona, Maria Teresa; Prager, Martin; Pacheco, Robinson; Diaz, Yira Rosalba; Tellez, Jair Alexander; Saravia, Nancy Gore

    2016-01-01

    Background Leishmania (Viannia) species are the principal cause of mucosal leishmaniasis. The natural history and pathogenesis of mucosal disease are enigmatic. Parasitological evaluation of mucosal tissues has been constrained by the invasiveness of conventional sampling methods. Methods We evaluated the presence ofLeishmania in the mucosa of 26 patients with cutaneous leishmaniasis and 2 patients with mucocutaneous leishmaniasis. Swab samples of the nasal mucosa, tonsils, and conjunctiva were analyzed using polymerase chain reaction with LV-B1 primers and Southern blot hybridization. Results Two patients with mucocutaneous leishmaniasis and 21 (81%) of 26 patients with cutaneous leishmaniasis had Leishmania kinetoplast minicircle DNA (kDNA) in mucosal tissues. kDNA was amplified from swab samples of nasal mucosa from 14 (58%) of 24 patients, tonsils from 13 (46%) of 28 patients, and conjunctiva from 6 (25%) of 24 patients. kDNA was detected in the mucosa of patients with cutaneous disease caused by Leishmania panamensis, Leishmania guyanensis, and Leishmania braziliensis. Conclusion The asymptomatic presence of parasites in mucosal tissues may be common in patients with Leishmania (Viannia) infection. PMID:19569974

  2. First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran

    PubMed Central

    Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat–Aldin; Mohebali, Mehdi

    2013-01-01

    Background Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. Objectives This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Materials and Methods Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents’ ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. Results All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Conclusions Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area. PMID:24616787

  3. Immunostimulatory cellular responses of cured Leishmania-infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovani

    PubMed Central

    Garg, R; Gupta, S K; Tripathi, P; Naik, S; Sundar, S; Dube, A

    2005-01-01

    Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non-membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (n = 9), endemic controls (n = 5) of visceral leishmaniasis (VL) and treated hamsters (n = 10). IMPs and NSPs induced significant proliferative responses (SI 6·3 ± 4·1 and 5·6 ± 2·3, respectively; P < 0·01) and IFN-γ production (356·3 ± 213·4 and 294·29 ± 107·6 pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2 ± 4·7 & 6·4 ± 4·1, respectively; P < 0·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A-1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL. PMID:15762886

  4. Sandflies (Diptera: Psychodidae) associated with opossum nests at urban sites in southeastern Brazil: a risk factor for urban and periurban zoonotic Leishmania transmission?

    PubMed

    Cutolo, Andre Antonio; Teodoro, Anna Karollina Menezes; Ovallos, Fredy Galvis; Allegretti, Silmara Marques; Galati, Eunice Aparecida Bianchi

    2014-06-01

    Sandflies associated with opossum nests are reported for the first time in the yards of residences located in the urban area of the municipality of Monte Mor, situated in the metropolitan region of Campinas, state of São Paulo, Brazil. Eleven specimens of Evandromyia cortelezzii and one of Evandromyia lenti were captured in two Didelphis albiventris nests. Ev. cortelezzii is considered a secondary vector species for the transmission of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum in the Neotropics. This association may contribute to the introduction, establishment and maintenance of urban and periurban zoonotic transmission outbreaks of Leishmania and should therefore be investigated further.

  5. Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

    PubMed

    Löfgren, S E; Miletti, L C; Steindel, M; Bachère, E; Barracco, M A

    2008-02-01

    Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent. PMID:17888907

  6. Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase.

    PubMed

    Waki, Kayoko; Dutta, Sujoy; Ray, Debalina; Kolli, Bala Krishna; Akman, Leyla; Kawazu, Shin-Ichiro; Lin, Chung-Ping; Chang, Kwang-Poo

    2007-02-01

    A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.

  7. Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania-Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum N-Acetylglucosamine-1-Phosphate Transferase▿ †

    PubMed Central

    Waki, Kayoko; Dutta, Sujoy; Ray, Debalina; Kolli, Bala Krishna; Akman, Leyla; Kawazu, Shin-Ichiro; Lin, Chung-Ping; Chang, Kwang-Poo

    2007-01-01

    A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes

  8. Miltefosine and Antimonial Drug Susceptibility of Leishmania Viannia Species and Populations in Regions of High Transmission in Colombia

    PubMed Central

    Fernández, Olga Lucía; Diaz-Toro, Yira; Muvdi, Sandra; Rodríguez, Isabel; Gomez, María Adelaida; Saravia, Nancy Gore

    2014-01-01

    Background Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance. Methodology/Principal Findings In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (SbV); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to SbV was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980–1989 and 2000–2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to SbV. Resistance to HePC and SbV occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to SbV. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to SbV were discerned among L. V. panamensis strains isolated during 1980–1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2. Conclusions/Significance Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution

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

    PubMed

    Mukherjee, Debarati; Singh, Chingakham Brajakishor; Dey, Somaditya; Mandal, Supratim; Ghosh, Joydip; Mallick, Suvadip; Hussain, Aabid; Swapana, Ningombam; Ross, Samir Anis; Pal, Chiranjib

    2016-01-01

    In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmania infection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovani infection and provided the basis for future research on the application of transitional medicinal plants. PMID:26643969

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

    PubMed

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

    2015-02-01

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

  11. Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

    PubMed Central

    2013-01-01

    Background Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis. Methods In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis. Results Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection. Conclusion Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis. PMID

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

    PubMed Central

    2011-01-01

    Background Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Methods Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. Findings The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. Conclusions We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species. PMID:22082050

  13. The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples.

    PubMed

    Mosleh, Ibrahim M; Shönian, Gabriele; Geith, Eid; Al-Jawabreh, Amer; Natsheh, Lina

    2015-01-01

    The identity of the causative species of cutaneous leishmaniasis (CL) in the endemic Jordanian Mid Jordan Valley (JMidJV) was investigated using the polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS-1) followed by the restriction fragment length polymorphism (RFLP). The geographical distribution of CL and the usefulness of ITS1 PCR in diagnosis of suspected CL in the study area were also addressed. Over the period from 2004 to 2009, 56 clinical isolates of Leishmania promastigotes and 185 lesion scrapings spotted on filter papers were obtained from suspected CL patients living in the JMidJV, which is divided into northern and southern districts. The majority (67.1%) of patients occurred in the populated eastern part of the southern district. Of the 185 suspected CL patients, 173 (93.5%) were confirmed positive using PCR. Leishmanial DNA was detected in 27 (90%) of 30 patients having clinically atypical lesions of CL and in 60 (92%) of 65 smear- and culture-negative cases having typical lesions of CL. The parasites in all of the 56 isolates and the 173 PCR-positive scrapings were classified as Leishmania major. In conclusion, PCR is useful in diagnosis of CL especially when smear and culture are negative. It is also recommended as a differential diagnostic tool of atypical lesions when CL is endemic. The identification of L. major as the causative species in such a considerable number of CL cases, representative of all mini foci of CL in the study area, shows that the JMidJV is a classic focus of L. major.

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

    PubMed

    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.

  15. Laboratory diagnosis of Leishmania.

    PubMed

    Palma, G; Gutierrez, Y

    1991-12-01

    Leishmaniasis should be considered in the differential diagnosis of individuals living in or with a history of having traveled to known endemic areas and who present with signs and symptoms of visceral infection or with cutaneous or mucosal lesions. Because leishmaniae are capable of producing a wide spectrum of disease in humans, the clinical manifestations overlap with many other diseases. Definitive diagnosis of Leishmania infection in the laboratory requires demonstration of the parasite in smears, in biopsies, or by isolation of the organism in culture media or in experimental animals. Many other methods for demonstration of parasites (histochemical and immunohistochemical) or for detecting the presence of antibodies against leishmaniae (serologic) have been described. Many advances have been made in these areas, but the methodology and the technology involved in immunohistochemistry and serology remain outside the reach of the standard clinical diagnostic laboratory, which both in developed and less developed countries still relies on demonstration of the parasites in smears stained with Giemsa stain and on biopsy specimens processed and stained with hematoxylin and eosin stain. The newer serologic techniques, such as ELISA with several variations, IFAT, and others, are largely research tools with the greatest use in seroepidemiologic surveys.

  16. Cross-resistance of Leishmania infantum isolates to nitric oxide from patients refractory to antimony treatment, and greater tolerance to antileishmanial responses by macrophages.

    PubMed

    de Moura, Tatiana R; Santos, Micheli Luize Barbosa; Braz, Juciene M; Santos, Luis Felipe V C; Aragão, Matheus T; de Oliveira, Fabricia A; Santos, Priscila L; da Silva, Ângela Maria; de Jesus, Amélia Ribeiro; de Almeida, Roque P

    2016-02-01

    Visceral leishmaniasis is a life-threatening disease characterized by intense parasitism of the spleen, liver, and bone marrow. Antimonials have served as front-line antileishmanial therapeutics for decades, but the increasing failure rates under antimonial treatment have challenged the continued use of these drugs. Pentavalent antimonials are known to reinforce the killing mechanisms of macrophages, although the associated mechanism remains unclear. Here, for the first time, we determined whether Leishmania infantum strains isolated from patients refractory to antimony treatment (relapse cases) were cross-resistant to antimonials, liposomal amphotericin B, and/or nitric oxide, and also whether these strains modulate macrophage infection. We selected four clinical isolates from relapse cases and two clinical isolates from antimony-responsive patients (control group) for the present study. The L. infantum promastigotes from all four relapse cases were resistant to trivalent antimonial treatment and nitric oxide, while only one isolate was resistant to liposomal amphotericin B. We evaluated whether the resistant strains from relapse cases showed enhanced infectivity and amastigote survival in macrophages, or macrophage-killing mechanisms in macrophages activated by lipopolysaccharide plus interferon gamma. Infection indexes calculated using macrophages infected with isolates from relapse were higher than those observed with control strains that were stimulated independently. Macrophage infection was higher with L. infantum isolates from relapse cases and correlated with enhanced interleukin 1-β production but showed similar nitrite production. Our results demonstrate that L. infantum field isolates from relapse cases were resistant to antimonials and nitric oxide and that these parasites stimulated inflammatory cytokines and were resistant to macrophage-killing mechanisms, factors that may contribute to disease severity.

  17. Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR-RFLP.

    PubMed

    Koarashi, Yu; Cáceres, Abraham G; Saca, Florencia Margarita Zúniga; Flores, Elsa Elvira Palacios; Trujillo, Adela Celis; Alvares, José Luis Abanto; Yoshimatsu, Kumiko; Arikawa, Jiro; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-06-01

    A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis.

  18. Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR-RFLP.

    PubMed

    Koarashi, Yu; Cáceres, Abraham G; Saca, Florencia Margarita Zúniga; Flores, Elsa Elvira Palacios; Trujillo, Adela Celis; Alvares, José Luis Abanto; Yoshimatsu, Kumiko; Arikawa, Jiro; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-06-01

    A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis. PMID:26943992

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

    PubMed

    Diaz, E; Zacarias, A K; Pérez, S; Vanegas, O; Köhidai, L; Padrón-Nieves, M; Ponte-Sucre, A

    2015-11-01

    In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

  20. Genetic homogeneity within Leishmania (L.) infantum isolated from human and dogs: the relationship with the sandfly fauna distribution in endemic areas of Nueva Esparta State, Venezuela.

    PubMed

    Rodriguez, N M; De Guglielmo, Z; Barrios, M A; Barrios, R M; Zerpa, O; Feliciangeli, M D

    2005-06-01

    Leishmania infantum has been described as a highly polymorphic group of parasites, responsible for visceral leishmaniasis and cutaneous leishmaniasis. In this paper we report the life-cycle of L. (L.) infantum in an endemic area of visceral leishmaniasis in Venezuela, by using molecular diagnosis and characterization of parasites isolated from dogs, humans with visceral leishmaniasis and sand flies. The molecular characterization was carried out by use of kDNA restriction analysis, dot-blot hybridization with species-specific probes and RFLP of the PCR products. The results demonstrated that L. (L.) infantum is the parasite responsible for VL in the island. The parasites were revealed to be genetically homogeneous with no intra-specific differences between isolates from different individuals. The highest homology of the isolates was with L. (L.) infantum from the Old World rather than with L. (L.) chagasi from the New World. Additionally, we report the geographical distribution of Lutzomyia longipalpis, and the relationship with the transmission of L. (L.) infantum in the studied area. PMID:15977897

  1. Natural infection of bats with Leishmania in Ethiopia.

    PubMed

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

    2015-10-01

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

  2. Molecular detection of Leishmania spp. isolated from cutaneous lesions of patients referred to Herat regional hospital, Afghanistan.

    PubMed

    Mosawi, S H; Dalimi, A

    2016-03-15

    Cutaneous leishmaniasis is one of the main public health problems in Afghanistan, particularly in Herat. To identify Leishmania spp., molecular techniques were applied to samples from 64 cutaneous leishmaniasis patients referred to Herat regional hospital during 2013. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the ribosomal RNA gene internal transcribed spacer-1 (ITS1) was used. Most of the patients demonstrated dry type single lesions on the head. The results of direct microscopy detection using Giemsastained skin scrapings were compared with that of ITS PCR-RFLP for the diagnosis of cutaneous leishmaniasis. Light microscopy examination showed 37/64 positive cases (58%). PCR revealed 50 positive cases (78%), from which ITS PCR-RFLP identified 48 cases (96%) as L. tropica and 2 cases (4%) as L. major. Cutaneous leishmaniasis in Herat appears to be endemic and of the clinically dry type, caused mainly by L. tropica and occasionally by L. major.

  3. Enhancement of Experimental Cutaneous Leishmaniasis by Leishmania Molecules Is Dependent on Interleukin-4, Serine Protease/Esterase Activity, and Parasite and Host Genetic Backgrounds ▿

    PubMed Central

    Silva, Virgínia M. G.; Larangeira, Daniela F.; Oliveira, Pablo R. S.; Sampaio, Romina B.; Suzart, Paula; Nihei, Jorge S.; Teixeira, Márcia C. A.; Mengel, José O.; dos-Santos, Washington L. C.; Pontes-de-Carvalho, Lain

    2011-01-01

    Most inbred strains of mice, like the BALB/c strain, are susceptible to Leishmania amazonensis infections and resistant to Leishmania braziliensis infections. This parasite-related difference could result from the activity of an L. amazonensis-specific virulence factor. In agreement with this hypothesis, it is shown here that the intravenous injection of BALB/c mice with L. amazonensis amastigote extract (LaE) but not the L. braziliensis extract confers susceptibility to L. braziliensis infection. This effect was associated with high circulating levels of IgG1 anti-L. amazonensis antibodies and with an increase in interleukin-4 (IL-4) production and a decrease in gamma interferon production by draining lymph node cells. Moreover, the effect was absent in IL-4-knockout mice. The biological activity in the LaE was not mediated by amphiphilic molecules and was inhibited by pretreatment of the extract with irreversible serine protease inhibitors. These findings indicate that the LaE contains a virulence-related factor that (i) enhances the Leishmania infection by promoting Th2-type immune responses, (ii) is not one of the immunomodulatory Leishmania molecules described so far, and (iii) is either a serine protease or has an effect that depends on that protease activity. In addition to being Leishmania species specific, the infection-enhancing activity was also shown to depend on the host genetic makeup, as LaE injections did not affect the susceptibility of C57BL/6 mice to L. braziliensis infection. The identification of Leishmania molecules with infection-enhancing activity could be important for the development of a vaccine, since the up- or downmodulation of the immune response against a virulence factor could well contribute to controlling the infection. PMID:21173308

  4. Genetic diversity of Leishmania tropica strains isolated from clinical forms of cutaneous leishmaniasis in rural districts of Herat province, Western Afghanistan, based on ITS1-rDNA.

    PubMed

    Fakhar, Mahdi; Pazoki Ghohe, Hossein; Rasooli, Sayed Abobakar; Karamian, Mehdi; Mohib, Abdul Satar; Ziaei Hezarjaribi, Hajar; Pagheh, Abdol Sattar; Ghatee, Mohammad Amin

    2016-07-01

    Despite the high incidence of cutaneous leishmaniasis (CL) in Afghanistan, there is a little information concerning epidemiological status of the disease and phylogenetic relationship and population structure of causative agents. This study was conducted to determine the prevalence and distribution of CL cases and investigate the Leishmania tropica population structure in rural districts of Heart province in the West of Afghanistan in comparison to neighboring foci. Overall, 4189 clinically suspected CL cases from 177 villages (including 12 districts) in Herat province were enrolled in the referral laboratory of WHO sub-office in Herat city from January 2012 to December 2013. 3861 cases were confirmed as CL by microscopic examination of Giemsa-stained slides. ITS1 PCR-RFLP analysis showed dominance of L. tropica (more than 98%) among 127 randomly chosen samples. Analysis of the ITS1 sequences revealed 4 sequence types among the 21 L. tropica isolates. Comparison of sequence types from Herat rural districts with the representatives of L. tropica from Iran, India, and Herat city showed two main population groups (cluster A and B). All isolates from Herat province, India and Southeast, East, and Central Iran were found exclusively in cluster A. The close proximity of West Afghanistan focus and Birjand county as the capital of Southern Khorasan province in East Iran can explain relatively equal to the genetic composition of L. tropica in these two neighboring regions. In addition, two populations were found among L. tropica isolates from Herat rural districts. Main population showed more similarity to some isolates from Birjand county in East Iran while minor population probably originated from the Southeast and East Iranian L. tropica. Recent study provided valuable information concerning the population structure of L. tropica and epidemiology of ACL in the West of Afghanistan, which could be the basis for molecular epidemiology studies in other regions of Afghanistan.

  5. Molecular characterization of the MRPA transporter and antimony uptake in four New World Leishmania spp. susceptible and resistant to antimony☆

    PubMed Central

    Moreira, Douglas S.; Monte Neto, Rubens L.; Andrade, Juvana M.; Santi, Ana Maria M.; Reis, Priscila G.; Frézard, Frédéric; Murta, Silvane M.F.

    2013-01-01

    ATP-binding cassette (ABC) transporters have been associated with drug resistance in various diseases. The MRPA gene, a transporter of ABCC subfamily, is involved in the resistance by sequestering metal-thiol conjugates in intracellular vesicles of Leishmania parasite. In this study, we performed the molecular characterization of the MRPA transporter, analysis of P-glycoprotein (Pgp) and aquaglyceroporin-1 (AQP1) expression, and determination of antimony level in antimony-susceptible and -resistant lines of L. (V.) guyanensis, L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum. PFGE analysis revealed an association of chromosomal amplification of MRPA gene with the drug resistance phenotype in all SbIII-resistant Leishmania lines analyzed. Levels of mRNA from MRPA gene determined by real-time quantitative RT-PCR showed an increased expression of two fold in SbIII-resistant lines of Leishmania guyanensis, Leishmania amazonensis and Leishmania braziliensis. Western blot analysis revealed that Pgp is increased in the SbIII-resistant L. guyanensis and L. amazonensis lines. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed a reduction in the accumulation of this element in SbIII-resistant L. guyanensis, L. amazonensis and L. braziliensis lines when compared to their susceptible counterparts. Interestingly, a down-regulation of AQP1 protein was observed in the SbIII-resistant L. guyanensis and L. amazonensis lines, contributing for decreasing of SbIII entry in these lines. In addition, efflux experiments revealed that the rates of SbIII efflux are higher in the SbIII-resistant lines of L. guyanensis and L. braziliensis, that may explain also the low SbIII concentration within of these parasites. The BSO, an inhibitor of γ-glutamylcysteine synthetase enzyme, reversed the SbIII-resistance phenotype of L. braziliensis and caused an increasing in the Sb intracellular level in the LbSbR line. Our data

  6. Antileishmanial activity and evaluation of the mechanism of action of strychnobiflavone flavonoid isolated from Strychnos pseudoquina against Leishmania infantum.

    PubMed

    Lage, Paula S; Chávez-Fumagalli, Miguel A; Mesquita, Juliana T; Mata, Laís M; Fernandes, Simone O A; Cardoso, Valbert N; Soto, Manuel; Tavares, Carlos A P; Leite, João P V; Tempone, Andre G; Coelho, Eduardo A F

    2015-12-01

    The present study aimed to investigate the in vitro antileishmanial activity of strychnobiflavone flavonoid against Leishmania infantum, as well as its mechanism of action, and evaluate the ex vivo biodistribution profile of the flavonoid in naive BALB/c mice. The antileishmanial activity (IC50 value) of strychnobiflavone against stationary promastigote and amastigote-like stages of the parasites was of 5.4 and 18.9 μM, respectively; with a 50% cytotoxic concentration (CC50) value of 125.0 μM on murine macrophages, resulting in selectivity index (SI) of 23.2 and 6.6, respectively. Amphotericin B, used as a positive control, presented SI values of 7.6 and 3.3 for promastigote and amastigote-like stages of L. infantum, respectively. The strychnobiflavone was also effective in reducing in significant levels the percentage of infected macrophages, as well as the number of amastigotes per macrophage, after the treatment of infected macrophages using the flavonoid. By using different fluorescent probes, we investigated the bioenergetics metabolism of L. infantum promastigotes and demonstrated that the flavonoid caused the depolarization of the mitochondrial membrane potential, without affecting the production of reactive oxygen species. In addition, using SYTOX(®) green as a fluorescent probe, the strychnobiflavone demonstrated no interference in plasma membrane permeability. For the ex vivo biodistribution assays, the flavonoid was labeled with technetium-(99m) and studied in a mouse model by intraperitoneal route. After a single dose administration, the scintigraphic images demonstrated a highest uptake by the liver and spleen of the animals within 60 min, resulting in low concentrations after 24 h. The present study therefore demonstrated, for the first time, the antileishmanial activity of the strychnobiflavone against L. infantum, and suggests that the mitochondria of the parasites may be the possible target organelle. The preferential distribution of this

  7. New hemiketal steroid from the introduced soft coral Chromonephthea braziliensis is a chemical defense against predatory fishes.

    PubMed

    Fleury, Beatriz G; Lages, Bruno G; Barbosa, Jussara P; Kaiser, Carlos R; Pinto, Angelo C

    2008-08-01

    Recent studies show that chemical defenses in the exotic soft coral Chromonephthea braziliensis Ofwegen (Nephtheidae, Alcyonacea) can be one of the reasons for the success of this introduced species. We report for the first time the detailed composition of the monohydroxylated sterol fraction and a new hemiketal steroid, 23-keto-cladiellin-A, isolated from the unpalatable hexane extract from C. braziliensis. Bioassay-guided fractionation of this extract revealed that this hemiketal steroid exhibits potent feeding deterrent properties against a natural assemblage of fishes at the natural concentration. The major sterol fraction, containing the monohydroxylated sterols, was inactive in the bioassay. The results suggest that this active molecule may be driving the observed success of the invasion of this soft coral along the Brazilian Atlantic coast.

  8. Distinct Leishmania Species Infecting Wild Caviomorph Rodents (Rodentia: Hystricognathi) from Brazil

    PubMed Central

    Cássia-Pires, Renata; Boité, Mariana C.; D'Andrea, Paulo S.; Herrera, Heitor M.; Cupolillo, Elisa; Jansen, Ana Maria; Roque, André Luiz R.

    2014-01-01

    Background Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. Methodology We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. Principal findings In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. Conclusions/Significance The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the

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

    PubMed

    Ureña, F

    1986-01-01

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

  10. Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients.

    PubMed

    Cantanhêde, Lilian Motta; da Silva Júnior, Cipriano Ferreira; Ito, Marcos Massayuki; Felipin, Kátia Paula; Nicolete, Roberto; Salcedo, Juan Miguel Villalobos; Porrozzi, Renato; Cupolillo, Elisa; Ferreira, Ricardo de Godoi Mattos

    2015-01-01

    Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71

  11. Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients.

    PubMed

    Cantanhêde, Lilian Motta; da Silva Júnior, Cipriano Ferreira; Ito, Marcos Massayuki; Felipin, Kátia Paula; Nicolete, Roberto; Salcedo, Juan Miguel Villalobos; Porrozzi, Renato; Cupolillo, Elisa; Ferreira, Ricardo de Godoi Mattos

    2015-01-01

    Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71

  12. Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients

    PubMed Central

    Cantanhêde, Lilian Motta; da Silva Júnior, Cipriano Ferreira; Ito, Marcos Massayuki; Felipin, Kátia Paula; Nicolete, Roberto; Salcedo, Juan Miguel Villalobos; Porrozzi, Renato; Cupolillo, Elisa; Ferreira, Ricardo de Godoi Mattos

    2015-01-01

    Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71

  13. Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

    PubMed Central

    Das, Sanchita; Shah, Priyanka; Tandon, Rati; Yadav, Narendra Kumar; Sahasrabuddhe, Amogh A.; Sundar, Shyam; Siddiqi, Mohammad Imran; Dube, Anuradha

    2015-01-01

    Background Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche

  14. Leishmania (Viannia) Infection in the Domestic Dog in Chaparral, Colombia

    PubMed Central

    Santaella, Julián; Ocampo, Clara B.; Saravia, Nancy G.; Méndez, Fabián; Góngora, Rafael; Gomez, Maria Adelaida; Munstermann, Leonard E.; Quinnell, Rupert J.

    2011-01-01

    Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction–Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction–Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited. PMID:21540374

  15. Reduced antimony accumulation in ARM58-overexpressing Leishmania infantum.

    PubMed

    Schäfer, Carola; Tejera Nevado, Paloma; Zander, Dorothea; Clos, Joachim

    2014-01-01

    Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins.

  16. Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia.

    PubMed

    Santaella, Julián; Ocampo, Clara B; Saravia, Nancy G; Méndez, Fabián; Góngora, Rafael; Gomez, Maria Adelaida; Munstermann, Leonard E; Quinnell, Rupert J

    2011-05-01

    Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.

  17. Molecular characterization and functional analysis of pteridine reductase in wild-type and antimony-resistant Leishmania lines.

    PubMed

    de Souza Moreira, Douglas; Ferreira, Rafael Fernandes; Murta, Silvane M F

    2016-01-01

    Pteridine reductase (PTR1) is an NADPH-dependent reductase that participates in the salvage of pteridines, which are essential to maintain growth of Leishmania. In this study, we performed the molecular characterization of ptr1 gene in wild-type (WTS) and SbIII-resistant (SbR) lines from Leishmania guyanensis (Lg), Leishmania amazonensis (La), Leishmania braziliensis (Lb) and Leishmania infantum (Li), evaluating the chromosomal location, mRNA levels of the ptr1 gene and PTR1 protein expression. PFGE results showed that the ptr1 gene is located in a 797 kb chromosomal band in all Leishmania lines analyzed. Interestingly, an additional chromosomal band of 1070 kb was observed only in LbSbR line. Northern blot results showed that the levels of ptr1 mRNA are increased in the LgSbR, LaSbR and LbSbR lines. Western blot assays using the polyclonal anti-LmPTR1 antibody demonstrated that PTR1 protein is more expressed in the LgSbR, LaSbR and LbSbR lines compared to their respective WTS counterparts. Nevertheless, no difference in the level of mRNA and protein was observed between the LiWTS and LiSbR lines. Functional analysis of PTR1 enzyme was performed to determine whether the overexpression of ptr1 gene in the WTS L. braziliensis and L. infantum lines would change the SbIII-resistance phenotype of transfected parasites. Western blot results showed that the expression level of PTR1 protein was increased in the transfected parasites compared to the non-transfected ones. IC50 analysis revealed that the overexpression of ptr1 gene in the WTS L. braziliensis line increased 2-fold the SbIII-resistance phenotype compared to the non-transfected counterpart. Furthermore, the overexpression of ptr1 gene in the WTS L. infantum line did not change the SbIII-resistance phenotype. These results suggest that the PTR1 enzyme may be implicated in the SbIII-resistance phenotype in L. braziliensis line.

  18. An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

    PubMed

    Costa Duarte, Mariana; dos Reis Lage, Letícia Martins; Lage, Daniela Pagliara; Mesquita, Juliana Tonini; Salles, Beatriz Cristina Silveira; Lavorato, Stefânia Neiva; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Alves, Ricardo José; Tavares, Carlos Alberto Pereira; Tempone, André Gustavo; Coelho, Eduardo Antonio Ferraz

    2016-02-15

    The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis.

  19. Leishmania (Leishmania) martiniquensis n. sp. (Kinetoplastida: Trypanosomatidae), description of the parasite responsible for cutaneous leishmaniasis in Martinique Island (French West Indies)

    PubMed Central

    2014-01-01

    The parasite responsible for autochthonous cutaneous leishmaniasis in Martinique island (French West Indies) was first isolated in 1995; its taxonomical position was established only in 2002, but it remained unnamed. In the present paper, the authors name this parasite Leishmania (Leishmania) martiniquensis Desbois, Pratlong & Dedet n. sp. and describe the type strain of this taxon, including its biological characteristics, biochemical and molecular identification, and pathogenicity. This parasite, clearly distinct from all other Euleishmania, and placed at the base of the Leishmania phylogenetic tree, is included in the subgenus Leishmania. PMID:24626346

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

    PubMed

    Monroy-Ostria, Amalia; Sanchez-Tejeda, Gustavo

    2002-04-01

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

  1. Screening Leishmania donovani complex-specific genes required for visceral disease.

    PubMed

    Zhang, Wen-Wei; Matlashewski, Greg

    2015-01-01

    Leishmania protozoan parasites are the causing agent of leishmaniasis. Depending on the infecting species, Leishmania infection can causes a wide variety of diseases such as self-healing cutaneous lesions by L. major and fatal visceral leishmaniasis by L. donovani and L. infantum. Comparison of the visceral disease causing L. infantum genome with cutaneous disease causing L. major and L. braziliensis genomes has identified 25 L. infantum (L. donovani complex) species-specific genes that are absent or pseudogenes in L. major and L. braziliensis. To investigate whether these L. donovani complex species-specific genes are involved in visceral infection, we cloned these genes from L. donovani and introduced them into L. major and then determined whether the transgenic L. major had an increased ability to survive in liver and spleen of BALB/c mice. Several of these L. donovani complex specific genes were found to significantly increase L. major survival in visceral organs in BALB/c mice including the A2 and Ld2834 genes, while down regulation of these genes in L. donovani by either antisense RNA or gene knockout dramatically reduced L. donovani virulence in BALB/c mice. This demonstrated that L. donovani complex species-specific genes play important roles in visceral infection. In this chapter, we describe procedures to screen L. donovani complex specific genes required for visceral infection by cross species transgenic expression, gene deletion targeting and measuring infection levels in mice. PMID:25388124

  2. DETC Induces Leishmania Parasite Killing in Human In Vitro and Murine In Vivo Models: A Promising Therapeutic Alternative in Leishmaniasis

    PubMed Central

    Khouri, Ricardo; Novais, Fernanda; Santana, Gisélia; de Oliveira, Camila Indiani; Vannier dos Santos, Marcos André; Barral, Aldina; Barral-Netto, Manoel; Van Weyenbergh, Johan

    2010-01-01

    Background Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. Methodology/Principal Findings We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p<0.001) and is able to “sterilize” Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p<0.001) and parasite destruction (p<0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p<0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. Conclusions/Significance Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection. PMID:21200432

  3. Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia.

    PubMed

    Beer, María Florencia; Frank, Fernanda Maria; Germán Elso, Orlando; Ernesto Bivona, Augusto; Cerny, Natacha; Giberti, Gustavo; Luis Malchiodi, Emilio; Susana Martino, Virginia; Alonso, María Rosario; Patricia Sülsen, Valeria; Cazorla, Silvia Ines

    2016-10-01

    Context Chagas' disease and leishmaniasis produce significant disability and mortality with great social and economic impact. The genus Stevia (Asteraceae) is a potential source of antiprotozoal compounds. Objective Aerial parts of four Stevia species were screened on Trypanosoma cruzi. Stevia satureiifolia (Lam.) Sch. Bip. var. satureiifolia (Asteraceae) dichloromethane extract was selected for a bioassay-guided fractionation in order to isolate its active compounds. Additionally, the antileishmanial activity and the cytotoxicity of these compounds on mammalian cells were assessed. Materials and methods The dichloromethane extract was fractionated by column chromatography. The isolated compounds were evaluated using concentrations of 0-100 μg/mL on T. cruzi epimastigotes and on Leishmania braziliensis promastigotes for 72 h, on trypomastigotes and amastigotes of T. cruzi for 24 h and 120 h, respectively. The compounds' cytotoxicity (12.5-500 μg/mL) was assessed on Vero cells by the MTT assay. The structure elucidation of each compound was performed by spectroscopic methods and HPLC analysis. Results The dichloromethane extracts of Stevia species showed significant activity on T. cruzi epimastigotes. The flavonoids eupatorin (1.3%), cirsimaritin (1.9%) and 5-desmethylsinensetin (1.5%) were isolated from S. satureiifolia var. satureiifolia extract. Eupatorin and 5-desmethylsinensetin showed IC50 values of 0.2 and 0.4 μg/mL on T. cruzi epimastigotes and 61.8 and 75.1 μg/mL on trypomastigotes, respectively. The flavonoid 5-desmethylsinensetin showed moderate activity against T. cruzi amastigotes (IC50  value = 78.7 μg/mL) and was the most active compound on L. braziliensis promastigotes (IC50  value = 37.0 μg/mL). Neither of the flavonoids showed cytotoxicity on Vero cells, up to a concentration of 500 μg/mL.

  4. Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia.

    PubMed

    Beer, María Florencia; Frank, Fernanda Maria; Germán Elso, Orlando; Ernesto Bivona, Augusto; Cerny, Natacha; Giberti, Gustavo; Luis Malchiodi, Emilio; Susana Martino, Virginia; Alonso, María Rosario; Patricia Sülsen, Valeria; Cazorla, Silvia Ines

    2016-10-01

    Context Chagas' disease and leishmaniasis produce significant disability and mortality with great social and economic impact. The genus Stevia (Asteraceae) is a potential source of antiprotozoal compounds. Objective Aerial parts of four Stevia species were screened on Trypanosoma cruzi. Stevia satureiifolia (Lam.) Sch. Bip. var. satureiifolia (Asteraceae) dichloromethane extract was selected for a bioassay-guided fractionation in order to isolate its active compounds. Additionally, the antileishmanial activity and the cytotoxicity of these compounds on mammalian cells were assessed. Materials and methods The dichloromethane extract was fractionated by column chromatography. The isolated compounds were evaluated using concentrations of 0-100 μg/mL on T. cruzi epimastigotes and on Leishmania braziliensis promastigotes for 72 h, on trypomastigotes and amastigotes of T. cruzi for 24 h and 120 h, respectively. The compounds' cytotoxicity (12.5-500 μg/mL) was assessed on Vero cells by the MTT assay. The structure elucidation of each compound was performed by spectroscopic methods and HPLC analysis. Results The dichloromethane extracts of Stevia species showed significant activity on T. cruzi epimastigotes. The flavonoids eupatorin (1.3%), cirsimaritin (1.9%) and 5-desmethylsinensetin (1.5%) were isolated from S. satureiifolia var. satureiifolia extract. Eupatorin and 5-desmethylsinensetin showed IC50 values of 0.2 and 0.4 μg/mL on T. cruzi epimastigotes and 61.8 and 75.1 μg/mL on trypomastigotes, respectively. The flavonoid 5-desmethylsinensetin showed moderate activity against T. cruzi amastigotes (IC50  value = 78.7 μg/mL) and was the most active compound on L. braziliensis promastigotes (IC50  value = 37.0 μg/mL). Neither of the flavonoids showed cytotoxicity on Vero cells, up to a concentration of 500 μg/mL. PMID:26983579

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

    PubMed Central

    LYRA, Marcelo Rosandiski; PIMENTEL, Maria Inês Fernandes; MADEIRA, Maria de Fátima; ANTONIO, Liliane de Fátima; LYRA, Janine Pontes de Miranda; FAGUNDES, Aline; SCHUBACH, Armando de Oliveira

    2015-01-01

    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. PMID:26603237

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

    PubMed Central

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

    2016-01-01

    Background 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. Methods/Principal Findings 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. Conclusions/Significance 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. PMID:26928050

  7. Comorbidity of Leishmania Major with Cutaneous Sarcoidosis

    PubMed Central

    Moravvej, Hamideh; Vesal, Parvaneh; Abolhasani, Ehsan; Nahidi, Shizar; Mahboudi, Fereidoun

    2014-01-01

    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. PMID:24891680

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

    PubMed

    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

    2016-04-01

    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

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

    PubMed Central

    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

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

    PubMed

    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

    2016-04-01

    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.

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

    PubMed

    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.

  12. Leishmania species: mechanisms of complement activation by five strains of promastigotes.

    PubMed

    Mosser, D M; Burke, S K; Coutavas, E E; Wedgwood, J F; Edelson, P J

    1986-12-01

    The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade

  13. Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples▿

    PubMed Central

    Weirather, Jason L.; Jeronimo, Selma M. B.; Gautam, Shalini; Sundar, Shyam; Kang, Mitchell; Kurtz, Melissa A.; Haque, Rashidul; Schriefer, Albert; Talhari, Sinésio; Carvalho, Edgar M.; Donelson, John E.; Wilson, Mary E.

    2011-01-01

    The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species. PMID:22042830

  14. A Novel Sterol Isolated from a Plant Used by Mayan Traditional Healers Is Effective in Treatment of Visceral Leishmaniasis Caused by Leishmania donovani.

    PubMed

    Gupta, Gaurav; Peine, Kevin J; Abdelhamid, Dalia; Snider, Heidi; Shelton, Andrew B; Rao, Latha; Kotha, Sainath R; Huntsman, Andrew C; Varikuti, Sanjay; Oghumu, Steve; Naman, C Benjamin; Pan, Li; Parinandi, Narasimham L; Papenfuss, Tracy L; Kinghorn, A Douglas; Bachelder, Eric M; Ainslie, Kristy M; Fuchs, James R; Satoskar, Abhay R

    2015-10-01

    Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani, is a global health problem affecting millions of people worldwide. Treatment of VL largely depends on therapeutic drugs such as pentavalent antimonials, amphotericin B, and others, which have major drawbacks due to drug resistance, toxicity, and high cost. In this study, for the first time, we have successfully demonstrated the synthesis and antileishmanial activity of the novel sterol pentalinonsterol (PEN), which occurs naturally in the root of a Mexican medicinal plant, Pentalinon andrieuxii. In the experimental BALB/c mouse model of VL induced by infection with L. donovani, intravenous treatment with liposome-encapsulated PEN (2.5 mg/kg) led to a significant reduction in parasite burden in the liver and spleen. Furthermore, infected mice treated with liposomal PEN showed a strong host-protective TH1 immune response characterized by IFN-γ production and formation of matured hepatic granulomas. These results indicate that PEN could be developed as a novel drug against VL. PMID:27623316

  15. A Novel Sterol Isolated from a Plant Used by Mayan Traditional Healers Is Effective in Treatment of Visceral Leishmaniasis Caused by Leishmania donovani.

    PubMed

    Gupta, Gaurav; Peine, Kevin J; Abdelhamid, Dalia; Snider, Heidi; Shelton, Andrew B; Rao, Latha; Kotha, Sainath R; Huntsman, Andrew C; Varikuti, Sanjay; Oghumu, Steve; Naman, C Benjamin; Pan, Li; Parinandi, Narasimham L; Papenfuss, Tracy L; Kinghorn, A Douglas; Bachelder, Eric M; Ainslie, Kristy M; Fuchs, James R; Satoskar, Abhay R

    2015-10-01

    Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani, is a global health problem affecting millions of people worldwide. Treatment of VL largely depends on therapeutic drugs such as pentavalent antimonials, amphotericin B, and others, which have major drawbacks due to drug resistance, toxicity, and high cost. In this study, for the first time, we have successfully demonstrated the synthesis and antileishmanial activity of the novel sterol pentalinonsterol (PEN), which occurs naturally in the root of a Mexican medicinal plant, Pentalinon andrieuxii. In the experimental BALB/c mouse model of VL induced by infection with L. donovani, intravenous treatment with liposome-encapsulated PEN (2.5 mg/kg) led to a significant reduction in parasite burden in the liver and spleen. Furthermore, infected mice treated with liposomal PEN showed a strong host-protective TH1 immune response characterized by IFN-γ production and formation of matured hepatic granulomas. These results indicate that PEN could be developed as a novel drug against VL.

  16. A Telomeric Cluster of Antimony Resistance Genes on Chromosome 34 of Leishmania infantum.

    PubMed

    Tejera Nevado, Paloma; Bifeld, Eugenia; Höhn, Katharina; Clos, Joachim

    2016-09-01

    The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu(2+) challenge but not under sodium arsenite, Cd(2+), or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance. PMID:27324767

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

    PubMed

    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. PMID:23228525

  18. Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.

    PubMed

    Dougall, Annette M; Alexander, Bruce; Holt, Deborah C; Harris, Tegan; Sultan, Amal H; Bates, Paul A; Rose, Karrie; Walton, Shelley F

    2011-04-01

    The first autochthonous Leishmania infection in Australia was reported by Rose et al. (2004) and the parasite was characterised as a unique species. The host was the red kangaroo (Macropus rufus) but the transmitting vector was unknown. To incriminate the biological vector, insect trapping by a variety of methods was undertaken at two field sites of known Leishmania transmission. Collected sand flies were identified to species level and were screened for Leishmania DNA using a semi-quantitative real-time PCR. Collections revealed four species of sand fly, with a predominance of the reptile biter Sergentomyia queenslandi (Hill). However, no Leishmania-positive flies were detected. Therefore, alternative vectors were investigated for infection, giving startling results. Screening revealed that an undescribed species of day-feeding midge, subgenus Forcipomyia (Lasiohelea) Kieffer, had a prevalence of up to 15% for Leishmania DNA, with high parasitemia in some individuals. Manual gut dissections confirmed the presence of promastigotes and in some midges material similar to promastigote secretory gel, including parasites with metacyclic-like morphology. Parasites were cultured from infected midges and sequence analysis of the Leishmania RNA polymerase subunit II gene confirmed infections were identical to the original isolated Leishmania sp. Phylogenetic analysis revealed the closest known species to be Leishmania enriettii, with this and the Australian species confirmed as members of Leishmania sensu stricto. Collectively the results strongly suggest that the day-feeding midge (F. (Lasiohelea) sp. 1) is a potential biological vector of Leishmania in northern Australia, which is to our knowledge the first evidence of a vector other than a phlebotomine sand fly anywhere in the world. These findings have considerable implications in the understanding of the Leishmania life cycle worldwide.

  19. Expression and characterization of a recombinant kinesin antigen from an old Indian strain (DD8) of Leishmania donovani and comparing it with a commercially available antigen from a newly isolated (KE16) strain of L. donovani.

    PubMed

    Sivakumar, Ramu; Dey, Ayan; Sharma, Pawan; Singh, Sarman

    2008-05-01

    Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of Leishmania donovani (MHOM/IN/KE16/1998) with high sensitivity and specificity and the same has been commercialized. While comparing the sequence data of kinesin gene of this (KE16) strain and its expressed protein with another commercially available recombinant antigen (Lc-rK39) from kinesin gene of L. chagasi we found significant genetic and amino acid variations. This prompted us to undertake the present study to unravel whether the kinesin gene and its expressed protein from another old but Indian isolate of L. donovani (MHOM/IN/DD8/1968) had any genetic and amino acid heterogeneity. Sequencing of the kinesin gene revealed that the kinesin gene of DD8 strain is 3016bp long and has immunodominant region consisting of 4.8 tandem repeats, 117 base pairs each. Further blast analysis of the immunodominant regions of 5 strains of L. donovani revealed that it has only 79% homology with L. chagasi, and 80% homology with L. infantum; while it had 82% homology with Sudan strain of L. donovani, 82% with another (Morena) strain of Indian L. donovani but highest homology of 83% with L. donovani KE16 strain of India. We also evaluated the diagnostic potential of the recombinant DD8 antigen (Ld-rDD8) and compared the results with that of Ld-rKE16. The study revealed that Ld-rKDD8 antigen was less sensitive and specific as compared to rKE16 antigen for the diagnosis of visceral and post-kala-azar dermal leishmaniasis. This was probably due to prolong in vitro culture maintenance of the DD8 strain. PMID:18374635

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

    PubMed

    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.

  1. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated.

  2. The Leishmania mexicana A600 genes are functionally required for amastigote replication.

    PubMed

    Murray, Angus S; Lynn, Miriam A; McMaster, W Robert

    2010-08-01

    Leishmania parasites, the causative agent of leishmaniasis, have a digenetic lifecycle consisting of the morphologically distinct insect vector stage (promastigote) and the mammalian infective amastigote stage. Differentiation of promastigotes to the amastigote stage involves significant morphological and biochemical changes, however, very few genes have been characterised as being differentially expressed in the two stages. The Leishmania A600 genes are one of the few gene families that exhibit stage-specific expression and, as such, they are of interest as potential virulent factors. In this study, we characterize the A600 family in several Leishmania species and investigate their role in amastigote differentiation and proliferation. Four open reading frames, A600-1, A600-2, A600-3, and A600-4, were identified at the multi-gene L. mexicana A600 locus via cloning and restriction mapping. Homology searching identified A600 homologues in other Leishmania species, L. major, L. braziliensis and L. infantum but not in the closely related Trypanosoma family. A targeted gene deletion approach was utilized to determine the cellular function of the L. mexicanaA600 genes. A600(-/-) promastigotes differentiated to axenic amastigotes in response to temperature shift and acidification of culture media, but showed significant growth arrest. Similarly, during in vitro macrophage infection studies, A600(-/-) promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600(-/-) amastigotes to proliferate in mouse peritoneal macrophages was restored by re-introduction of the A600-1 gene, but not the A600-4 gene. The results from these experiments show that the A600-1 gene is essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Furthermore, these results suggest a potential role for the L. mexicana A600-deficient mutant as a vaccine candidate

  3. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated. PMID:25290356

  4. Characterization of Leishmania (Leishmania) waltoni n.sp. (Kinetoplastida: Trypanosomatidae), the Parasite Responsible for Diffuse Cutaneous Leishmaniasis in the Dominican Republic.

    PubMed

    Shaw, Jeffrey; Pratlong, Francine; Floeter-Winter, Lucile; Ishikawa, Edna; El Baidouri, Fouad; Ravel, Christophe; Dedet, Jean-Pierre

    2015-09-01

    Leishmania parasites isolated, between 1979 and 1988 by the late Bryce Walton, from Dominican Republic (DR) patients with diffuse cutaneous leishmaniasis, were characterized using a panel of 12 isoenzymes, 23 monoclonal antibodies, small subunit ribosomal DNA (SSu rDNA), and multilocus sequence analysis (MLSA). The isoenzyme and monoclonal antibody profiles and the MLSA results showed that the Dominican Republic parasites were distinct from other described Leishmania species. This new species belongs to the mexicana complex, which is distributed in central and parts of northern South America. It is suggested that the parasites uniqueness from other members of the mexicana complex is related to it being isolated on an island for millions of years. If Leishmania (Leishmania) waltoni fails to adapt to some imported mammal, such as the house rat, it will be the only Leishmania to be classified as an endangered species. The excessive destruction of habitats on Hispaniola threatens the survival of its vectors and presumed natural reservoirs, such as the rodent hutias and the small insectivorous mammal solenodon. The concept of Leishmania species is discussed in the light of recent evaluations on criteria for defining bacterial species.

  5. Characterization of Leishmania (Leishmania) waltoni n.sp. (Kinetoplastida: Trypanosomatidae), the Parasite Responsible for Diffuse Cutaneous Leishmaniasis in the Dominican Republic.

    PubMed

    Shaw, Jeffrey; Pratlong, Francine; Floeter-Winter, Lucile; Ishikawa, Edna; El Baidouri, Fouad; Ravel, Christophe; Dedet, Jean-Pierre

    2015-09-01

    Leishmania parasites isolated, between 1979 and 1988 by the late Bryce Walton, from Dominican Republic (DR) patients with diffuse cutaneous leishmaniasis, were characterized using a panel of 12 isoenzymes, 23 monoclonal antibodies, small subunit ribosomal DNA (SSu rDNA), and multilocus sequence analysis (MLSA). The isoenzyme and monoclonal antibody profiles and the MLSA results showed that the Dominican Republic parasites were distinct from other described Leishmania species. This new species belongs to the mexicana complex, which is distributed in central and parts of northern South America. It is suggested that the parasites uniqueness from other members of the mexicana complex is related to it being isolated on an island for millions of years. If Leishmania (Leishmania) waltoni fails to adapt to some imported mammal, such as the house rat, it will be the only Leishmania to be classified as an endangered species. The excessive destruction of habitats on Hispaniola threatens the survival of its vectors and presumed natural reservoirs, such as the rodent hutias and the small insectivorous mammal solenodon. The concept of Leishmania species is discussed in the light of recent evaluations on criteria for defining bacterial species. PMID:26149864

  6. Characterization of Leishmania (Leishmania) waltoni n.sp. (Kinetoplastida: Trypanosomatidae), the Parasite Responsible for Diffuse Cutaneous Leishmaniasis in the Dominican Republic

    PubMed Central

    Shaw, Jeffrey; Pratlong, Francine; Floeter-Winter, Lucile; Ishikawa, Edna; El Baidouri, Fouad; Ravel, Christophe; Dedet, Jean-Pierre

    2015-01-01

    Leishmania parasites isolated, between 1979 and 1988 by the late Bryce Walton, from Dominican Republic (DR) patients with diffuse cutaneous leishmaniasis, were characterized using a panel of 12 isoenzymes, 23 monoclonal antibodies, small subunit ribosomal DNA (SSu rDNA), and multilocus sequence analysis (MLSA). The isoenzyme and monoclonal antibody profiles and the MLSA results showed that the Dominican Republic parasites were distinct from other described Leishmania species. This new species belongs to the mexicana complex, which is distributed in central and parts of northern South America. It is suggested that the parasites uniqueness from other members of the mexicana complex is related to it being isolated on an island for millions of years. If Leishmania (Leishmania) waltoni fails to adapt to some imported mammal, such as the house rat, it will be the only Leishmania to be classified as an endangered species. The excessive destruction of habitats on Hispaniola threatens the survival of its vectors and presumed natural reservoirs, such as the rodent hutias and the small insectivorous mammal solenodon. The concept of Leishmania species is discussed in the light of recent evaluations on criteria for defining bacterial species. PMID:26149864

  7. First description of Migonemyia migonei (França) and Nyssomyia whitmani (Antunes & Coutinho) (Psychodidae: Phlebotominae) natural infected by Leishmania infantum in Argentina.

    PubMed

    Moya, Sofía L; Giuliani, Magalí G; Manteca Acosta, Mariana; Salomón, Oscar D; Liotta, Domingo J

    2015-12-01

    Leishmania infantum is the etiological agent of the Visceral Leishmaniasis (VL) disease in America, with Lutzomyia longipalpis phlebotomine sandflies as its proven vectors in Argentina, and infected dogs as its main urban reservoir. In Puerto Iguazú City (Misiones province, Argentina), human and canine cases of VL were recorded. Additionally, in the rural area known as "2000 Hectáreas", less than 10km away from the city, several human cases of Tegumentary Leishmaniasis (TL) were registered determining an endemic area with Leishmania braziliensis as the etiological agent. Because of this, several phlebotomine captures were done in this site showing that Nyssomyia whitmani is the most abundant sandfly followed by Migonemyia migonei. In this study, three of the sandflies captured were found infected whit L. infantum parasites, detected by PCR and sequencing. Two of them were N. whitmani and the other one was a M. migonei specimen, being this the first report of L. infantum natural infection for Argentina in these sandfly species. N. whitmani is the main vector of L. braziliensis in this area, and M. migonei has been suggested as a putative vector in other locations where human and canine cases of VL where reported with L. longipalpis apparently absent. In this context, we consider necessary further studies that could define the role of M. migonei and N. whitmani as specific or permissive vectors of L. infantum, their vectorial competence and capacity, and their actual role in the transmission of both Tegumentary and Visceral Leishmaniasis in the study area.

  8. First description of Migonemyia migonei (França) and Nyssomyia whitmani (Antunes & Coutinho) (Psychodidae: Phlebotominae) natural infected by Leishmania infantum in Argentina.

    PubMed

    Moya, Sofía L; Giuliani, Magalí G; Manteca Acosta, Mariana; Salomón, Oscar D; Liotta, Domingo J

    2015-12-01

    Leishmania infantum is the etiological agent of the Visceral Leishmaniasis (VL) disease in America, with Lutzomyia longipalpis phlebotomine sandflies as its proven vectors in Argentina, and infected dogs as its main urban reservoir. In Puerto Iguazú City (Misiones province, Argentina), human and canine cases of VL were recorded. Additionally, in the rural area known as "2000 Hectáreas", less than 10km away from the city, several human cases of Tegumentary Leishmaniasis (TL) were registered determining an endemic area with Leishmania braziliensis as the etiological agent. Because of this, several phlebotomine captures were done in this site showing that Nyssomyia whitmani is the most abundant sandfly followed by Migonemyia migonei. In this study, three of the sandflies captured were found infected whit L. infantum parasites, detected by PCR and sequencing. Two of them were N. whitmani and the other one was a M. migonei specimen, being this the first report of L. infantum natural infection for Argentina in these sandfly species. N. whitmani is the main vector of L. braziliensis in this area, and M. migonei has been suggested as a putative vector in other locations where human and canine cases of VL where reported with L. longipalpis apparently absent. In this context, we consider necessary further studies that could define the role of M. migonei and N. whitmani as specific or permissive vectors of L. infantum, their vectorial competence and capacity, and their actual role in the transmission of both Tegumentary and Visceral Leishmaniasis in the study area. PMID:26409011

  9. Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge

    PubMed Central

    Resende, Lucilene Aparecida; Aguiar-Soares, Rodrigo Dian de Oliveira; Gama-Ker, Henrique; Roatt, Bruno Mendes; de Mendonça, Ludmila Zanandreis; Alves, Marina Luiza Rodrigues; da Silveira-Lemos, Denise; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Araújo, Márcio Sobreira Silva; Fujiwara, Ricardo Toshio; Gontijo, Nelder Figueiredo; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2016-01-01

    Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the “LbSapSal” vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with “LbSapSal” is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-β), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after “LbSapSal” immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the “LbSapSal” vaccination is a potential tool to control the Leishmania chagasi infection. PMID:27556586

  10. Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge.

    PubMed

    Resende, Lucilene Aparecida; Aguiar-Soares, Rodrigo Dian de Oliveira; Gama-Ker, Henrique; Roatt, Bruno Mendes; Mendonça, Ludmila Zanandreis de; Alves, Marina Luiza Rodrigues; Silveira-Lemos, Denise da; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Araújo, Márcio Sobreira Silva; Fujiwara, Ricardo Toshio; Gontijo, Nelder Figueiredo; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2016-01-01

    Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the "LbSapSal" vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with "LbSapSal" is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-β), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after "LbSapSal" immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the "LbSapSal" vaccination is a potential tool to control the Leishmania chagasi infection.

  11. Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge.

    PubMed

    Resende, Lucilene Aparecida; Aguiar-Soares, Rodrigo Dian de Oliveira; Gama-Ker, Henrique; Roatt, Bruno Mendes; Mendonça, Ludmila Zanandreis de; Alves, Marina Luiza Rodrigues; Silveira-Lemos, Denise da; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Araújo, Márcio Sobreira Silva; Fujiwara, Ricardo Toshio; Gontijo, Nelder Figueiredo; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2016-01-01

    Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the "LbSapSal" vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with "LbSapSal" is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-β), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after "LbSapSal" immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the "LbSapSal" vaccination is a potential tool to control the Leishmania chagasi infection. PMID:27556586

  12. Reproductive biology of the isopod Excirolana braziliensis at the southern edge of its geographical range

    NASA Astrophysics Data System (ADS)

    Martínez, Gastón; Defeo, Omar

    2006-12-01

    A full analysis of the reproductive biology of the isopod Excirolana braziliensis Richardson 1912 was conducted in a sandy beach of Uruguay, located at the southernmost edge of its distributional range in the Atlantic Ocean. Reproductive and recruitment periods of E. braziliensis were concentrated in austral summer. Females with oostegites appeared in November, whereas total biomass, individual sizes and fecundity of ovigerous females peaked between December and January. These concurrent traits were responsible for the significant peak of juveniles in January. The size at maturity was 9.88 mm. Four embryonic developmental stages were described and identified: mean length linearly increased from stages I to III, whereas dry weight exponentially decreased from stages I to IV. The high reproductive output (0.41-0.58), reported for the first time in this isopod, exceeds the rates documented for other isopods. Reproduction of E. braziliensis at the southern edge of its range is semelparous: females produce one brood during the reproductive season, exhaust their energy reserves during incubation, and probably die at the end of the reproductive season. A macroscale comparison suggests that E. braziliensis at the southern edge of its range counteracts its narrow reproductive period by a short incubation period with larger individual mature female and embryo sizes, higher fecundity and a higher percentage of ovigerous females than in subtropical and tropical populations. These extreme reproductive indicators could be attributed to the internal retention of embryos that assures offspring survival, coupled with a high adaptation capability to environmental variations across its range.

  13. The genus Leishmania

    PubMed Central

    Garnham, P. C. C.

    1971-01-01

    The systematic position of the so-called ”species” of Leishmania is examined and an attempt made to determine their phylogenetic relationships. The morphology of the organisms as seen by light- and electron-microscopy is described; neither method provides useful criteria for the determination of species. The behaviour of the parasites in insect and in vertebrate hosts offers a better method of classification. In this way, the species may be divided into 4 main groups, comprising the mammalian species involving man, the distinctive species L. enriettii in the guinea-pig, those infecting lizards, and species apparently in various stages of evolution in phlebotomines. The so-called ”human” group is divided into visceral forms (originating chiefly in wild canidae) and cutaneous forms (probably of rodent origin). The named species of the former group include L. donovani and L. infantum. The cutaneous species include L. tropica tropica (=minor), L. tropica major, L. brasiliensis, L. peruana, L. guyanensis, and L. mexicana. L. pifanoi is probably not a distinct species but represents various forms as modified by the failure of cell-mediated immunity in the host. Leishmanial infections can be identified first by ascertaining the geographical area where the infection was acquired, and then by more or less complicated laboratory investigations including characteristics in culture, serological tests, the response of special hosts in terms of symptomatology, and the behaviour of the parasite in the phlebotomine host. No test is infallible, and an effective simple test is urgently needed. The preservation of Leishmania strains is an important research procedure and a method for conserving parasites by lyophilization is described briefly. PMID:5316250

  14. Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.

    PubMed

    Resende, Lucilene Aparecida; Roatt, Bruno Mendes; Aguiar-Soares, Rodrigo Dian de Oliveira; Viana, Kelvinson Fernandes; Mendonça, Ludmila Zanandreis; Lanna, Mariana Ferreira; Silveira-Lemos, Denise; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Fujiwara, Ricardo Toshio; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2013-12-01

    In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-γ and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-β decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection. PMID:24129068

  15. Evaluation of Organelle Changes in Promastigotes of Unresponsive Leishmania Tropica to Meglumine Antimoniate in Comparison with Sensitive and Standard Isolates by Electron Microscopy

    PubMed Central

    Bahreini, Mitra; Bolorizadeh, Mehdi; Dabiri, Shahriar; Sharifi, Iraj

    2015-01-01

    Background: The control of leishmaniasis faces serious challenges because of resistance to the first-line antimonial drugs. We aimed to evaluate the differences in organelle changes of cultivated promastigotes obtained from skin lesions of sensitive and unresponsive isolates to meglumine antimoniate (Glucantime) by electron microscopy. Material and Methods: This study was done in Bam city, southeastern Iran, in which the incidence of disease has sharply increased since the earthquake in 2003. The samples were taken from 66 patients who were referred to the cutaneous leishmaniasis (CL) treatment center in Bam. A questionnaire was completed for each individual, recording their demographic characteristics and CL status. The scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by Giemsa, and examined under a compound light microscope for amastigote form simultaneously. To prepare the specimens for transmission electron imaging, promastigotes were centrifuged and resuspened. Results: Transmission electron microscopic study of the cultivated promastigotes revealed that there were alterations in the organelles and structures of sensitive isolates compared with unresponsive and standard ones. Organelles and structures such as mitochondria, kinetoplast, microtubules, cytoplasmic vacuoles, plasma membrane and vesicles were studied. The alterations such as disintegration of kinetoplast into thin filaments and condensation of kinetoplast DNA core, changes in size, number and location of vesicles and microtubules were observed. We noted intense cytoplasmic vacuolization, and considerable swelling of mitochondria. Conclusion: The significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism. PMID:26120175

  16. Leishmania and human immunodeficiency virus coinfection: the first 10 years.

    PubMed Central

    Alvar, J; Cañavate, C; Gutiérrez-Solar, B; Jiménez, M; Laguna, F; López-Vélez, R; Molina, R; Moreno, J

    1997-01-01

    Over 850 Leishmania-human immunodeficiency virus (HIV) coinfection cases have been recorded, the majority in Europe, where 7 to 17% of HIV-positive individuals with fever have amastigotes, suggesting that Leishmania-infected individuals without symptoms will express symptoms of leishmaniasis if they become immunosuppressed. However, there are indirect reasons and statistical data demonstrating that intravenous drug addiction plays a specific role in Leishmania infantum transmission: an anthroponotic cycle complementary to the zoonotic one has been suggested. Due to anergy in patients with coinfection, L. infantum dermotropic zymodemes are isolated from patient viscera and a higher L. infantum phenotypic variability is seen. Moreover, insect trypanosomatids that are currently considered nonpathogenic have been isolated from coinfected patients. HIV infection and Leishmania infection each induce important analogous immunological changes whose effects are multiplied if they occur concomitantly, such as a Th1-to-Th2 response switch; however, the consequences of the viral infection predominate. In fact, a large proportion of coinfected patients have no detectable anti-Leishmania antibodies. The microorganisms share target cells, and it has been demonstrated in vitro how L. infantum induces the expression of latent HIV-1. Bone marrow culture is the most useful diagnostic technique, but it is invasive. Blood smears and culture are good alternatives. PCR, xenodiagnosis, and circulating-antigen detection are available only in specialized laboratories. The relationship with low levels of CD4+ cells conditions the clinical presentation and evolution of disease. Most patients have visceral leishmaniasis, but asymptomatic, cutaneous, mucocutaneous, diffuse cutaneous, and post-kala-azar dermal leishmaniasis can be produced by L. infantum. The digestive and respiratory tracts are frequently parasitized. The course of coinfection is marked by a high relapse rate. There is a lack

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

    PubMed

    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

    2015-09-01

    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 of L. (V.) naiffi patients (8 cases) was observed, thus demonstrating that this species is not uncommon in the region. No patient infected with L. (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.

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

    PubMed Central

    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

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

    PubMed

    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.

  20. Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

    PubMed

    Andrade, Juvana M; Baba, Elio H; Machado-de-Avila, Ricardo A; Chavez-Olortegui, Carlos; Demicheli, Cynthia P; Frézard, Frédéric; Monte-Neto, Rubens L; Murta, Silvane M F

    2016-08-01

    Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III). PMID:27161624

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

    PubMed Central

    2008-01-01

    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. PMID:18577226

  2. Leishmania (Leishmania) hertigi in a porcupine (Coendou sp.) found in Brasília, Federal District, Brazil.

    PubMed

    Silva, Denise Amaro da; Madeira, Maria de Fatima; Barbosa Filho, Carlos José Lima; Schubach, Edvar Yuri Paheco; Barros, Juliana Helena da Silva; Figueiredo, Fabiano Borges

    2013-01-01

    Studies report the occurrence of Leishmania (Leishmania) hertigi in northern states of Brazil. In the present investigation, we describe the isolation of L. (L.) hertigi from a porcupine (Coendou sp.) found in Brasília, Federal District, center-west region of Brazil. During a study on canine visceral leishmaniasis conducted in the city of Brasília, Federal District, a porcupine was found dead on a public road. The animal was identified and fragments of intact skin and spleen were collected for isolation of parasite in the culture. This report of the occurrence of L. hertigi in another part of Brazil may help establish the distribution of this parasite in the country. Further studies are needed to better understand the role of L. hertigi in the pathology and pathogenesis of leishmaniasis and its survival in mammals and possible vectors.

  3. Leishmania (Leishmania) hertigi in a porcupine (Coendou sp.) found in Brasília, Federal District, Brazil.

    PubMed

    Silva, Denise Amaro da; Madeira, Maria de Fatima; Barbosa Filho, Carlos José Lima; Schubach, Edvar Yuri Paheco; Barros, Juliana Helena da Silva; Figueiredo, Fabiano Borges

    2013-01-01

    Studies report the occurrence of Leishmania (Leishmania) hertigi in northern states of Brazil. In the present investigation, we describe the isolation of L. (L.) hertigi from a porcupine (Coendou sp.) found in Brasília, Federal District, center-west region of Brazil. During a study on canine visceral leishmaniasis conducted in the city of Brasília, Federal District, a porcupine was found dead on a public road. The animal was identified and fragments of intact skin and spleen were collected for isolation of parasite in the culture. This report of the occurrence of L. hertigi in another part of Brazil may help establish the distribution of this parasite in the country. Further studies are needed to better understand the role of L. hertigi in the pathology and pathogenesis of leishmaniasis and its survival in mammals and possible vectors. PMID:23538497

  4. Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats.

    PubMed

    Jegede, Olorunfemi Cornelius; Ajanusi, Joseph Ologunja; Adaudi, Ambrose O; Agbede, Rowland I S

    2006-09-01

    Spigelia anthelmia Linn is used as a herb and is a common annual weed of cultivation in open re-growths, on unused land in towns as well as on road sides. The plant can grow to approximately 30 cm in height. The aim of this study was to screen extracts of Spigelia anthelmia for their anthelmintic activity against an experimental Nippostrongylus braziliensis infection in rats. Acute oral toxicity occurred at a dose of 1,140 mg/kg, while anthelmintic trials against Nippostrongylus braziliensis in rats using the aqueous fraction showed a progressive decrease in worm count with increasing dose (10, 13, 16, 20 and 25 mg per kg body weight) (p < 0.05). At 25 mg per kg body weight, the worm count was significantly lower than that at 10 mg per kg body weight (p < 0.05).

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

    PubMed

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

    2014-11-01

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

  6. Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia

    PubMed Central

    Teles, Carolina Bioni Garcia; dos Santos, Ana Paula de Azevedo; Freitas, Rui Alves; de Oliveira, Arley Faria José; Ogawa, Guilherme Maerschner; Rodrigues, Moreno Souza; Pessoa, Felipe Arley Costa; Medeiros, Jansen Fernandes; Camargo, Luís Marcelo Aranha

    2016-01-01

    In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality. PMID:27304023

  7. Natural Leishmania Infection of Lutzomyia auraensis in Madre de Dios, Peru, Detected by a Fluorescence Resonance Energy Transfer–Based Real-Time Polymerase Chain Reaction

    PubMed Central

    Valdivia, Hugo O.; De Los Santos, Maxy B.; Fernandez, Roberto; Baldeviano, G. Christian; Zorrilla, Victor O.; Vera, Hubert; Lucas, Carmen M.; Edgel, Kimberly A.; Lescano, Andrés G.; Mundal, Kirk D.; Graf, Paul C. F.

    2012-01-01

    Leishmania species of the Viannia subgenus are responsible for most cases of New World tegumentary leishmaniasis. However, little is known about the vectors involved in disease transmission in the Amazon regions of Peru. We used a novel real-time polymerase chain reaction (PCR) to assess Leishmania infections in phlebotomines collected in rural areas of Madre de Dios, Peru. A total of 1,299 non-blood fed female sand flies from 33 species were captured by using miniature CDC light traps. Lutzomyia auraensis was the most abundant species (63%) in this area. Seven of 164 pools were positive by PCR for Leishmania by kinetoplast DNA. The real-time PCR identified four Lu. auraensis pools as positive for L. (Viannia) lainsoni and L. (V.) braziliensis. The minimum infection prevalence for Lu. auraensis was estimated to be 0.6% (95% confidence interval = 0.20–1.42%). Further studies are needed to assess the importance of Lu. auraensis in the transmission of New World tegumentary leishmaniasis in hyperendemic areas of Peru. PMID:22802444

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

    PubMed Central

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

    2016-01-01

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

  9. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  10. Sheep blood-LB agar base medium (SLM) as a simple and suitable medium for the cultivation of Leishmania major promastigotes.

    PubMed

    Nasiri, Vahid

    2013-11-01

    Several methods of cultivation of Leishmania parasites are available, and among them, biphasic media like Nicolle-Novy-MacNeal medium are considered to be superior for the initial isolation of the parasites and their in vivo infectivity. In the present work, the efficacy of sheep blood-LB agar base medium (SLM) was evaluated for the cultivation of Leishmania major promastigotes. The SLM supports the proliferation of the parasites and can be used for routine isolation and cultivation of Leishmania parasites with acceptable in vivo infectivity for research purposes.

  11. Leishmania-based expression systems.

    PubMed

    Taheri, Tahereh; Seyed, Negar; Mizbani, Amir; Rafati, Sima

    2016-09-01

    Production of therapeutic or medical recombinant proteins, such as monoclonal antibodies, proteins, or active enzymes, requires a highly efficient system allowing natural folding and perfect post-translation modifications of the expressed protein. These requirements lead to the generation of a variety of gene expression systems from bacteria to eukaryotes. To achieve the best form of eukaryotic proteins, two factors need to be taken into consideration: choosing a suitable organism to express the protein of interest, and selecting an efficient delivery system. For this reason, the expression of recombinant proteins in eukaryotic nonpathogenic Leishmania parasites is an interesting approach which meets both criteria. Here, new Leishmania-based expression systems are compared with current systems that have long histories in research and industry. PMID:27435294

  12. In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

    PubMed

    Chamakh-Ayari, Rym; Bras-Gonçalves, Rachel; Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection. PMID:24786587

  13. Folate metabolic pathways in Leishmania.

    PubMed

    Vickers, Tim J; Beverley, Stephen M

    2011-01-01

    Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

  14. LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4+ and CD8+) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis

    PubMed Central

    2014-01-01

    Background The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production. Methods We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. Results The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. Conclusions These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector. PMID:24507702

  15. COMPARATIVE STUDIES OF HERPETOMONADS AND LEISHMANIAS

    PubMed Central

    Noguchi, Hideyo; Tilden, Evelyn B.

    1926-01-01

    obtained in culture, all the strains grow well on leptospira medium, as well as on blood slants. Growth takes place both at 26°C. and at 37°. The morphology of the organisms is considerably modified by cultivation. This is especially true of the plant flagellates. In the latex they have ribbon-like bodies, often twisted, and comparatively short flagella; the protoplasm is clear, almost hyaline. The flagellates seen in the gut and feces of insects are usually large, slender organisms, with flagella as long as or even longer than the body, which contains numerous volutin granules in the cytoplasm. In cultures under parallel conditions the flagellates from both these sources become shorter and thicker, the plant forms no longer appear flat and ribbon-like, and in general the organisms approach one another in morphological features. Even in the case of the least modified insect flagellates, i.e. those from flies, there is never exact correspondence between the natural and the cultivated forms. The morphological features of the cultivated flagellates vary according to the medium on which the organisms are grown and the age of the culture. The flagellates grown on the surface of blood slants are pyriform, with truncated anterior portion, and short flagellum; in the condensation water, however, the individuals are elongated and have long active flagella. On the leptospira medium the slender active forms with long flagella predominate. In the presence of fermentable carbohydrate, or in medium containing considerable acid, peculiar bifurcated or multifurcated individuals are seen. Similar forms have been seen under natural conditions. Cultures of Leishmania behave in the same way under the conditions described. There is a striking difference in rapidity of growth between the organisms isolated by us and the leishmanias, H. ctenocephali, and T. rotatorium. While the stock cultures of the group first mentioned multiply rapidly at 37°C., growth becoming visible within 24 hours, the

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

    SciTech Connect

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

    1986-12-01

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

  17. Comprehensive proteomics analysis of glycosomes from Leishmania donovani.

    PubMed

    Jamdhade, Mahendra D; Pawar, Harsh; Chavan, Sandip; Sathe, Gajanan; Umasankar, P K; Mahale, Kiran N; Dixit, Tanwi; Madugundu, Anil K; Prasad, T S Keshava; Gowda, Harsha; Pandey, Akhilesh; Patole, Milind S

    2015-03-01

    Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes. PMID:25748437

  18. Comprehensive Proteomics Analysis of Glycosomes from Leishmania donovani

    PubMed Central

    Jamdhade, Mahendra D.; Pawar, Harsh; Chavan, Sandip; Sathe, Gajanan; Umasankar, P.K.; Mahale, Kiran N.; Dixit, Tanwi; Madugundu, Anil K.; Prasad, T.S. Keshava; Gowda, Harsha

    2015-01-01

    Abstract Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host–parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes. PMID:25748437

  19. Leishmania RNA virus: when the host pays the toll

    PubMed Central

    Hartley, Mary-Anne; Ronet, Catherine; Zangger, Haroun; Beverley, Stephen M.; Fasel, Nicolas

    2012-01-01

    The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic “metastatic factors” or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically

  20. Anthroponotic cutaneous leishmaniasis in Tunisia: presence of Leishmania killicki outside its original focus of Tataouine.

    PubMed

    Haouas, N; Chargui, N; Chaker, E; Ben Said, M; Babba, H; Belhadj, S; Kallel, K; Pratlong, F; Dedet, J-P; Mezhoud, H; Azaiez, R

    2005-07-01

    The first three documented cases of anthroponotic cutaneous leishmaniasis due to Leishmania killicki are reported from locations outside the original focus of Tataouine in southeast Tunisia. Three strains were isolated from three patients from Gafsa, Sidi Bouzid and Seliana indicating an extension of this parasite's range towards the centre and the north of Tunisia. PMID:15869775

  1. A first case of cutaneous leishmaniasis due to Leishmania (Viannia) lainsoni in Bolivia.

    PubMed

    Martinez, E; Le Pont, F; Mollinedo, S; Cupolillo, E

    2001-01-01

    We present the first known case of cutaneous leishmaniasis due to Leishmania (Viannia) lainsoni detected in Bolivia. The parasite was isolated from a young girl living in the subtropical region of Carrasco (900-1000 m above sea level, Caranavi Province, Department of La Paz, Bolivia). The parasite identification was confirmed by multilocus enzyme electrophoresis. PMID:11579876

  2. Leishmania molecules that mediate intracellular pathogenesis.

    PubMed

    Kima, Peter E

    2014-09-01

    Parasites of the Leishmania genus are the causative agents of a complex disease called leishmaniasis. Many activities of infected cells including their responses to a range of stimuli are modulated by Leishmania parasites. This review will profile some of the parasite molecules that target host cell processes for which there has been recent progress.

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-10-01

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

  5. Leishmanial Excreted Factor: Protein-Bound and Free Forms from Promastigote Cultures of Leishmania tropica and Leishmania donovani

    PubMed Central

    Slutzky, Gerald M.; El-On, Joseph; Greenblatt, Charles L.

    1979-01-01

    Leishmania spp. growing in culture produce an immunologically active substance called excreted factor (EF), which precipitates antibodies raised against intact cells and has been implicated as the conditioning agent for parasite infection of host macrophages. An improved method for isolation of the material is described, based on Sephadex column chromatography of growth medium which had been boiled at pH 5.0. This procedure allows the detection of differences among the EF molecules of different species, and it overcomes previous shortcomings through the monitoring of immunological activity throughout. Analysis of the products of this procedure revealed that EFs from Leishmania tropica and Leishmania donovani share a common carrier protein, identified as rabbit serum albumin, and are chemically quite similar. Growth medium from L. tropica boiled at acidic pH contains primarily an EF-albumin complex of 75,000 molecular weight. Treated growth medium from L. donovani, on the other hand, contains both the albumin complex and a smaller molecule (less than 27,000 molecular weight) that is not associated with rabbit protein. This material accounts for nearly 20% of the EF of one L. donovani strain, but constitutes only a minute fraction of L. tropica EF. Treatment of the EF-albumin complex with trichloroacetic acid separates the molecule into two major subunits, one having a molecular weight of about 61,000 (without anti-Leishmania activity) and the other having a molecular weight of about 18,000 (with no anti-rabbit activity). The protein-free EF of L. tropica differs from that released by trichloroacetic acid extraction in that it is capable of precipitating antisera of nonhomologous serotypes, whereas the albumin complex and the trichloroacetic acid-treated EF fragment are not. EFs from both species display pH-dependent affinity for certain lectins. Images PMID:118936

  6. Phlebotomus sergenti (Parrot, 1917) identified as Leishmania killicki host in Ghardaïa, south Algeria.

    PubMed

    Boubidi, S C; Benallal, K; Boudrissa, A; Bouiba, L; Bouchareb, B; Garni, R; Bouratbine, A; Ravel, C; Dvorak, V; Votypka, J; Volf, P; Harrat, Z

    2011-07-01

    Since 2005, an outbreak of human cutaneous leishmaniasis (CL) in Ghardaïa, south Algeria, was studied and one output of these investigations was the identification of two Leishmania species, Leishmania major and Leishmania killicki, as the CL causative agents. In the present study, we were curious to focus on sand fly fauna present in this area and detection of Leishmania-positive sand fly females. Sand flies (3717) were collected during two seasons using sticky papers and CDC light traps in urban, rural and sylvatic sites. Twelve Phlebotomus species were identified. Phlebotomus papatasi was dominant in the urban site while Phlebotomus sergenti and Phlebotomus riouxi/chabaudi were dominant in the sylvatic site. Out of 74 P. sergenti females captured by CDC light traps in the sylvatic site populated by Ghardaïas' Gundi (Massoutiera mzabi), three ones were hosting Leishmania promastigotes. PCR-RFLP and sequencing of seven single-copy coding DNA sequences identified the promastigotes as L. killicki. Furthermore, laboratory experiments revealed that L. killicki isolate sampled from a CL patient inhabiting the studied region develop well in P. sergenti females. Our findings strongly suggest that the human cutaneous leishmaniases caused by L. killicki is a zoonotic disease with P. sergenti sand flies acting as hosts and vectors and gundi rodents as reservoirs.

  7. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

    PubMed

    Losada-Barragán, Monica; Cavalcanti, Amanda; Umaña-Pérez, Adriana; Porrozzi, Renato; Cuervo-Escobar, Sergio; Vallejo, Andrés Felipe; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2016-08-15

    Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.

  8. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

    PubMed

    Losada-Barragán, Monica; Cavalcanti, Amanda; Umaña-Pérez, Adriana; Porrozzi, Renato; Cuervo-Escobar, Sergio; Vallejo, Andrés Felipe; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2016-08-15

    Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites. PMID:27514885

  9. Biogeographic patterns in life history traits of the Pan-American sandy beach isopod Excirolana braziliensis

    NASA Astrophysics Data System (ADS)

    Cardoso, Ricardo S.; Defeo, Omar

    2004-11-01

    Biogeographic patterns in life history traits of the Pan-American sandy beach isopod Excirolana braziliensis were analyzed to determine latitudinal variations along its distribution, from tropical (9°N) to temperate (39°S) sandy beaches in Atlantic and Pacific oceans. Population features exhibited systematic geographical patterns of variation: (1) an increase in individual sizes and growth rates towards temperate beaches, following an inverse relationship with mean water temperature of the surf zone; (2) a shift from almost continuous to seasonal growth from subtropical to temperate Atlantic beaches and a positive relationship between amplitude of intra-annual growth oscillations and temperature range; (3) a linear decrease in life span and an increase in natural mortality from temperate to subtropical beaches; and (4) an increase in the individual mass-at-size (length-mass relationship) from subtropical to temperate beaches. Analyses discriminated by sex were consistent with the patterns illustrated above. Local effects of temperature and beach morphodynamics are discussed. Our results demonstrate that the population dynamics of E. braziliensis is highly plastic over latitudinal gradients, with large-scale variations in temperature and concurrent environmental variables leading to an adjustment of the phenotype-environment relationship.

  10. The Genetic Relationship between Leishmania aethiopica and Leishmania tropica Revealed by Comparing Microsatellite Profiles

    PubMed Central

    Krayter, Lena; Schnur, Lionel F.; Schönian, Gabriele

    2015-01-01

    Background Leishmania (Leishmania) aethiopica and L. (L.) tropica cause cutaneous leishmaniases and appear to be related. L. aethiopica is geographically restricted to Ethiopia and Kenya; L. tropica is widely dispersed from the Eastern Mediterranean, through the Middle East into eastern India and in north, east and south Africa. Their phylogenetic inter-relationship is only partially revealed. Some studies indicate a close relationship. Here, eight strains of L. aethiopica were characterized genetically and compared with 156 strains of L. tropica from most of the latter species' geographical range to discern the closeness. Methodology/Principal Findings Twelve unlinked microsatellite markers previously used to genotype strains of L. tropica were successfully applied to the eight strains of L. aethiopica and their microsatellite profiles were compared to those of 156 strains of L. tropica from various geographical locations that were isolated from human cases of cutaneous and visceral leishmaniasis, hyraxes and sand fly vectors. All the microsatellite profiles were subjected to various analytical algorithms: Bayesian statistics, distance-based and factorial correspondence analysis, revealing: (i) the species L. aethiopica, though geographically restricted, is genetically very heterogeneous; (ii) the strains of L. aethiopica formed a distinct genetic cluster; and (iii) strains of L. aethiopica are closely related to strains of L. tropica and more so to the African ones, although, by factorial correspondence analysis, clearly separate from them. Conclusions/Significance The successful application of the 12 microsatellite markers, originally considered species-specific for the species L. tropica, to strains of L. aethiopica confirmed the close relationship between these two species. The Bayesian and distance-based methods clustered the strains of L. aethiopica among African strains of L. tropica, while the factorial correspondence analysis indicated a clear separation

  11. Co-infection by Leishmania amazonensis and L. infantum/L. chagasi in a case of diffuse cutaneous leishmaniasis in Bolivia.

    PubMed

    Martinez, E; Mollinedo, S; Torrez, M; Muñoz, M; Bañuls, A L; Le Pont, F

    2002-01-01

    We present the first report of a co-infection by Leishmania amazonensis and L. infantum/L. chagasi isolated in 1993 from a patient with diffuse cutaneous leishmaniasis (DCL), living in the sub-Andean region of Bolivia. This is the third reported case of DCL in Bolivia, but the first one with isoenzymatic identification of the aetiological agents involved and the first one giving evidence for a mixed infection by 2 Leishmania parasites in the same lesion. PMID:12474481

  12. Phylogenomic reconstruction supports supercontinent origins for Leishmania.

    PubMed

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

    2016-03-01

    Leishmania, a genus of parasites transmitted to human hosts and mammalian/reptilian reservoirs by an insect vector, is the causative agent of the human disease complex leishmaniasis. The evolutionary relationships within the genus Leishmania and its origins are the source of ongoing debate, reflected in conflicting phylogenetic and biogeographic reconstructions. This study employs a recently described bioinformatics method, SISRS, to identify over 200,000 informative sites across the genome from newly sequenced and publicly available Leishmania data. This dataset is used to reconstruct the evolutionary relationships of this genus. Additionally, we constructed a large multi-gene dataset, using it to reconstruct the phylogeny and estimate divergence dates for species. We conclude that the genus Leishmania evolved at least 90-100 million years ago, supporting a modified version of the Multiple Origins hypothesis that we call the Supercontinent hypothesis. According to this scenario, separate Leishmania clades emerged prior to, and during, the breakup of Gondwana. Additionally, we confirm that reptile-infecting Leishmania are derived from mammalian forms and that the species that infect porcupines and sloths form a clade long separated from other species. Finally, we firmly place the guinea-pig infecting species, Leishmaniaenriettii, the globally dispersed Leishmaniasiamensis, and the newly identified Australian species from a kangaroo, as sibling species whose distribution arises from the ancient connection between Australia, Antarctica, and South America. PMID:26708057

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

    PubMed Central

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

    1994-01-01

    This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen compared with values for untreated control animals. The [3H]thymidine uptake by the parasites isolated from the treated hamsters was only about 1% of that observed in parasites isolated from the controls. Oral administration of licochalcone A at concentrations of 5 to 150 mg/kg of body weight per day for 6 consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases. PMID:8092835

  14. Metaproteomics reveals metabolic transitions between healthy and diseased stony coral Mussismilia braziliensis.

    PubMed

    Garcia, Gizele D; Santos, Eidy de O; Sousa, Gabriele V; Zingali, Russolina B; Thompson, Cristiane C; Thompson, Fabiano L

    2016-09-01

    Infectious diseases such as white plague syndrome (WPS) and black band disease (BBD) have caused massive coral loss worldwide. We performed a metaproteomic study on the Abrolhos coral Mussismilia braziliensis to define the types of proteins expressed in healthy corals compared to WPS- and BBD-affected corals. A total of 6363 MS/MS spectra were identified as 361 different proteins. Healthy corals had a set of proteins that may be considered markers of holobiont homoeostasis, including tubulin, histone, Rab family, ribosomal, peridinin-chlorophyll a-binding protein, F0F1-type ATP synthase, alpha-iG protein, calmodulin and ADP-ribosylation factor. Cnidaria proteins found in healthy M. braziliensis were associated with Cnidaria-Symbiodinium endosymbiosis and included chaperones (hsp70, hsp90 and calreticulin), structural and membrane modelling proteins (actin) and proteins with functions related to intracellular vesicular traffic (Rab7 and ADP-ribosylation factor 1) and signal transduction (14-3-3 protein and calmodulin). WPS resulted in a clear shift in the predominance of proteins, from those related to aerobic nitrogen-fixing bacteria (i.e. Rhizobiales, Sphingomonadales and Actinomycetales) in healthy corals to those produced by facultative/anaerobic sulphate-reducing bacteria (i.e. Enterobacteriales, Alteromonadales, Clostridiales and Bacteroidetes) in WPS corals. BBD corals developed a diverse community dominated by cyanobacteria and sulphur cycle bacteria. Hsp60, hsp90 and adenosylhomocysteinase proteins were produced mainly by cyanobacteria in BBD corals, which is consistent with elevated oxidative stress in hydrogen sulphide- and cyanotoxin-rich environments. This study demonstrates the usefulness of metaproteomics for gaining better comprehension of coral metabolic status in health and disease, especially in reef systems such as the Abrolhos that are suffering from the increase in global and local threatening events.

  15. Metaproteomics reveals metabolic transitions between healthy and diseased stony coral Mussismilia braziliensis.

    PubMed

    Garcia, Gizele D; Santos, Eidy de O; Sousa, Gabriele V; Zingali, Russolina B; Thompson, Cristiane C; Thompson, Fabiano L

    2016-09-01

    Infectious diseases such as white plague syndrome (WPS) and black band disease (BBD) have caused massive coral loss worldwide. We performed a metaproteomic study on the Abrolhos coral Mussismilia braziliensis to define the types of proteins expressed in healthy corals compared to WPS- and BBD-affected corals. A total of 6363 MS/MS spectra were identified as 361 different proteins. Healthy corals had a set of proteins that may be considered markers of holobiont homoeostasis, including tubulin, histone, Rab family, ribosomal, peridinin-chlorophyll a-binding protein, F0F1-type ATP synthase, alpha-iG protein, calmodulin and ADP-ribosylation factor. Cnidaria proteins found in healthy M. braziliensis were associated with Cnidaria-Symbiodinium endosymbiosis and included chaperones (hsp70, hsp90 and calreticulin), structural and membrane modelling proteins (actin) and proteins with functions related to intracellular vesicular traffic (Rab7 and ADP-ribosylation factor 1) and signal transduction (14-3-3 protein and calmodulin). WPS resulted in a clear shift in the predominance of proteins, from those related to aerobic nitrogen-fixing bacteria (i.e. Rhizobiales, Sphingomonadales and Actinomycetales) in healthy corals to those produced by facultative/anaerobic sulphate-reducing bacteria (i.e. Enterobacteriales, Alteromonadales, Clostridiales and Bacteroidetes) in WPS corals. BBD corals developed a diverse community dominated by cyanobacteria and sulphur cycle bacteria. Hsp60, hsp90 and adenosylhomocysteinase proteins were produced mainly by cyanobacteria in BBD corals, which is consistent with elevated oxidative stress in hydrogen sulphide- and cyanotoxin-rich environments. This study demonstrates the usefulness of metaproteomics for gaining better comprehension of coral metabolic status in health and disease, especially in reef systems such as the Abrolhos that are suffering from the increase in global and local threatening events. PMID:27492757

  16. Vaccine Development Against Leishmania donovani

    PubMed Central

    Das, Amrita; Ali, Nahid

    2012-01-01

    Visceral leishmaniasis (VL) caused by Leishmania donovani and Leishmania infantum/chagasi represents the second most challenging infectious disease worldwide, leading to nearly 500,000 new cases and 60,000 deaths annually. Zoonotic VL caused by L. infantum is a re-emergent canid zoonoses which represents a complex epidemiological cycle in the New world where domestic dogs serve as a reservoir host responsible for potentially fatal human infection and where dog culling is the only measure for reservoir control. Life-long immunity to VL has motivated development of prophylactic vaccines against the disease but very few have progressed beyond the experimental stage. No licensed vaccine is available till date against any form of leishmaniasis. High toxicity and increasing resistance to the current chemotherapeutic regimens have further complicated the situation in VL endemic regions of the world. Advances in vaccinology, including recombinant proteins, novel antigen-delivery systems/adjuvants, heterologous prime-boost regimens and strategies for intracellular antigen presentation, have contributed to recent advances in vaccine development against VL. Attempts to develop an effective vaccine for use in domestic dogs in areas of canine VL should be pursued for preventing human infection. Studies in animal models and human patients have revealed the pathogenic mechanisms of disease progression and features of protective immunity. This review will summarize the accumulated knowledge of pathogenesis, immune response, and prerequisites for protective immunity against human VL. Authors will discuss promising vaccine candidates, their developmental status and future prospects in a quest for rational vaccine development against the disease. In addition, several challenges such as safety issues, renewed and coordinated commitment to basic research, preclinical studies and trial design will be addressed to overcome the problems faced in developing prophylactic strategies for

  17. Leishmania major, the predominant Leishmania species responsible for cutaneous leishmaniasis in Mali.

    PubMed

    Paz, Carlos; Samake, Sibiry; Anderson, Jennifer M; Faye, Ousmane; Traore, Pierre; Tall, Koureishi; Cisse, Moumine; Keita, Somita; Valenzuela, Jesus G; Doumbia, Seydou

    2013-03-01

    Leishmania major is the only species of Leishmania known to cause cutaneous leishmanisis (CL) in Mali. We amplified Leishmania DNA stored on archived Giemsa-stained dermal scraping slides obtained from self-referral patients with clinically suspected CL seen in the Center National d'Appui A La Lutte Contre La Maladie (CNAM) in Bamako, Mali, to determine if any other Leishmania species were responsible for CL in Mali and evaluate its geographic distribution. Polymerase chain reaction (PCR) amplification was performed using a Leishmania species-specific primer pair that can amplify DNA from L. major, L. tropica, L. infantum, and L. donovani parasites, possible causative agents of CL in Mali. L. major was the only species detected in 41 microscopically confirmed cases of CL from five regions of Mali (Kayes, Koulikoro, Ségou, Mopti, and Tombouctou). These results implicate L. major as the predominant, possibly exclusive species responsible for CL in Mali.

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

    PubMed

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

    2013-03-01

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

  19. Detection of a broad range of Leishmania species and determination of parasite load of infected mouse by real-time PCR targeting the arginine permease gene AAP3.

    PubMed

    Tellevik, Marit Gjerde; Muller, Karl Erik; Løkken, Karen Rebbestad; Nerland, Audun Helge

    2014-09-01

    Leishmaniasis is one of the world's most neglected infectious diseases, affecting around 12 million people and more than 350 million at risk of infection. The clinical picture varies from self-healing cutaneous lesions to severe visceral infections, but still no commercial vaccines for humans are available and the currently used drugs have unpleasant side effects. Here we report a real-time PCR assay targeting the arginine permease gene AAP3 that can be applied for all the nine different species of the Leishmania genus tested; 4 Old World species and 5 New World species, from both L. (Leishmania) and L. (Viannia) subgenera. No cross-reaction was seen with Trypanosoma cruzi, Trypanosoma brucei, human or mouse genomic DNA. The assay has a high sensitivity, with a limit of detection of 10fg DNA for L. (L.) major and L. (L.) donovani, and 100fg DNA for L. (V.) braziliensis, and can be used for both qualitative and quantitative purposes. This AAP3-Assay, run in duplex with a host specific gene-assay, was also successfully used for quantification of parasite load of footpads from L. (L.) major-infected mice. It can therefore be a valuable tool in applications like monitoring effects of drugs, the selection of vaccine candidates and in screening patients, including asymptomatic carriers. PMID:24859532

  20. Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

    PubMed

    Belaz, Sorya; Rattier, Thibault; Lafite, Pierre; Moreau, Philippe; Routier, Françoise H; Robert-Gangneux, Florence; Gangneux, Jean-Pierre; Daniellou, Richard

    2015-10-13

    The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism. PMID:26279524

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

    PubMed Central

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

    2012-01-01

    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 in order 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. Herein, 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 (LPG) isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX (CoPP), a 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 to those from wild type mice. Furthermore, upregulation of HO-1 by CoPP 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. PMID:22461696

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

    PubMed

    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.

  3. Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites.

    PubMed

    Chenik, M; Lakhal, S; Ben Khalef, N; Zribi, L; Louzir, H; Dellagi, K

    2006-04-01

    Leishmania parasites are able to survive in host macrophages despite the harsh phagolysosomal vacuoles conditions. This could reflect, in part, their capacity to secrete proteins that may play an essential role in the establishment of infection and serve as targets for cellular immune responses. To characterize Leishmania major proteins excreted/secreted early after promastigote entry into the host macrophage, we have generated antibodies against culture supernatants of stationary-phase promastigotes collected 6 h after incubation in conditions that partially reproduce those prevailing in the parasitophorous vacuole. The screening of an L. major cDNA library with these antibodies led us to isolate 33 different cDNA clones that we report here. Sequence analysis revealed that the corresponding proteins could be classified in 3 groups: 9 proteins have been previously described as excreted/secreted in Leishmania and/or other species; 11 correspond to known proteins already characterized in Leishmania and/or other species although it is unknown whether they are excreted/secreted and 13 code for unknown proteins. Interestingly, the latter are transcribed as shown by RT-PCR and some of them are stage regulated. The L. major excreted/secreted proteins may constitute putative virulence factors, vaccine candidates and/or new drug targets.

  4. Leishmania is not prone to develop resistance to tamoxifen.

    PubMed

    Coelho, Adriano C; Trinconi, Cristiana T; Senra, Luisa; Yokoyama-Yasunaka, Jenicer K U; Uliana, Silvia R B

    2015-12-01

    Tamoxifen, an antineoplastic agent, is active in vitro and in vivo against the parasitic protozoa Leishmania. As part of our efforts to unravel this drug's mechanisms of action against the parasite and understand how resistance could arise, we tried to select tamoxifen-resistant Leishmania amazonensis. Three different strategies to generate tamoxifen resistant mutants were used: stepwise increase in drug concentration applied to promastigote cultures, chemical mutagenesis followed by drug selection and treatment of infected mice followed by selection of amastigotes. For amastigote selection, we employed a method with direct plating of parasites recovered from lesions into semi-solid media. Tamoxifen resistant parasites were not rescued by any of these methods. Miltefosine was used as a control in selection experiments and both stepwise selection and chemical mutagenesis allowed successful isolation of miltefosine resistant mutants. These findings are consistent with a multi-target mode of action to explain tamoxifen's leishmanicidal properties. Considering that drug resistance is a major concern in anti-parasitic chemotherapy, these findings support the proposition of using tamoxifen as a partner in drug combination schemes for the treatment of leishmaniasis.

  5. Sensitivity of Leishmania viannia panamensis to Pentavalent Antimony Is Correlated with the Formation of Cleavable DNA-Protein Complexes

    PubMed Central

    Lucumi, Armando; Robledo, Sara; Gama, Vivian; Saravia, Nancy G.

    1998-01-01

    The emergence of Leishmania less sensitive to pentavalent antimonial agents (SbVs), the report of inhibition of purified topoisomerase I of Leishmania donovani by sodium stibogluconate (Pentostam), and the uncertain mechanism of action of antimonial drugs prompted an evaluation of SbVs in the stabilization of cleavable complexes in promastigotes of Leishmania (Viannia). The effect of camptothecin, an inhibitor of topoisomerase, and additive-free meglumine antimoniate (Glucantime) on the stabilization of cleavable DNA-protein complexes associated with the inhibition of topoisomerase was assessed in the human promonocytic cell line U-937, promastigotes of L. (Viannia) panamensis selected for SbV resistance in vitro, and the corresponding wild-type strain. The stabilization of cleavable complexes and the 50% effective dose (ED50) of SbVs for parasites isolated from patients with relapses were also evaluated. The median ED50 for the wild-type strain was 16.7 μg of SbV/ml, while that of the line selected for resistance was 209.5 μg of SbV/ml. Treatment with both meglumine antimoniate and sodium stibogluconate (20 to 200 μg of SbV/ml) stabilized DNA-protein complexes in the wild-type strain but not the resistant line. The ED50s of the SbVs for Leishmania strains from patients with relapses was comparable to those for the line selected for in vitro resistance, and DNA-protein complexes were not stabilized by exposure to meglumine antimoniate. Cleavable complexes were observed in all Leishmania strains treated with camptothecin. Camptothecin stabilized cleavable complexes in U-937 cells; SbVs did not. The selective effect of the SbVs on the stabilization of DNA-protein complexes in Leishmania and the loss of this effect in naturally resistant or experimentally derived SbV-resistant Leishmania suggest that topoisomerase may be a target of antimonial drugs. PMID:9687395

  6. Glycoinositol phospholipids from American Leishmania and Trypanosoma spp: partial characterization of the glycan cores and the human humoral immune response to them.

    PubMed Central

    Avila, J L; Rojas, M; Acosta, A

    1991-01-01

    The glycoinositol phospholipid (GIPL) profiles of American Leishmania spp. (L. mexicana and L. braziliensis), Leishmania donovani, and American Trypanosoma spp. (T. cruzi and T. rangeli) were compared. The major GIPLs in these parasites include tetraglycosyl-, pentaglycosyl-, and hexaglycosylphosphatidylinositol. These were partially identified by their comigration by high-performance thin-layer chromatography with purified L. major GIPLs, gas-liquid chromatography of the monosaccharides released after aqueous HF treatment, N-acetylation and methanolysis, sensitivity to exoglycosidases, and antibody absorption on several specific natural haptens. Members of the genus Leishmania have two other highly polar glycolipids, while the T. rangeli glycolipid profile was quite different from those of other kinetoplastids that were studied. On a weight basis, the glycan core of L. major GIPL-1 is the most reactive, followed by GIPL-3 and GIPL-2. Antibodies to the core glycans of GIPL-1, GIPL-2, and GIPL-3 were present at a low titer in the serum of every normal individual studied, while elevated GIPL-2 antibody levels were present in 80 to 100% of T. cruzi-, T. rangeli-, or L. donovani-infected patients, with lower values being found for GIPL-3 (30 to 60%) and GIPL-1 (30 to 50%). Except for GIPL-2 antibodies, which were mainly located on immunoglobulin G (IgG) and IgM, GIPL-1 and GIPL-3 antibodies were mainly distributed in IgM, with lower reactivity present in IgG. Antigen-antibody binding was very selectively blocked with Gal(alpha 1-3)Man, or Gal(beta 1-4)Man, Gal(alpha 1-3)Gal, and Gal(alpha 1-6)Gal for GIPL-1, GIPL-2, and GIPL-3 antibodies, respectively. Images PMID:1719024

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

    PubMed Central

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

    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 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. PMID:26645812

  8. T cell responses to crude and defined leishmanial antigens in patients from the lower Amazon region of Brazil infected with different species of Leishmania of the subgenera Leishmania and Viannia.

    PubMed

    Silveira, F T; Blackwell, J M; Ishikawa, E A; Braga, R; Shaw, J J; Quinnell, R J; Soong, L; Kima, P; McMahon-Pratt, D; Black, G F; Shaw, M A

    1998-01-01

    Amazonian localized cutaneous leishmaniasis (LCL) is caused by parasites of the subgenera Leishmania and Viannia. Respectively, these parasites may cause diffuse cutaneous leishmaniasis (DCL) and mucocutaneous leishmaniasis (MCL). This, together with differing skin test responses, suggests some species-specificity in cell mediated immunity. In this study, T cell responses (proliferative and interferon-gamma) to crude and defined antigens were examined in paired samples pre and post chemotherapy. Untreated L. (L.) amazonensis LCL patients showed lower responses to crude leishmanial antigens than the L. (V.) spp. group. L. (V.) braziliensis antigen was a more potent stimulator of T cell responses than L. (L.) amazonensis antigen in all patient groups. Few positive responses were seen to the L. (L.) amazonensis glycoprotein GP46. A substantial proportion of LCL patients did respond to the L. (L.) pifanoi amastigote antigens A2, and the surface membrane glycoprotein P8. DCL patients were poor responders to all leishmanial antigens, except GP46. In contrast, MCL patients were good responders to all antigens except GP46 and A2. A significant rise in the response to P8 and A2 antigen was seen post treatment across all LCL and MCL patients, indicating that these antigens might provide suitable vaccine candidates.

  9. Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

    PubMed Central

    Suárez, Milagros; Valencia, Braulio M.; Jara, Marlene; Alba, Milena; Boggild, Andrea K.; Dujardin, Jean-Claude; Llanos-Cuentas, Alejandro; Arevalo, Jorge; Adaui, Vanessa

    2015-01-01

    Background Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion. Methodology/Principal Findings We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4). Conclusion/Significance Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is

  10. Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis.

    PubMed

    McCall, Laura-Isobel; Zhang, Wen-Wei; Dejgaard, Kurt; Atayde, Vanessa Diniz; Mazur, Alexander; Ranasinghe, Shalindra; Liu, Jing; Olivier, Martin; Nilsson, Tommy; Matlashewski, Greg

    2015-02-01

    Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

  11. Protein turnover and differentiation in Leishmania

    PubMed Central

    Besteiro, Sébastien; Williams, Roderick A.M.; Coombs, Graham H.; Mottram, Jeremy C.

    2007-01-01

    Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facilitated the identification of the parasite’s vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks. PMID:17493624

  12. Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations.

    PubMed Central

    Davies, E V; Singleton, A M; Blackwell, J M

    1988-01-01

    Earlier studies had shown that the viscerotropic NIH 173 strain of cutaneous Leishmania major fails to come under Lsh gene control. Visceral Leishmania donovani LV9 and another viscerotropic cutaneous strain, Leishmania mexicana mexicana LV4, are controlled by Lsh. The results of double-infection experiments presented here show that expression of Lsh resistance against L. mexicana mexicana was enhanced in the presence of L. donovani, whereas L. major still failed to come under Lsh gene control, even in the presence of L. donovani. Prior irradiation (850 rads) of mice showed that in the absence of infiltrating monocytes, Lsh did exert some influence over L. major. The presence of a higher infiltrate of fresh monocytes after L. major infection was confirmed in liver macrophage populations isolated from mice after infection in vivo and in liver cryosections immunostained with monoclonal antibody M1/70 directed against the type 3 complement receptor CR3. The results support the hypothesis that Lsh is expressed maximally in the resident tissue macrophages and poorly in the immature macrophages preferentially infected by L. major amastigotes. Images PMID:3356462

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

    PubMed

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

    2009-08-01

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

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

    PubMed

    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.

  15. Lectin-binding properties of different Leishmania species.

    PubMed

    Andrade, A F; Saraiva, E M

    1999-07-01

    Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 x 10(6) receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 x 10(6) M(-1).

  16. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  17. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs.

    PubMed

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

    2016-06-01

    The objectives of this study were to evaluate the occurrence of Leishmania infantum in the male and female genital tract and female mammary glands of dogs and the parasite burden and to identify histological alterations associated with this protozoan. Twenty male and 20 female Leishmania-seropositive dogs with isolation of L. infantum were examined. Tissue samples of the prepuce, glans, epididymis, testes, prostate, vulva, vagina, uterus, uterine tubes, and mammary glands were analyzed by immunohistochemistry and histopathology. For parasitological culture and in situ hybridization, samples were collected from the testis, epididymis, and uterus. Additionally, seminal fluid was aspirated from the epididymis for parasitological culture. In the genital tract, 34 (85 %) dogs, including 18 males and 16 females, were positive for Leishmania. Of these, 27 (79 %) animals were symptomatic. Leishmania was detected in the mammary glands of 13 (65 %) females. L. infantum was isolated for the first time from the seminal fluid and uterus of naturally infected dogs. The parasite burden and intensity of the inflammatory reaction were greater in the prepuce and glans of males and in the vulva and mammary glands of females. In addition to inflammation, testicular degeneration, atrophy, absence of spermatogenesis, and necrosis were observed. Detection of amastigote forms in the mammary gland lumen indicates possible elimination of this parasite in milk. The frequent parasitism observed in the genital tract of infected males and females and the viability of L. infantum in seminal fluid and uterus suggest the possibility of bidirectional venereal and vertical transmission. PMID:26979730

  18. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs.

    PubMed

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

    2016-06-01

    The objectives of this study were to evaluate the occurrence of Leishmania infantum in the male and female genital tract and female mammary glands of dogs and the parasite burden and to identify histological alterations associated with this protozoan. Twenty male and 20 female Leishmania-seropositive dogs with isolation of L. infantum were examined. Tissue samples of the prepuce, glans, epididymis, testes, prostate, vulva, vagina, uterus, uterine tubes, and mammary glands were analyzed by immunohistochemistry and histopathology. For parasitological culture and in situ hybridization, samples were collected from the testis, epididymis, and uterus. Additionally, seminal fluid was aspirated from the epididymis for parasitological culture. In the genital tract, 34 (85 %) dogs, including 18 males and 16 females, were positive for Leishmania. Of these, 27 (79 %) animals were symptomatic. Leishmania was detected in the mammary glands of 13 (65 %) females. L. infantum was isolated for the first time from the seminal fluid and uterus of naturally infected dogs. The parasite burden and intensity of the inflammatory reaction were greater in the prepuce and glans of males and in the vulva and mammary glands of females. In addition to inflammation, testicular degeneration, atrophy, absence of spermatogenesis, and necrosis were observed. Detection of amastigote forms in the mammary gland lumen indicates possible elimination of this parasite in milk. The frequent parasitism observed in the genital tract of infected males and females and the viability of L. infantum in seminal fluid and uterus suggest the possibility of bidirectional venereal and vertical transmission.

  19. Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis.

    PubMed

    Ramírez, Laura; Santos, Diego M; Souza, Ana P; Coelho, Eduardo A F; Barral, Aldina; Alonso, Carlos; Escutia, Marta R; Bonay, Pedro; de Oliveira, Camila I; Soto, Manuel

    2013-02-18

    Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.

  20. Genetic and clinical characterization of canine leishmaniasis caused by Leishmania (Leishmania) infantum in northeastern Argentina.

    PubMed

    Barroso, Paola A; Nevot, M Cecilia; Hoyos, Carlos L; Locatelli, Fabricio M; Lauthier, Juan J; Ruybal, Paula; Cardozo, Rubén M; Russo, Pablo D; Vassiliades, Carola N; Mora, María C; Estévez, J Octavio; Hashiguchi, Yoshihisa; Korenaga, Masataka; Basombrío, Miguel A; Marco, Jorge D

    2015-10-01

    Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.

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

    PubMed

    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.

  2. Mutagenicity, genotoxicity, and scavenging activities of extracts from the soft coral Chromonephthea braziliensis: a possibility of new bioactive compounds.

    PubMed

    Carpes, R M; Fleury, B G; Lages, B G; Pinto, A C; Aiub, C A F; Felzenszwalb, I

    2013-09-13

    Coral reefs are diverse ecosystems that have a high density of biodiversity leading to intense competition among species. These species may produce unknown substances, many with pharmacological value. Chromonephthea braziliensis is an invasive soft coral from the Indo-Pacific Ocean that is possibly transported by oil platforms and whose presence can be a threat to a region's biodiversity. This species produces secondary metabolites that are responsible for inducing damage to the local ecosystem. In the present study, extracts were prepared from dried colonies of C. braziliensis (solvents: hexane, dichloromethane, ethyl acetate, and methanol). We evaluated their mutagenicity using the Salmonella reverse mutation assay (TA97, TA98, TA100, and TA102 strains), their genotoxicity using the DNA breakage analysis and micronucleus assay, and scavenging activity using the 1,1-diphenyl-2-picrylhydrazyl-free radical assay. Cytotoxicity and mutagenicity were not observed for any of the extracts. Genotoxicity was observed for the dichloromethane, ethyl acetate, and methanol extracts at high concentrations, but no DNA damage was observed in the micronucleus assay. Scavenging activity was not detected.

  3. Biochemical effects of riluzole on Leishmania parasites.

    PubMed

    Guerrieri, Davide; Ferrari, Stefania; Costi, M Paola; Michels, Paul A M

    2013-03-01

    We have previously shown that riluzole (6-(trifluoromethoxy)benzothiazol-2-amine), an agent used to treat CNS disorders, possesses inhibitory activity against pteridine reductase (PTR1) in pathogenic protists at low micromolar concentrations. Therefore, the potential use of this drug in anti-parasitic chemotherapy deserves evaluation. In this study, we report the effect of this compound on cell cultures of Leishmania mexicana and L. major. The anti-parasitic activity of riluzole was confirmed, with the largest effect observed when the drug was administered to cells during their exponential growth phase. Moreover, a remarkable decrease in PTR1 activity was observed in the lysates of cells pretreated with the compound, which is due to impairment of the enzyme's preferential reaction with biopterin as a cofactor. In addition, the treatment increased the parasites' susceptibility to oxidative stress, affecting the ability of Leishmania to survive under severe oxidative conditions. These results suggest that the inhibitory effect of riluzole on PTR1 is not the only mechanism through which it induces the death of Leishmania parasites.

  4. Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: risk of emergence in humans?

    PubMed

    Faye, B; Bañuls, A L; Bucheton, B; Dione, M M; Bassanganam, O; Hide, M; Dereure, J; Choisy, M; Ndiaye, J L; Konaté, O; Claire, M; Senghor, M W; Faye, M N; Sy, I; Niang, A A; Molez, J F; Victoir, K; Marty, P; Delaunay, P; Knecht, R; Mellul, S; Diedhiou, S; Gaye, O

    2010-12-01

    In the context of global warming and the risk of spreading arthropod-borne diseases, the emergence and reemergence of leishmaniasis should not be neglected. In Senegal, over the past few years, cases of canine leishmaniasis have been observed. We aim to improve the understanding of the transmission cycle of this zoonosis, to determine the responsible species and to evaluate the risk for human health. An epidemiological and serological study on canine and human populations in the community of Mont Rolland (Thiès area) was conducted. The data showed a high seroprevalence of canine leishmaniasis (>40%) and more than 30% seropositive people. The dogs' seroprevalence was confirmed by PCR data (concordance > 0.85, Kappa > 0.7). The statistical analysis showed strong statistical associations between the health status of dogs and seropositivity, the number of positive PCRs, clinical signs and the number of Leishmania isolates. For the first time, the discriminative PCRs performed on canine Leishmania strains clearly evidenced that the pathogenic agent is Leishmania infantum. The results obtained show that transmission of this species is well established in this area. That the high incidence of seropositivity in humans may be a consequence of infection with this species is discussed.

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

    PubMed Central

    Chen, M; Christensen, S B; Blom, J; Lemmich, E; Nadelmann, L; Fich, K; Theander, T G; Kharazmi, A

    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 and by synthesis, and its purity was verified by high-pressure liquid chromatography. The 50% inhibition of growth of logarithmic- and stationary-phase promastigotes of L. major, as measured by [3H]thymidine uptake, were 4 and 2.5 micrograms/ml, respectively. The growth of L. major promastigotes was totally inhibited after a 20-h incubation period with licochalcone A at 5 micrograms/ml. At a concentration of 0.5 microgram/ml, licochalcone A markedly reduced the infection rate of human peripheral blood monocyte-derived macrophages and U937 cells with L. major promastigotes and exhibited a strong intracellular killing of the parasite. These data show that intracellular Leishmania amastigotes are more susceptible than promastigotes to licochalcone A. Results of studies on the site of action of licochalcone A indicate that the target organelle appears to be the parasite mitochondria. These findings demonstrate 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. Images PMID:8109916

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

    PubMed

    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. PMID:26386969

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

    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.

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

    PubMed Central

    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

  10. Multilocus Microsatellite Typing as a New Tool for Discrimination of Leishmania infantum MON-1 Strains

    PubMed Central

    Ochsenreither, Sebastian; Kuhls, Katrin; Schaar, Matthias; Presber, Wolfgang; Schönian, Gabriele

    2006-01-01

    The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations. PMID:16455904

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

    PubMed

    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.

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

    PubMed

    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

  13. The genome sequence of Leishmania (Leishmania) amazonensis: functional annotation and extended analysis of gene models.

    PubMed

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

    2013-12-01

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

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

    PubMed

    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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Loría-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Villanueva-Lizama, Liliana Estefanía; Van Wynsberghe, Nicole Raymonde; Canto-Lara, Silvia Beatriz; Batún-Cutz, José Luis; Andrade-Narváez, Fernando José

    2013-01-01

    Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 102 and 2.5 x 106 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection. PMID:23579796

  17. A possible role for Phlebotomus (Anaphlebotomus) rodhaini (Parrot, 1930) in transmission of Leishmania donovani

    PubMed Central

    2011-01-01

    Background Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani is a major health problem in Sudan and other East African countries. In this region the only proven vectors of L. donovani are Phlebotomus orientalis in eastern Sudan, Ethiopia and Upper Nile areas of Southern Sudan and Phlebotomus martini in Ethiopia, Kenya and Southern Sudan. In this report, we present the first evidence that Phlebotomus rodhaini may also play a role in maintaining transmission of L. donovani between animal reservoir hosts in eastern Sudan. The study was conducted in a zoonotic focus of visceral leishmaniasis in Dinder National Park, eastern Sudan, where previous work showed high infection rates of L. donovani in P. orientalis. Sand flies, captured by CDC traps were dissected and examined for infection with Leishmania parasites. Parasite isolates were subjected to L. donovani specific PCR. Field experiments were also carried out to compare efficiency of rodent baited and un-baited CDC traps in collection of P. rodhaini and determine its man-biting rate. Results Three female P. rodhaini were found infected with Leishmania parasites in an astonishingly small number of flies captured in three separate field trips. Two of these isolates were typed by molecular methods as L. donovani, while the third isolate was inoculated into a hamster that was subsequently lost. Although P. rodhaini is generally considered a rare species, results obtained in this study indicate that it can readily be captured by rodent-baited traps. Results of human landing collection showed that it rarely bites humans in the area. Conclusion It is concluded that P. rodhaini is a possible vector of L. donovani between animal reservoir hosts but is not responsible for infecting humans. It is suggested that the role of P. rodhaini in transmission of L. donovani in other zoonotic foci of visceral leishmaniasis in Africa should be re-examined. PMID:22188864

  18. Cutaneous infection with Leishmania major mediates heterologous protection against visceral infection with Leishmania infantum

    PubMed Central

    Romano, Audrey; Doria, Nicole A.; Mendez, Jonatan; Sacks, David L.; Peters, Nathan C.

    2015-01-01

    Visceral Leishmaniasis (VL) is a fatal disease of the internal organs caused by the eukaryotic parasite Leishmania. Control of VL would best be achieved through vaccination. However, this has proven to be difficult partly because the correlates of protective immunity are not fully understood. In contrast, protective immunity against non-fatal cutaneous Leishmaniasis (CL) is well defined and mediated by rapidly recruited, IFN-γ-producing, Ly6C+CD4+ T cells at the dermal challenge site. Protection against CL is best achieved by prior infection or live vaccination with Leishmania major, termed leishmanization. A long-standing question is whether prior CL or leishmanization can protect against VL. Employing an intra-dermal challenge model in mice, we report that cutaneous infection with Leishmania major provides heterologous protection against visceral infection with Leishmania infantum. Protection was associated with a robust CD4+ T cell response at the dermal challenge site and in the viscera. In-vivo labeling of circulating cells revealed that increased frequencies of IFN-γ+CD4+ T cells at sites of infection is due to recruitment or retention of cells in the tissue, rather than increased numbers of cells trapped in the vasculature. Shortly after challenge IFN-γ producing cells were highly enriched for Ly6C+T-bet+ cells in the viscera. Surprisingly, this heterologous immunity was superior to homologous immunity mediated by prior infection with Leishmania infantum. Our observations demonstrate a common mechanism of protection against different clinical forms of leishmaniasis. The efficacy of leishmanization against VL may warrant the introduction of the practice in VL endemic areas or during outbreaks of disease. PMID:26371247

  19. Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs.

    PubMed

    Colombo, Fabio A; Odorizzi, Rosa M F N; Laurenti, Marcia D; Galati, Eunice A B; Canavez, Flavio; Pereira-Chioccola, Vera L

    2011-08-01

    The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination. PMID:21221638

  20. 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

    PubMed Central

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

    2015-01-01

    Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection. PMID:26513474

  1. 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.

    PubMed

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

    2015-01-01

    Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection. PMID:26513474

  2. Genetic Analysis of Leishmania donovani Tropism Using a Naturally Attenuated Cutaneous Strain

    PubMed Central

    Haydock, Andrew; Ranasinghe, Shalindra; Abeygunasekara, Priyanka; Sirimanna, Ganga; Wickremasinghe, Renu; Myler, Peter; Matlashewski, Greg

    2014-01-01

    A central question in Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection. PMID:24992200

  3. Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

    PubMed

    Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

    2015-01-01

    In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991-1992 and 2008-2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991-1992 and 2008-2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008-2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations. PMID:26302440

  4. Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

    PubMed

    Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

    2015-01-01

    In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991-1992 and 2008-2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991-1992 and 2008-2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008-2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.

  5. Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis

    PubMed Central

    Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

    2015-01-01

    In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations. PMID:26302440

  6. Cytotoxicity and antileishmanial activity of Annona muricata pericarp.

    PubMed

    Jaramillo, M C; Arango, G J; González, M C; Robledo, S M; Velez, I D

    2000-04-01

    Hexane, ethyl acetate and methanol extracts of Annona muricata pericarp were tested in vitro against Leishmania braziliensis and L. panamensis promastigotes, and against cell line U-937. The ethyl acetate extract was more active than the other extracts and even of Glucantime used as reference substance. Its fractionation led to the isolation of three acetogenins--annonacin, annonacin A and annomuricin A.

  7. Activity of Cuban Plants Extracts against Leishmania amazonensis

    PubMed Central

    García, Marley; Monzote, Lianet; Scull, Ramón; Herrera, Pedro

    2012-01-01

    Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a major health problem worldwide that affects millions of people especially in the developing nations. There is no immunoprophylaxis (vaccination) available for Leishmania infections, and conventional treatments are unsatisfactory; therefore, antileishmanial drugs are urgently needed. In this work, 48 alcoholic extracts from 46 Cuban plants were evaluated by an in vitro bioassay against Leishmania amazonensis. Furthermore, their toxicity was assayed against murine macrophage. The three most potent extracts against the amastigote stage of Leishmania amazonensis were from Hura crepitans, Bambusa vulgaris, and Simarouba glauca. PMID:22530133

  8. Rapid fluorescent assay for screening drugs on Leishmania amastigotes.

    PubMed

    Shimony, Orly; Jaffe, Charles L

    2008-10-01

    A rapid fluorescent viability assay employing alamarBlue was optimized for use with Leishmania axenic amastigotes, the stage of the parasite responsible for disease pathology. The activity of two protein kinase inhibitors, Staurosporine and H-89, as well as Amphotericin B, on promastigotes and amastigotes of Leishmania donovani and Leishmania tropica was compared. Both protein kinase inhibitors inhibited promastigote growth at lower concentrations than amastigotes, while the GI(50) for Amphotericin B on both stages was similar. This assay only requires a limited number of axenic amastigotes (50,000 cells/well) and can be used to rapidly screen large chemical or natural product libraries for activity against amastigotes.

  9. Occurrence of Leishmania infantum cutaneous leishmaniasis in central Tunisia.

    PubMed

    BenSaid, Moncef; Guerbouj, Souheila; Saghrouni, Fatma; Fathallah-Mili, Akila; Guizani, Ikram

    2006-06-01

    Cutaneous leishmaniasis (CL) due to Leishmania infantum occurs sporadically in Tunisia where its distribution is confined to the northern parts of the country. However, during the past decade there have been occasional repeated reports of cases from areas in central Tunisia, known to be free of CL. Epidemiological, clinical and parasitological data regarding these patients were collected and analysed. Data were very suggestive of the sporadic form of CL due to L. infantum. The parasites contained within the lesions of some of the patients were characterised by two different previously described PCR assays, each having different resolutive powers. The first assay, which amplified complete kDNA minicircles, showed a fragment size characteristic of the L. donovani complex; whilst the second consisted of a PCR-RFLP analysis targeting the gp63 coding sequences that confirmed assignment of the parasites to L. infantum species while illustrating its differences from the reference isolate. These findings confirm the aetiology of CL in the concerned areas in central Tunisia and suggest that L. infantum CL might be more prevalent and widespread than previously thought, or possibly emerging in these areas. PMID:16356518

  10. Leishmania tropica in Stray Dogs in Southeast Iran

    PubMed Central

    BAMOROVAT, Mehdi; SHARIFI, Iraj; DABIRI, Shahriar; MOHAMMADI, Mohammad Ali; FASIHI HARANDI, Majid; MOHEBALI, Mehdi; AFLATOONIAN, Mohammad Reza; KEYHANI, Alireza

    2015-01-01

    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. PMID:26576349

  11. Anti-Leishmania Activity of Osthole

    PubMed Central

    Kermani, Elaheh Kordzadeh; Sajjadi, Seyed Ebrahim; Hejazi, Seyed Hossein; Arjmand, Reza; Saberi, Sedigheh; Eskandarian, Abbas Ali

    2016-01-01

    Background: Treatment of cutaneous leishmaniasis (CL) is occasionally highly resistant to pentavalent antimonials, the gold standard in pharmacotherapy of CL. Since there is no effective vaccine, the discovery of natural antileishmanial products as complementary therapeutic agents could be used to improve the current regimens. Objective: In this study in vitro and in vivo antileishmanial activities of osthole, a natural coumarin known to possess antibacterial and parasiticidal activities are evaluated. Materials and Methods: Leishmania major infected J774.A1 macrophages were treated with increasing concentrations of osthole. CL lesions of BALB/c mice were treated topically with 0.2% osthole. Results: Osthole exhibited dose-dependent leishmanicidal activity against intracellular amastigotes with IC50 value of 14.95 μg/ml. Treatment of CL lesions in BALB/c mice with osthole significantly declined lesion progression compared to untreated mice (P < 0.05), however did not result in recovery. Conclusion: Osthole demonstrated remarkable leishmanicidal activity in vitro. Higher concentrations of osthole may demonstrate the therapeutic property in vivo. SUMMARY In vitro and in vivo antileishmanial activities of osthole, a pernylated coumarin extracted from Prangos asperula Boiss., are studied against Leishmania major. PMID:27114685

  12. The Leishmania ARL-1 and Golgi Traffic

    PubMed Central

    Tetaud, Emmanuel; Cuvillier, Armelle; Lartigue, Lydia; Ambit, Audrey; Robinson, Derrick R.; Merlin, Gilles

    2008-01-01

    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. PMID:18286177

  13. Proteome profiling of Leishmania infantum promastigotes.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; Larraga, Vicente

    2011-01-01

    A proteome analysis of the promastigote stage of the trypanosomatid parasite Leishmania infantum (MON-1 zymodeme) is described here for the first time. Total protein extracts were prepared at early logarithmic and stationary phases of replicate axenic cultures and processed by 2D electrophoresis (pH 3-10). A total of 28 differentially regulated proteins were identified by matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry. This approach has revealed that the electron transfer flavoprotein (ETF) and the eukaryotic elongation factor 1α (eEF1α) subunit have the same differential expression pattern at the protein and mRNA levels, up-regulation in the stationary phase. A low-molecular-weight isoform and an alternatively processed form of the eEF1α subunit have been detected. A 51 kDa subunit of replication factor A is up-regulated in dividing logarithmic promastigotes. None of the proteins described here shows opposite differential regulation values with the corresponding mRNA levels. Taken together with previous approaches to the proteome and the transcriptome, this report contributes to the elucidation of the differential regulation patterns of the ETF, the eEF1α subunit, the 40S ribosomal protein S12, α-tubulin and the T-complex protein 1 subunit γ throughout the life cycle of the parasites from the genus Leishmania. PMID:21569158

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

    PubMed Central

    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

  15. Successful vaccination against Leishmania chagasi infection in BALB/c mice with freeze-thawed Leishmania antigen and Corynebacterium parvum.

    PubMed

    Vilela, Márcia de Carvalho; Gomes, Daniel Cláudio de Oliveira; Marques-da-Silva, Eduardo de Almeida; Serafim, Tiago Donatelli; Afonso, Luis Carlos Crocco; Rezende, Simone Aparecida

    2007-01-01

    This study evaluated the potential of a Leishmania antigen vaccine in protecting BALB/c mice against Leishmania chagasi. Mice received two subcutaneous doses of L. amazonensis vaccine with Corynebacterium parvum and subsequent boost was done without adjuvant. One week later, mice were challenged with L. chagasi. We observed that this vaccine caused a significant reduction in parasite load in liver and spleen and induced a high production of IFN-gamma and IL-4 by spleen cells from vaccinated mice in response to Leishmania antigen. Together, our data show that this vaccine is capable of inducing a Th1/Th2 response that is important to control parasite replication. PMID:17919443

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

    PubMed

    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.

  17. Cell structure and cytokinesis alterations in multidrug-resistant Leishmania (Leishmania) amazonensis.

    PubMed

    Borges, V M; Lopes, U G; De Souza, W; Vannier-Santos, M A

    2005-01-01

    Multidrug-resistant Leishmania (Leishmania) amazonensis may be obtained by in vitro selection with vinblastine. In order to determine whether this phenotype is linked to structural alterations, we analyzed the cell architecture by electron microscopy. The vinblastine resistant CL2 clone of L. (L.) amazonensis, but not wild-type parasites, showed a cytokinesis dysfunction. The CL2 promastigotes had multiple nuclei, kinetoplasts and flagella, suggesting that vinblastine resistance may be associated with truncated cell division. The subpellicular microtubule plasma membrane connection was also affected. Wild-type parasites treated with vinblastine displayed similar alterations, presenting lobulated and multinucleated cells. Taken together, these data indicate that antimicrotubule drug-selected parasites may show evidence of the mutation of cytoskeleton proteins, impairing normal cell function. PMID:15592939

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

    PubMed Central

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

    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. PMID:23569768

  19. Melatonin attenuates Leishmania (L.) amazonensis infection by modulating arginine metabolism.

    PubMed

    Laranjeira-Silva, Maria Fernanda; Zampieri, Ricardo A; Muxel, Sandra M; Floeter-Winter, Lucile Maria; Markus, Regina P

    2015-11-01

    Acute inflammatory responses induced by bacteria or fungi block nocturnal melatonin synthesis by rodent pineal glands. Here, we show Leishmania infection does not impair daily melatonin rhythm in hamsters. Remarkably, the attenuated parasite burden and lesion progression in hamsters infected at nighttime was impaired by blockage of melatonin receptors with luzindole, whereas melatonin treatment during the light phase attenuated Leishmania infection. In vitro studies corroborated in vivo observations. Melatonin treatment reduced macrophage expression of Cat-2b, Cat1, and ArgI, genes involved in arginine uptake and polyamine synthesis. Indeed, melatonin reduced macrophage arginine uptake by 40%. Putrescine supplementation reverted the attenuation of infectivity by melatonin indicating that its effect was due to the arrest of parasite replication. This study shows that the Leishmania/host interaction varies in a circadian manner according to nocturnal melatonin pineal synthesis. Our results provide new data regarding Leishmania infectiveness and show new approaches for applying agonists of melatonin receptors in Leishmaniasis therapy.

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

    PubMed Central

    Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    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. PMID:26162094

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

    PubMed

    Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    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. PMID:26162094

  2. Hepatic extracellular matrix alterations in dogs naturally infected with Leishmania (Leishmania) chagasi

    PubMed Central

    Melo, Ferdinan Almeida; Moura, Eliane Perlatto; Ribeiro, Raul Rio; Alves, Cíntia Fontes; Caliari, Marcelo Vidigal; Tafuri, Washington Luiz; da Silva Calabrese, Kátia; Tafuri, Wagner Luiz

    2009-01-01

    The aim of this work was to study alterations in the extracellular matrix of liver in dogs naturally infected with Leishmania (Leishmania) chagasi that are correlated with clinical aspects and with histological, parasitological and immunological findings. The study was carried out on 30 dogs, 10 uninfected (control group) and 20 infected. The infected animals were further divided into two groups: an asymptomatic group of 10 dogs without clinical signs of the disease; and a symptomatic group of 10 dogs with classical clinical signs. All thirty animals were mongrel dogs of undefined age, obtained from the municipality of Belo Horizonte, MG, metropolitan area. During necropsy, liver fragments were collected and fixed in 10% buffered formaldehyde for histological examination. Paraffined sections of the tissues were stained with haematoxylin–eosin, Gomori’s ammoniacal silver stain for reticular fibres and strepto-avidin peroxidase for immunohistochemical detection of Leishmania amastigotes. Frozen tissue sections were stained by immunofluorescence for fibronectin (FN) and laminin (LN). Liver collagen deposition was significantly greater in the infected than the control animals and differed significantly between the symptomatic and asymptomatic dogs. There was a positive correlation between the parasite load and liver collagen deposition. The increased collagen deposition in infected animal livers may be associated with the parasite burden. Adhesive FN and LN fibres were significantly more highly expressed in the livers of symptomatic than of asymptomatic dogs. Our results demonstrate that canine visceral leishmaniasis causes fibrogenesis in liver, associated with the parasite load and degenerative processes. PMID:19765108

  3. Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

    PubMed Central

    Serafim, Tiago Donatelli; Figueiredo, Amanda Braga; Costa, Pedro Augusto Carvalho; Marques-da-Silva, Eduardo Almeida; Gonçalves, Ricardo; de Moura, Sandra Aparecida Lima; Gontijo, Nelder Figueiredo; da Silva, Sydnei Magno; Michalick, Marilene Suzan Marques; Meyer-Fernandes, José Roberto; de Carvalho, Roberto Paes; Uliana, Silvia Reni Bortolin; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos Crocco

    2012-01-01

    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. PMID:23050028

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

    PubMed

    Serafim, Tiago Donatelli; Figueiredo, Amanda Braga; Costa, Pedro Augusto Carvalho; Marques-da-Silva, Eduardo Almeida; Gonçalves, Ricardo; de Moura, Sandra Aparecida Lima; Gontijo, Nelder Figueiredo; da Silva, Sydnei Magno; Michalick, Marilene Suzan Marques; Meyer-Fernandes, José Roberto; de Carvalho, Roberto Paes; Uliana, Silvia Reni Bortolin; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos Crocco

    2012-01-01

    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.

  5. Performance of commercially available serological diagnostic tests to detect Leishmania infantum infection on experimentally infected dogs.

    PubMed

    Rodríguez-Cortés, Alhelí; Ojeda, Ana; Todolí, Felicitat; Alberola, Jordi

    2013-01-31

    Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of a widespread serious zoonotic disease that affects both humans and dogs. Prevalence and incidence of the canine infection are important parameters to determine the risk and the ways to control this reemergent zoonosis. Unfortunately, there is not a gold standard test for Leishmania infection. Our aim was to assess the operative validity of commercial tests used to detect antibodies to Leishmania in serum samples from experimental infections. Three ELISA tests (LEISCAN(®) Leishmania ELISA Test, INGEZIM(®) LEISHMANIA, and INGEZIM(®) LEISHMANIA VET), three immunochromatographic tests (INGEZIM(®) LEISHMACROM, SNAP(®) Leishmania, and WITNESS(®) Leishmania), and one IFAT were evaluated. LEISCAN(®) Leishmania ELISA test achieved the highest sensitivity and accuracy (both 0.98). Specificity was 1 for all tests except for IFAT. All tests but IFAT obtained a positive predictive value of 1, while the maximum negative predictive value was achieved by LEISCAN(®) Leishmania ELISA Test (0.93). The best positive likelihood ratio was obtained by INGEZIM(®) LEISHMANIA VET (30.26), while the best negative likelihood ratio was obtained by LEISCAN(®) Leishmania ELISA Test (0.02). The highest diagnostic odds ratio was achieved by LEISCAN(®) Leishmania ELISA Test (729.00). The largest area under the ROC curve was obtained by LEISCAN(®) Leishmania ELISA Test (0.981). Quantitative ELISA based tests performmed better than qualitative tests ("Rapid Tests"), and the test best suited to detect Leishmania in infected dogs and to provide clinically useful information was LEISCAN(®) Leishmania ELISA Test. This and other results point also to the need of revising the status of IFAT as a gold standard for the diagnosis of leishmaniasis.

  6. Structures of the Leishmania infantum polymerase beta

    PubMed Central

    Mejia, Edison; Burak, Matthew; Alonso, Ana; Larraga, Vicente; Kunkel, Thomas A.; Bebenek, Katarzyna; Garcia-Diaz, Miguel

    2014-01-01

    Protozoans of the genus Leishmania, the pathogenic agent causing leishmaniasis, encode the family X DNA polymerase Li Pol β. Here, we report the first crystal structures of Li Pol β. Our pre- and post-catalytic structures show that the polymerase adopts the common family X DNA polymerase fold. However, in contrast to other family X DNA polymerases, the dNTP-induced conformational changes in Li Pol β are much more subtle. Moreover, pre- and post-catalytic structures reveal that Li Pol β interacts with the template strand through a nonconserved, variable region known as loop3. Li Pol β Δloop3 mutants display a higher catalytic rate, catalytic efficiency and overall error rates with respect to WT Li Pol β. These results further demonstrate the subtle structural variability that exists within this family of enzymes and provides insight into how this variability underlies the substantial functional differences among their members. PMID:24666693

  7. Leishmaniosis (Leishmania infantum infection) in dogs.

    PubMed

    Gharbi, M; Mhadhbi, M; Rejeb, A; Jaouadi, K; Rouatbi, M; Darghouth, M A

    2015-08-01

    The authors present an overview of canine leishmaniosis due to Leishmania infantum. This protozoan is transmitted by sandflies and the disease is frequently characterised by chronic evolution. Cutaneous and visceral clinical signs appear as the infection progresses. Lymph node enlargement, emaciation and skin lesions are the main signs observed in the classical forms of the disease. Control is difficult since infected dogs remain carriers for years and may relapse at any time. The mass screening of infected animals and their treatment or euthanasia represent the best way to reduce the prevalence of this disease in endemic regions. Further research is needed to improve the efficiency of the vaccines available to protect dogs against infection. This disease is zoonotic; in humans, clinical cases are reported mainly in elderly people, the young and those whose immune systems have been compromised. PMID:26601461

  8. Delayed culture of Leishmania in skin biopsies.

    PubMed

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

    1999-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-03-01

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

  11. Trypanosoma cruzi and Leishmania infantum chagasi Infection in Wild Mammals from Maranhão State, Brazil.

    PubMed

    da Costa, Andréa Pereira; Costa, Francisco Borges; Soares, Herbert Sousa; Ramirez, Diego Garcia; Mesquita, Eric Takashi Kamakura de Carvalho; Gennari, Solange Maria; Marcili, Arlei

    2015-11-01

    Trypanosoma and Leishmania are obligate parasites that cause important diseases in human and domestic animals. Wild mammals are the natural reservoirs of these parasites, which are transmitted by hematophagous arthropods. The present study aimed to detect the natural occurrence of trypanosomatids through serological diagnosis, PCR of whole blood and blood culture (hemoculture), and phylogenetic relationships using small subunit ribosomal DNA (SSU rDNA), cytochrome b, and glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. Samples from 131 wild animals, including rodents, marsupials, and bats, were sampled in six areas in the state of Maranhão, in a transition zone of semiarid climates northeast of the equatorial humid Amazon. Serological analysis for Leishmania (Leishmania) infantum chagasi was performed in opossums by indirect fluorescent antibody test (IFAT), and all animals were serologically negative. Nine positive hemocultures (6.77%) were isolated and cryopreserved and from mammals of the Didelphimorphia and Chiroptera orders and positioned in phylogenies on the basis of sequences from different genes with reference strains of Trypanosoma cruzi marinkellei and T. cruzi. From primary samples (blood and tissues) only one bat, Pteronotus parnellii, was positive to SSU rDNA and gGAPDH genes and grouped with the L. infantum chagasi branch. The studies conducted in Maranhão State provide knowledge of parasite diversity. It is important to determine the presence of trypanosomatids in wild mammals with synanthropic habits. PMID:26501369

  12. Trypanosoma cruzi and Leishmania infantum chagasi Infection in Wild Mammals from Maranhão State, Brazil.

    PubMed

    da Costa, Andréa Pereira; Costa, Francisco Borges; Soares, Herbert Sousa; Ramirez, Diego Garcia; Mesquita, Eric Takashi Kamakura de Carvalho; Gennari, Solange Maria; Marcili, Arlei

    2015-11-01

    Trypanosoma and Leishmania are obligate parasites that cause important diseases in human and domestic animals. Wild mammals are the natural reservoirs of these parasites, which are transmitted by hematophagous arthropods. The present study aimed to detect the natural occurrence of trypanosomatids through serological diagnosis, PCR of whole blood and blood culture (hemoculture), and phylogenetic relationships using small subunit ribosomal DNA (SSU rDNA), cytochrome b, and glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. Samples from 131 wild animals, including rodents, marsupials, and bats, were sampled in six areas in the state of Maranhão, in a transition zone of semiarid climates northeast of the equatorial humid Amazon. Serological analysis for Leishmania (Leishmania) infantum chagasi was performed in opossums by indirect fluorescent antibody test (IFAT), and all animals were serologically negative. Nine positive hemocultures (6.77%) were isolated and cryopreserved and from mammals of the Didelphimorphia and Chiroptera orders and positioned in phylogenies on the basis of sequences from different genes with reference strains of Trypanosoma cruzi marinkellei and T. cruzi. From primary samples (blood and tissues) only one bat, Pteronotus parnellii, was positive to SSU rDNA and gGAPDH genes and grouped with the L. infantum chagasi branch. The studies conducted in Maranhão State provide knowledge of parasite diversity. It is important to determine the presence of trypanosomatids in wild mammals with synanthropic habits.

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

    PubMed

    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. PMID:25340465

  14. Molecular Detection of Leishmania major and L. turanica in Phlebotomus papatasi and First Natural Infection of P. salehi to L. major in North-East of Iran

    PubMed Central

    Rafizadeh, Sayena; Saraei, Mehrzad; Abaei, Mohammad Reza; Oshaghi, Mohammad Ali; Mohebali, Mehdi; Peymani, Amir; Naserpour-Farivar, Taghi; Bakhshi, Hassan; Rassi, Yavar

    2016-01-01

    Background: Leishmaniasis is an important public health disease in many developing countries as well in Iran. The main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in Esfarayen district, north east of Iran. Methods: Sand flies were collected by sticky papers and mounted in a drop of Puri’s medium for species identification. Polymerase chain reaction techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism were used for identification of DNA of Leishmania parasites within infected sand flies. Results: Among the collected female sand flies, two species of Phlebotomus papatasi and Phlebotomus salehi were found naturally infected with Leishmania major. Furthermore, mixed infection of Leishmania turanica and L. major was observed in one specimen of P. papatasi. Sequence analysis revealed two parasite ITS1 haplotypes including three L. major with accession numbers: KJ425408, KJ425407, KM056403 and one L. turanica. (KJ425406). The haplotype of L. major was identical (100%) to several L. major sequences deposited in GenBank, including isolates from Iran, (Gen Bank accession nos.AY573187, KC505421, KJ194178) and Uzbekistan (Accession no.FN677357). Conclusion: To our knowledge, this is the first detection of L. major within wild caught P. salehi in northeast of Iran. PMID:27308272

  15. Experimental model for reproduction of canine visceral leishmaniosis by Leishmania infantum.

    PubMed

    Fernández-Cotrina, J; Iniesta, V; Belinchón-Lorenzo, S; Muñoz-Madrid, R; Serrano, F; Parejo, J C; Gómez-Gordo, L; Soto, M; Alonso, C; Gómez-Nieto, L C

    2013-02-18

    In this report an experimental model of Leishmania infantum (L. infantum) infection in dogs is described. The data presented are derived from an overall and comparative analysis of the clinical outcomes of three groups of dogs intravenously infected with 500,000 promastigotes on different dates (2003, 2006 and 2008). The parasites used for challenge were isolated from a dog having a patent form of leishmaniosis, classified as MCAN/ES/1996/BCN150 zymodeme MON-1. Late-log-phase promastigote forms derived from cultured amastigotes obtained from the spleen of the heavily infected hamsters were used for infection. Only one single infective dose was administered to each dog. After challenge, the animals were monitored for 12 months. To analyze the disease outcome, several biopathological, immunological and parasitological end-points were considered. The analysis of the infected dogs indicated that the development of the clinical disease was very similar in the three experimental challenges, as shown by the immune response, the parasite load and the clinical and histopathological lesions detected at necropsy. A high similarity was also observed between the disease development after the experimental challenge and the one reported to occur in endemic natural infection areas, as various degrees of susceptibility to the disease and even resistance were observed in the experimentally infected animals. We believe that this challenge model faithfully reproduces and mimics the course of a natural infection and that it could be used as a suitable tool for analyzing the efficacy of anti-Leishmania drugs and vaccines.

  16. Characterization of Metabolically Quiescent Leishmania Parasites in Murine Lesions Using Heavy Water Labeling

    PubMed Central

    Kloehn, Joachim; Saunders, Eleanor C.; O’Callaghan, Sean; Dagley, Michael J.; McConville, Malcolm J.

    2015-01-01

    Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using 2H2O labeling. Infected BALB/c mice were labeled with 2H2O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ. PMID:25714830

  17. Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots.

    PubMed

    Andreadou, Margarita; Liandris, Emmanouil; Gazouli, Maria; Mataragka, Antonia; Tachtsidis, Ilias; Goutas, Nikolaοs; Vlachodimitropoulos, Dimitrios; Ikonomopoulos, John

    2016-04-01

    Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/μl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings. PMID:26658854

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

    PubMed

    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.

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

    PubMed Central

    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 < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. 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. PMID:25815025

  20. Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana.

    PubMed

    Loria-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Van Wynsberghe, Nicole Raymonde; Saldarriaga, Omar Abdul; Melby, Peter C; Andrade-Narvaez, Fernando Jose

    2016-07-01

    Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5×10(6) or 1×10(2) promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5×10(6) promastigotes developed signs of LCL at 12wpi while the mice inoculated with 1×10(2) remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5×10(6)) compared with subclinical ones (1×10(2)). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12wpi. PMID:27155064

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

    PubMed Central

    Lara-Silva, Fabiana de Oliveira; Michalsky, Érika 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. PMID:24707492

  2. Chronic infection by Leishmania amazonensis mediated through MAPK ERK mechanisms

    PubMed Central

    Martinez, Pedro A.; Petersen, Christine A.

    2014-01-01

    Leishmania amazonensis is an intracellular protozoan parasite responsible for chronic cutaneous leishmaniasis (CL). CL is a neglected tropical disease responsible for infecting millions of people worldwide. L. amazonensis promotes alteration of various signaling pathways that are essential for host cell survival. Specifically, through parasite-mediated phosphorylation of extracellular signal regulated kinase (ERK), L. amazonensis inhibits cell-mediated parasite killing and promotes its own survival by co-opting multiple host cell functions. In this review we highlight Leishmania-host cell signaling alterations focusing on those specific to 1) motor proteins, 2) prevention of NADPH subunit phosphorylation impairing reactive oxygen species production (ROS), and 3) localized endosomal signaling to up-regulate ERK phosphorylation. This review will focus upon mechanisms and possible explanations as to how Leishmania spp. evades the various layers of defense employed by the host immune response. PMID:24838145

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

    PubMed Central

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

    2015-01-01

    A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments.  How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania. PMID:26594352

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

    PubMed

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

    2015-01-01

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

  5. Drug resistance analysis by next generation sequencing in Leishmania

    PubMed Central

    Leprohon, Philippe; Fernandez-Prada, Christopher; Gazanion, Élodie; Monte-Neto, Rubens; Ouellette, Marc

    2014-01-01

    The use of next generation sequencing has the power to expedite the identification of drug resistance determinants and biomarkers and was applied successfully to drug resistance studies in Leishmania. This allowed the identification of modulation in gene expression, gene dosage alterations, changes in chromosome copy numbers and single nucleotide polymorphisms that correlated with resistance in Leishmania strains derived from the laboratory and from the field. An impressive heterogeneity at the population level was also observed, individual clones within populations often differing in both genotypes and phenotypes, hence complicating the elucidation of resistance mechanisms. This review summarizes the most recent highlights that whole genome sequencing brought to our understanding of Leishmania drug resistance and likely new directions. PMID:25941624

  6. Characterization of prenylated protein methyltransferase in Leishmania.

    PubMed Central

    Hasne, M P; Lawrence, F

    1999-01-01

    Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-l-[methyl-(3)H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans, trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, but N-acetyl-S-trans, trans-geranyl-l-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5'-[gamma-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-l-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5'-[gamma-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa. PMID:10477261

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

    PubMed

    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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    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

  10. CD8+ T Cells in Leishmania Infections: Friends or Foes?

    PubMed Central

    Stäger, Simona; Rafati, Sima

    2012-01-01

    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 of Leishmania infections, following vaccination, and as potential immunotherapeutic targets. PMID:22566891

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

    PubMed

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

    2009-07-01

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

  12. Cell surface origin of antigens shed by Leishmania donovani during growth in axenic culture.

    PubMed Central

    Kaneshiro, E S; Gottlieb, M; Dwyer, D M

    1982-01-01

    Antisera against isolated cell surface preparations (PCSP-As) of Leishmania donovani promastigotes were used to detect extracellular antigens produced during the growth of these organisms in four different growth media. The PCSP-As precipitated two major antigenically identical but electrophoretically distinct components, in addition to several minor antigens. Immunoelectrophoretic studies employing PCSP-As, PCSP-As absorbed with intact, live promastigotes, and PCSP-As absorbed with a major extracellular antigen demonstrated the antigenic identity between the major extracellular antigens and two major components externally disposed at the surface of promastigotes. Growth curve kinetic investigations suggested that the major extracellular antigens did not appear in the growth media primarily as a result of cell lysis or damage. The carbohydrate nature of the major extracellular antigens was indicated by physicochemical characterization. Images PMID:7118250

  13. First report of natural infection of a bush dog (Speothos venaticus) with Leishmania (Leishmania) chagasi in Brazil.

    PubMed

    Figueiredo, F B; Gremião, I D F; Pereira, S A; Fedulo, L P; Menezes, R C; Balthazar, D A; Schubach, T M P; Madeira, M F

    2008-02-01

    We report here the first known case of natural infection of a bush dog with Leishmania (Leishmania) chagasi in Brazil. The specimen was captured in the wild in the State of Mato Grosso and is currently being held in captivity at Fundação Jardim Zoológico, Rio de Janeiro, Brazil. The leishmaniasis was diagnosed by culture of promastigote forms in intact skin fragments and their characterization by isoenzyme electrophoresis. This report calls attention to the parasitological and etiological control of certain zoonoses, such as leishmaniasis, in wild animals kept in captivity, especially when animals are exchanged between zoos in Brazil.

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

    PubMed

    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.

  15. Immunoregulatory pathways in murine leishmaniasis: different regulatory control during Leishmania mexicana mexicana and Leishmania major infections.

    PubMed Central

    Alexander, J; Kaye, P M

    1985-01-01

    The effect of whole body sublethal gamma irradiation on the subsequent growth of Leishmania mexicana mexicana and Leishmania major was studied in CBA/Ca and BALB/c mice. Whereas BALB/c mice are highly susceptible to both parasites developing non healing progressively growing lesions at the site of cutaneous infection, CBA/Ca mice develop small healing cutaneous ulcers following subcutaneous infection with L. major but non healing lesions following subcutaneous infection with L.m. mexicana. Prior whole body sublethal irradiation of CBA/Ca mice, but not BALB/c mice, resulted in strong resistance against infection with L.m. mexicana: no lesions developed at the site of cutaneous infection. Irradiated BALB/c mice did, however, develop small lesions which healed when infected with L. major. The protective effects of irradiation coincided with the development of delayed type hypersensitivity. Both naive and sensitized nylon wool purified lymphocytes could restore susceptibility to L. major in irradiated BALB/c mice but only lymphocytes from long term infected donor mice adoptively transferred a non healing response to irradiated CBA/Ca mice infected with L.m. mexicana. Non-irradiated, L. major infected, CBA/Ca mice, but not similarly treated BALB/c mice, were found to be resistant to subsequent infection with L.m. mexicana. On the other hand, irradiated BALB/c mice infected with L. major were resistant to subsequent infectious challenge with L.m. mexicana. We suggest that the susceptibility of CBA/Ca mice to L.m. mexicana is under the control of an as yet unidentified gene which is not dependent on the generation of T suppressor cells and is bypassed by previous infection with L. major. Therefore, BALB/c mice immunized against L. major by prior sublethal irradiation are also resistant to L.m. mexicana. PMID:3907906

  16. Host Cell Signalling and Leishmania Mechanisms of Evasion

    PubMed Central

    Shio, Marina Tiemi; Hassani, Kasra; Isnard, Amandine; Ralph, Benjamin; Contreras, Irazu; Gomez, Maria Adelaida; Abu-Dayyeh, Issa; Olivier, Martin

    2012-01-01

    Leishmania parasites are able to secure their survival and propagation within their host by altering signalling pathways involved in the ability of macrophages to kill pathogens or to engage adaptive immune system. An important step in this immune evasion process is the activation of host protein tyrosine phosphatase SHP-1 by Leishmania. SHP-1 has been shown to directly inactivate JAK2 and Erk1/2 and to play a role in the negative regulation of several transcription factors involved in macrophage activation. These signalling alterations contribute to the inactivation of critical macrophage functions (e.g., Nitric oxide, IL-12, and TNF-α). Additionally, to interfere with IFN-γ receptor signalling, Leishmania also alters several LPS-mediated responses. Recent findings from our laboratory revealed a pivotal role for SHP-1 in the inhibition of TLR-induced macrophage activation through binding to and inactivating IL-1-receptor-associated kinase 1 (IRAK-1). Furthermore, we identified the binding site as an evolutionarily conserved ITIM-like motif, which we named kinase tyrosine-based inhibitory motif (KTIM). Collectively, a better understanding of the evasion mechanisms utilized by Leishmania parasite could help to develop more efficient antileishmanial therapies in the near future. PMID:22131998

  17. Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    PubMed Central

    Gaur Dixit, Upasna; Barker, Jason H.; Teesch, Lynn M.; Love-Homan, Laurie; Donelson, John E.; Wilson, Mary E.

    2013-01-01

    The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. PMID:23630964

  18. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation

    PubMed Central

    Okuda, Kendi; Tong, Mei; Dempsey, Brian; Moore, Kathryn J.; Gazzinelli, Ricardo T.; Silverman, Neal

    2016-01-01

    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. PMID:27280707

  19. Leishmania tropica infection in golden jackals and red foxes, Israel.

    PubMed

    Talmi-Frank, Dalit; Kedem-Vaanunu, Noa; King, Roni; Bar-Gal, Gila Kahila; Edery, Nir; Jaffe, Charles L; Baneth, Gad

    2010-12-01

    During a survey of wild canids, internal transcribed spacer 1 real-time PCR and high-resolution melt analysis identified Leishmania tropica in samples from jackals and foxes. Infection was most prevalent in ear and spleen samples. Jackals and foxes may play a role in the spread of zoonotic L. tropica. PMID:21122235

  20. Photoacoustic monitoring of life cycles of Leishmania Mexicana

    NASA Astrophysics Data System (ADS)

    Arguello, C.; Acosta-Avalos, D.; Alvarado-Gil, J. J.; Vargas, H.

    1999-03-01

    Photoacoustic spectroscopy is used to monitor in situ, the difference between the two forms of the protozoan Leishmania Mexicana. Differences are the result of changes in the respiratory chain and could be attributed, according to our results, to the presence of cytochrome b in promastigotes and cytochrome c in amastigotes.

  1. Vectorborne Transmission of Leishmania infantum from Hounds, United States

    PubMed Central

    Schaut, Robert G.; Robles-Murguia, Maricela; Juelsgaard, Rachel; Esch, Kevin J.; Bartholomay, Lyric C.; Ramalho-Ortigao, Marcelo

    2015-01-01

    Leishmaniasis is a zoonotic disease caused by predominantly vectorborne Leishmania spp. In the United States, canine visceral leishmaniasis is common among hounds, and L. infantum vertical transmission among hounds has been confirmed. We found that L. infantum from hounds remains infective in sandflies, underscoring the risk for human exposure by vectorborne transmission. PMID:26583260

  2. Leishmania tropica infection in golden jackals and red foxes, Israel.

    PubMed

    Talmi-Frank, Dalit; Kedem-Vaanunu, Noa; King, Roni; Bar-Gal, Gila Kahila; Edery, Nir; Jaffe, Charles L; Baneth, Gad

    2010-12-01

    During a survey of wild canids, internal transcribed spacer 1 real-time PCR and high-resolution melt analysis identified Leishmania tropica in samples from jackals and foxes. Infection was most prevalent in ear and spleen samples. Jackals and foxes may play a role in the spread of zoonotic L. tropica.

  3. Wild and synanthropic reservoirs of Leishmania species in the Americas

    PubMed Central

    Roque, André Luiz R.; Jansen, Ana Maria

    2014-01-01

    The definition of a reservoir has changed significantly in the last century, making it necessary to study zoonosis from a broader perspective. One important example is that of Leishmania, zoonotic multi-host parasites maintained by several mammal species in nature. The magnitude of the health problem represented by leishmaniasis combined with the complexity of its epidemiology make it necessary to clarify all of the links in transmission net, including non-human mammalian hosts, to develop effective control strategies. Although some studies have described dozens of species infected with these parasites, only a minority have related their findings to the ecological scenario to indicate a possible role of that host in parasite maintenance and transmission. Currently, it is accepted that a reservoir may be one or a complex of species responsible for maintaining the parasite in nature. A reservoir system should be considered unique on a given spatiotemporal scale. In fact, the transmission of Leishmania species in the wild still represents an complex enzootic “puzzle”, as several links have not been identified. This review presents the mammalian species known to be infected with Leishmania spp. in the Americas, highlighting those that are able to maintain and act as a source of the parasite in nature (and are thus considered potential reservoirs). These host/reservoirs are presented separately in each of seven mammal orders – Marsupialia, Cingulata, Pilosa, Rodentia, Primata, Carnivora, and Chiroptera – responsible for maintaining Leishmania species in the wild. PMID:25426421

  4. The LPG1 gene family of Leishmania major

    PubMed Central

    Zhang, Kai; Barron, Tamara; Turco, Salvatore J.; Beverley, Stephen M.

    2013-01-01

    In Leishmania major, the core of the abundant surface lipophosphoglycan (LPG) is structurally related to that of the smaller glycosylinositolphospholipids (GIPLs) in containing galactosylfuranose (Galf ) residues in a Galf (β1, 3)Man motif. However, deletion of the putative Galf-transferase (Galf T) LPG1 affected Galf incorporation in LPG but not GIPLs. We hypothesized that the presumptive GIPL Galf-transferases could be homologous to LPG1, and identified three related genes in the L. major genome. These were termed LPG1L, LPG1R, and LPG1G, the latter of which was found in three identical copies located at the telomeres of chromosomes 5, 19, and 32 based on Leishmania genome project data. Neither LPG1 nor its homologues LPG1L and LPG1R were involved in the biosynthesis of GIPLs, as an lpg1−/lpg1l−/lpg1r− triple knockout (the first such in Leishmania) grew normally and made wild-type levels of Galf-containing GIPLs. In contrast, overexpression of these three led to elevated galactose incorporation in glycoproteins. Galf-containing glycoproteins had not been described in Leishmania but occur at high levels in other closely related trypanosomatids including Trypanosoma cruzi, Crithidia, Leptomonas, and Endotrypanum, and LPG1L and LPG1R homologs were detected in these species. These data suggest that the glyco-synthetic capabilities of Leishmania and perhaps other trypanosomatids may be larger than previously thought, with some activities being ‘cryptic’ in different lineages and potentially serving as reservoirs for glycoconjugate variation during evolution. Future tests will address whether the LPG1G family encodes the hypothesized GIPL-specific Galf T. PMID:15138063

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

    PubMed

    Subramanian, Abhishek; Jhawar, Jitesh; Sarkar, Ram Rup

    2015-01-01

    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 energy metabolism

  6. Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

    PubMed

    Prati, Federica; Goldman-Pinkovich, Adele; Lizzi, Federica; Belluti, Federica; Koren, Roni; Zilberstein, Dan; Bolognesi, Maria Laura

    2014-01-01

    The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1-15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives. PMID:25254495

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

    PubMed

    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.

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

    PubMed

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

    2015-02-01

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

  9. Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran

    PubMed Central

    Dabirzadeh, Mansour; Hashemi, Mohammad; Maroufi, Yahya

    2016-01-01

    Background Zoonotic cutaneous leishmaniasis (ZCL) is polymorphic disease that may show various clinical manifestations. Objectives This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. Materials and Methods A variety of nucleic acid detection methods that target both DNA and RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. Results Of the 24 isolates, 21 had 350 bp bands (87.5%) and three had 450 bp bands (12.5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. Conclusions The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for

  10. Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome

    PubMed Central

    2014-01-01

    Background This study describes immunological responses, diagnostic features, follow up and treatment outcomes from seventeen dogs with papular dermatitis due to Leishmania infection diagnosed by cytology or real time-PCR. Methods Specific Leishmania humoral and cellular immune responses were evaluated by means of an immunofluorescence antibody test in all cases and a delayed-type hypersensitivity (DTH) reaction to leishmanin in eight cases. The extent of infection was studied in several tissues including blood, lymph node, conjunctival and oral swabs, by means of PCR, at the time of diagnosis and during follow-up. Culture was performed on nine dogs from cutaneous lesions and lymph node aspirates and molecular typing was carried out on isolates based on ITS-1, ITS-2 and Haspb gene sequencing analysis. Results Cytological and molecular results from fine needle aspirates of papules were diagnostic in 8 out of 13 (61.5%) cases and in 14 out of 15 dogs (93.3%), respectively. In all dogs, specific anti-Leishmania antibody levels were low or absent. Blood and lymph node PCRs and lymph node culture were negative in all dogs. Three out of the nine dogs (33%) were positive by culture from cutaneous lesions. The three isolates were identified as ITS type A, however, polymorphism was observed in the Haspb gene (PCR products of 626 bp, 962 bp and 371 bp). DTH response was positive in all tested dogs at the time of diagnosis. The majority of dogs were successfully treated with only N-methylglucamine antimoniate, after which cutaneous lesions disappeared or were reduced to depigmented, flattened scars. All dogs remained seronegative and the majority of dogs were negative by PCR in several tissues during follow-up. Conclusions This study points out that papular dermatitis due to L. infantum is probably an underestimated benign cutaneous problem, associated with a parasite specific cell mediated immunity and a poor humoral immune response. Papular dermatitis is seen in young dogs

  11. Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran

    PubMed Central

    Dabirzadeh, Mansour; Hashemi, Mohammad; Maroufi, Yahya

    2016-01-01

    Background Zoonotic cutaneous leishmaniasis (ZCL) is polymorphic disease that may show various clinical manifestations. Objectives This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. Materials and Methods A variety of nucleic acid detection methods that target both DNA and RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. Results Of the 24 isolates, 21 had 350 bp bands (87.5%) and three had 450 bp bands (12.5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. Conclusions The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for

  12. A Newly Emerged Cutaneous Leishmaniasis Focus in Northern Israel and Two New Reservoir Hosts of Leishmania major

    PubMed Central

    Faiman, Roy; Abbasi, Ibrahim; Jaffe, Charles; Motro, Yoav; Nasereddin, Abdelmagid; Schnur, Lionel F.; Torem, Moshe; Pratlong, Francine; Dedet, Jean-Pierre; Warburg, Alon

    2013-01-01

    In 2006/7, 18 cases of cutaneous leishmaniasis (CL) were reported for the first time from Sde Eliyahu (pop. 650), a village in the Beit She'an valley of Israel. Between 2007–2011, a further 88 CL cases were diagnosed bringing the total to 106 (16.3% of the population of Sde Eliyahu). The majority of cases resided in the south-western part of the village along the perimeter fence. The causative parasite was identified as Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae). Phlebotomus papatasi (Scopoli), 1786 (Diptera: Psychodidae) was found to be the most abundant phlebotomine species comprising 97% of the sand flies trapped inside the village, and an average of 7.9% of the females were positive for Leishmania ITS1 DNA. Parasite isolates from CL cases and a sand fly were characterized using several methods and shown to be L. major. During a comprehensive survey of rodents 164 Levant voles Microtus guentheri Danford & Alston, 1880 (Rodentia: Cricetidae) were captured in alfalfa fields bordering the village. Of these 27 (16.5%) tested positive for Leishmania ITS1 DNA and shown to be L. major by reverse line blotting. A very high percentage (58.3% - 21/36) of Tristram's jirds Meriones tristrami Thomas, 1892 (Rodentia: Muridae), found further away from the village also tested positive for ITS1 by PCR. Isolates of L. major were successfully cultured from the ear of a wild jird found positive by ITS1 PCR. Although none of the wild PCR-positive voles exhibited external pathology, laboratory-reared voles that were infected by intradermal L. major inoculation, developed patent lesions and sand flies became infected by feeding on the ears of these laboratory-infected voles. This is the first report implicating M. guentheri and M. tristrami as reservoirs of Leishmania. The widespread co-distribution of M. guentheri and P. papatasi, suggests a significant threat from the spread of CL caused by L. major in the Middle East, central Asia and southern

  13. Structure, functions, and biosynthesis of glycoconjugates of Leishmania spp. cell surface.

    PubMed

    Novozhilova, N M; Bovin, N V

    2010-06-01

    Cell surface of leishmaniasis causal agent, a parasitic member of Protozoa of Leishmania genus, is covered by thick glycocalix consisting of various phosphatidylinositol-anchored molecules. This review deals with the structure and biosynthesis of the main phosphoglycans and glycoproteins of Leishmania cell surface, many of which incorporate the rare natural D-arabinopyranose, and the problem concerning the involvement of these molecules in support of Leishmania survival during their intricate life cycle is discussed.

  14. First detection of Leishmania spp. DNA in Brazilian bats captured strictly in urban areas.

    PubMed

    de Oliveira, Fernanda Müller; Costa, Luis Henrique Camargo; de Barros, Thainá Landim; Ito, Pier Kenji Rauschkolb Katsuda; Colombo, Fábio Antonio; de Carvalho, Cristiano; Pedro, Wagner André; Queiroz, Luzia Helena; Nunes, Cáris Maroni

    2015-10-01

    Leishmania spp. is a protozoan that maintains its life cycle in domestic and wild animals and it may include bats, a population that has increased in urban environments. This study aimed to investigate the presence of Leishmania spp. in bats captured strictly in urban areas that are endemic for visceral leishmaniasis. The spleen and skin samples of 488 bats from 21 endemic cities in northwestern São Paulo State, Brazil, were tested for the presence of Leishmania kDNA using real-time PCR. Differentiation from Trypanosoma spp. was achieved by amplifying a DNA fragment of the ribosomal RNA gene. The presence of Leishmania spp. kDNA was verified in 23.9% of bats and Trypanosoma spp. DNA was identified in 3.9%. Leishmania species differentiation revealed the presence of Leishmania amazonensis in 78.3% of the bats; L. infantum in 17.4%, and 1 sample (4.3%) showed a mix pattern of L. infantum and L. amazonensis. We also detected, for the first time, L. infantum and L. amazonensis DNA in Desmodus rotundus, the hematophagous bat. The presence of Leishmania spp. DNA in bats strictly from urban areas endemic for visceral leishmaniasis in the State of São Paulo, Brazil indicates that these wild and abundant animals are capable of harboring Leishmania spp. in this new scenario. Due to their longevity, high dispersion capacity and adaptability to synanthropic environments, they may play a role in the maintenance of the life cycle of Leishmania parasites.

  15. Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro

    PubMed Central

    Freitas, Elisangela Oliveira; Nico, Dirlei; Guan, Rong; Meyer-Fernandes, José Roberto; Clinch, Keith; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.; Palatnik-de-Sousa, Clarisa B.

    2015-01-01

    Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results

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

    PubMed Central

    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

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

    PubMed

    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

  18. Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

    PubMed Central

    Lemrani, Meryem; Mouna, Idrissi; Mohammed, Hida; Mostafa, Sabri; Rhajaoui, Mohamed; Hamarsheh, Omar; Schönian, Gabriele

    2013-01-01

    Leishmania infantum causes Visceral and cutaneous leishmaniasis in northern Morocco. It predominantly affects children under 5 years with incidence of 150 cases/year. Genetic variability and population structure have been investigated for 33 strains isolated from infected dogs and humans in Morocco. A multilocus microsatellite typing (MLMT) approach was used in which a MLMtype based on size variation in 14 independent microsatellite markers was compiled for each strain. MLMT profiles of 10 Tunisian, 10 Algerian and 21 European strains which belonged to zymodeme MON-1 and non-MON-1 according to multilocus enzyme electrophoresis (MLEE) were included for comparison. A Bayesian model-based approach and phylogenetic analysis inferred two L.infantum sub-populations; Sub-population A consists of 13 Moroccan strains grouped with all European strains of MON-1 type; and sub-population B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses sub-populations were significantly different from each other and from the Tunisian, Algerian and European non MON-1 strains which constructed one separate population. The presence of these two sub-populations co-existing in Moroccan endemics suggests multiple introduction of L. infantum from/to Morocco; (1) Introduction from/to the neighboring North African countries, (2) Introduction from/to the Europe. These scenarios are supported by the presence of sub-population B and sub-population A respectively. Gene flow was noticed between sub-populations A and B. Five strains showed mixed A/B genotypes indicating possible recombination between the two populations. MLMT has proven to be a powerful tool for eco-epidemiological and population genetic investigations of Leishmania. PMID:24147078

  19. Biochemical and biological characterization of the protective Leishmania pifanoi amastigote antigen P-8.

    PubMed

    Colmenares, M; Tiemeyer, M; Kima, P; McMahon-Pratt, D

    2001-11-01

    The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559-3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with the L. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously described Leishmania glycolipids (glycosylinositol-phospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.

  20. Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

    PubMed Central

    2010-01-01

    Background The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Results Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. Conclusions The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology. PMID:20074347

  1. MAPK1 of Leishmania donovani modulates antimony susceptibility by downregulating P-glycoprotein efflux pumps.

    PubMed

    Garg, Mansi; Goyal, Neena

    2015-07-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug.

  2. Differential surface deposition of complement proteins on logarithmic and stationary phase Leishmania chagasi promastigotes.

    PubMed

    Ramer-Tait, Amanda E; Lei, Soi Meng; Bellaire, Bryan H; Beetham, Jeffrey K

    2012-12-01

    Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHS-exposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of

  3. Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues.

    PubMed

    Bestman-Smith, J; Schmit, I; Papadopoulou, B; Boivin, G

    2001-04-01

    Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite Leishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in Leishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of Leishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in Leishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.

  4. Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.

    PubMed

    Rose, K; Curtis, J; Baldwin, T; Mathis, A; Kumar, B; Sakthianandeswaren, A; Spurck, T; Low Choy, J; Handman, E

    2004-05-01

    This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.

  5. Testicular amyloidosis in hamsters experimentally infected with Leishmania donovani.

    PubMed Central

    Gonzalez, J. L.; Gallego, E.; Castaño, M.; Rueda, A.

    1983-01-01

    Thirty hamsters were inoculated intraperitoneally with Leishmania donovani. Testes were examined grossly and histologically by light and electron microscopy. Progressive testicular atrophy developed. Spermatogenic cells of the seminiferous tubules showed vacuolar degeneration and decreased in number leading to a total azoospermia in the final weeks of the pathological process. Lymphoplasmocytic infiltrates with macrophages containing leishmanias appeared in the intertubular space. Amyloid deposits in the intertubular space and tubular basement membrane were identified by optical and ultrastructural methods. It has been suggested that testicular amyloidosis may have a pathogenic mechanism related to a dysfunction of plasma cells and stimulation of the reticuloendothial system, due to the antigenic character of the parasite. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6639870

  6. An agent-based model for Leishmania major infection

    NASA Astrophysics Data System (ADS)

    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 left 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].

  7. An agent-based model for Leishmania major infection

    NASA Astrophysics Data System (ADS)

    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].

  8. Leishmania promastigotes: building a safe niche within macrophages.

    PubMed

    Moradin, Neda; Descoteaux, Albert

    2012-01-01

    Upon their internalization by macrophages, Leishmania promastigotes inhibit phagolysosome biogenesis. The main factor responsible for this inhibition is the promastigote surface glycolipid lipophosphoglycan (LPG). This glycolipid has a profound impact on the phagosome, causing periphagosomal accumulation of F-actin and disruption of phagosomal lipid microdomains. Functionally, this LPG-mediated inhibition of phagosome maturation is characterized by an impaired assembly of the NADPH oxidase and the exclusion of the vesicular proton-ATPase from phagosomes. In this chapter, we review the current knowledge concerning the nature of the intra-macrophage compartment in which Leishmania donovani promastigotes establish infection. We also describe how LPG enables this parasite to remodel the parasitophorous vacuole. PMID:23050244

  9. Post-Genomics and Vaccine Improvement for Leishmania

    PubMed Central

    Seyed, Negar; Taheri, Tahereh; Rafati, Sima

    2016-01-01

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

  10. Production of eukaryotic cell-free lysate from Leishmania tarentolae.

    PubMed

    Johnston, Wayne A; Alexandrov, Kirill

    2014-01-01

    In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathogenic to humans and does not require special laboratory precautions. An additional reason it is a convenient source organism for cell-free lysate production is that all endogenous protein expression can be suppressed by a single antisense oligonucleotide targeting splice leader sequence on the 5'-end of all protein coding RNAs. We describe simple procedures for cell disruption and lysate processing starting from bioreactor culture. We also describe introduction of genetic information via vectors containing species-independent translation initiation sites (SITS). We consider that such an inexpensive eukaryotic cell-free production system has many advantages when expressing multi-subunit proteins or difficult to express proteins. PMID:24395406

  11. Metabolic Variation during Development in Culture of Leishmania donovani Promastigotes

    PubMed Central

    Silva, Ana Marta; Cordeiro-da-Silva, Anabela; Coombs, Graham H.

    2011-01-01

    The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3–6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase. PMID:22206037

  12. In Vitro Activities of Hexaazatrinaphthylenes against Leishmania spp.

    PubMed Central

    García-Velázquez, Daniel; Martín-Navarro, Carmen M.; Sifaoui, Ines; Reyes-Batlle, María; Lorenzo-Morales, Jacob; Gutiérrez-Ravelo, Ángel; Piñero, José E.

    2015-01-01

    The in vitro activity of a novel group of compounds, hexaazatrinaphthylene derivatives, against two species of Leishmania is described in this study. These compounds showed a significant dose-dependent inhibition effect on the proliferation of the parasites, with 50% inhibitory concentrations (IC50s) ranging from 1.23 to 25.05 μM against the promastigote stage and 0.5 to 0.7 μM against intracellular amastigotes. Also, a cytotoxicity assay was carried out to in order to evaluate the possible toxic effects of these compounds. Moreover, different assays were performed to determine the type of cell death induced after incubation with these compounds. The obtained results highlight the potential use of hexaazatrinaphthylene derivatives against Leishmania species, and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents. PMID:25753635

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

    PubMed

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

    2016-01-01

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

  14. Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

    PubMed

    Manhas, Reetika; Gowri, Venkatraman Subramanian; Madhubala, Rentala

    2016-01-15

    The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different. PMID:26586914

  15. DNA Aptamers Selectively Target Leishmania infantum H2A Protein

    PubMed Central

    Martín, M. Elena; García-Hernández, Marta; García-Recio, Eva M.; Gómez-Chacón, Gerónimo F.; Sánchez-López, Marta; González, Víctor M.

    2013-01-01

    Parasites of the genus Leishmania produce leishmaniasis which affects millions people around the world. Understanding the molecular characteristics of the parasite can increase the knowledge about the mechanisms underlying disease development and progression. Thus, the study of the molecular features of histones has been considered of particular interest because Leishmania does not condense the chromatin during mitosis and, consequently, a different role for these proteins in the biology of the parasite can be expected. Furthermore, the sequence divergences in the amino and in the carboxy-terminal domains of the kinetoplastid core histones convert them in potential diagnostic and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania histones is essential for the progress of this kind of study. Two aptamers which specifically recognize Leishmania infantum H2A histone were cloned from a previously obtained ssDNA enriched population. These aptamers were sequenced and subjected to an in silico analysis. ELONA, slot blot and Western blot were performed to establish aptamer affinity and specificity for LiH2A histone and ELONA assays using peptides corresponding to overlapped sequences of LiH2A were made mapping the aptamers:LiH2A interaction. As “proofs of concept”, aptamers were used to determine the number of parasites in an ELONA platform and to purify LiH2A from complex mixtures. The aptamers showed different secondary structures among them; however, both of them were able to recognize the same peptides located in a side of the protein. In addition, we demonstrate that these aptamers are useful for LiH2A identification and also may be of potential application as diagnostic

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

    PubMed Central

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

    2010-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H4B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H4B 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 H4B 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 H4B-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. PMID:20887755

  17. Glycosome turnover in Leishmania major is mediated by autophagy

    PubMed Central

    Cull, Benjamin; Prado Godinho, Joseane Lima; Fernandes Rodrigues, Juliany Cola; Frank, Benjamin; Schurigt, Uta; Williams, Roderick AM; Coombs, Graham H; Mottram, Jeremy C

    2015-01-01

    Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the

  18. In Vitro susceptibilities of wild and drug resistant Leishmania donovani amastigotes to piperolactam A loaded hydroxypropyl-β-cyclodextrin nanoparticles.

    PubMed

    Bhattacharya, Plaban; Mondal, Subhasish; Basak, Souvik; Das, Pradeep; Saha, Achintya; Bera, Tanmoy

    2016-06-01

    Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of piperolactam A (PL), isolated after bioactivity guided fractionation from root extracts of Piper betle was accentuated in detail. Activity potentiation was achieved via cyclodextrin complexation. Crude hydro-ethanolic extract (PB) and three fractions obtained from PB and fabricated PL-hydroxypropyl-β-cyclodextrin (HPBCD) nanoparticles were evaluated for antileishmanial activity. Tests were performed against L. donovani wild-type, sodium stibogluconate, paromomycin and field isolated (GE1) resistant strains in axenic amastigote and amastigote in macrophage models. PL-HPBCD complex was characterized and FITC loaded HPBCD nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Isolated and purified PL from most potent, alkaloid rich ethyl acetate fraction of PB showed high level of antileishmanial activities in wild-type (IC50=36 μM), sodium stibogluconate resistant (IC50=103 μM), paromomycin resistant (IC50=91 μM) and field isolated resistant (IC50=72 μM) strains together with cytotoxicity (CC50=900 μM) in mouse peritoneal macrophage cells. Inclusion of PL in HPBCD nanoparticles resulted in 10-fold and 4-10-fold increase in selectivity indexes (CC50/IC50) for wild-type and drug resistant strains, respectively. Drug-carrier interactions were clearly visualized in FT-IR studies. Complete incorporation of PL in HPBCD cavity was ascertained in DSC and XRD analyses. 180nm size stable nanospheres showed macrophage internalization within 1h of incubation. Piperolactam A (PL), a representative of the inchoate skeleton of aristolactam chassis might be the source of safe and affordable antileishmanial agents for the cure of deadly Leishmania infections. PMID:26940000

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

    PubMed

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

    PubMed Central

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

    2016-01-01

    Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they

  1. The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification

    PubMed Central

    Vikeved, Elisabet; Backlund, Anders; Alsmark, Cecilia

    2016-01-01

    Background The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT). Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania. Methodology/Principal Findings To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species. Conclusions/Significance LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets. PMID:26730948

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

    PubMed

    Alexander, Bruce; Oliveria, Emerson Barbosa de; Haigh, Emily; Almeida, Lourenço Leal de

    2002-07-01

    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. PMID:12219123

  3. Leishmania mexicana mexicana: quantitative analysis of the intracellular cycle.

    PubMed

    Doyle, P S; Engel, J C; Gam, A A; Dvorak, J A

    1989-12-01

    The complete intracellular cycle of the Leishmania mexicana mexicana G. S. strain was quantified in human macrophages and in the mouse IC-21 macrophage line utilizing a culture system that allows the direct observation of individual intracellular parasites. A wide range of pre-replicative lag periods exists, implying that promastigotes may be in any phase of their DNA synthetic cycle when phagocytosed by the macrophage. Amastigotes replicated 2-3 times, after which the host cell died and liberated amastigotes that were taken up by other macrophages and continued to replicate. The mean amastigote population-doubling time in human macrophages (17.5 h) was not statistically different from promastigotes growing in axenic culture (16.4 h), but was nearly 2-fold less than amastigotes growing in mouse-derived IC-21 macrophages (33.7 h). These observations are markedly different from cover-glass culture assays of Leishmania-macrophage interactions and provide an unambiguous description of the intracellular cycle of Leishmania mexicana mexicana. PMID:2608309

  4. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    PubMed

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

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

    PubMed

    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. PMID:27413751

  6. Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages.

    PubMed Central

    Kelleher, M; Moody, S F; Mirabile, P; Osborn, A H; Bacic, A; Handman, E

    1995-01-01

    Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors. PMID:7806383

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

    PubMed Central

    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

  8. Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion.

    PubMed

    Maclean, Lorna M; O'Toole, Peter J; Stark, Meg; Marrison, Jo; Seelenmeyer, Claudia; Nickel, Walter; Smith, Deborah F

    2012-05-01

    Proteins of the Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra- and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'non-classically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages. PMID:22256896

  9. Survey of Trypanosoma and Leishmania in wild and domestic animals in an Atlantic rainforest fragment and surroundings in the state of Espírito Santo, Brazil.

    PubMed

    Acosta, Igor da C L; Da Costa, Andrea P; Gennari, Solange M; Marcili, Arlei

    2014-05-01

    Trypanosoma and Leishmania infections affect wild and domestic animals and human populations. The growing process of deforestation and urbanization of Atlantic Rainforest areas has given rise to introduction of humans and domestic animals to the sylvatic cycles of Trypanosoma and Leishmania species. Serological, parasitological, and molecular surveys among wild and domestic animals in the Corrego do Veado Biological Reserve, which is an Atlantic Rainforest fragment in the state of Espírito Santo, southeastern Brazil, were evaluated. In total, 154 wild animals of 25 species and 67 domestic animals (47 dogs and 20 horses) were sampled. All the domestic animals were serologically negative for anti-Leishmania infantum chagasi antibodies and negative in parasitological approaches. Only the Order Chiroptera presented positive blood cultures and cryopreserved isolates. The phylogenetic trees based on SSU rDNA and gGAPDH genes confirmed the occurrence of Trypanosoma dionisii and provided the first record of Trypanosoma cruzi marinkellei in southeastern Brazil. The studies conducted in Atlantic Rainforest remaining trees provide the knowledge of parasite diversity or detect parasites that can accelerate the loss of hosts diversity.

  10. [Infection of skin fibroblasts in animals with different levels of sensitivity to Leishmania infantum and Leishmania mexicana (Kinetoplastida: Trypanosomatidae)].

    PubMed

    Minero, Miguel Angel; Chinchilla, Misael; Guerrero, Olga Marta; Castro, Alfredo

    2004-03-01

    Infection and multiplication of Leishmania infantum and L. mexicana inside of skin fibroblasts from hamsters, mice and rats was achieved. This process was demonstrated either by counting parasites inside the stained cells or by electronic microscopy studies. In addition multiplication rate differences in the cells from these rodent species were determined, for L. infantum as well as for L. mexicana. Parasite development in hamsters and mice fibroblasts was evident but there was not multiplication in rat cells showing that apparently they are refractory to Leishmania infection. These results suggest that the parasite affinity for each animal, as well as any intracellular environment resistance, could involve genetic factors in the parasite multiplication. On the other hand, presence of amastigote multiplication inside of parasitophorus vacuole, showed by electronic microscopy images, probes a true parasite transformation. Therefore it is suggested that fibroblasts could work as host cells for parasite survival and permanency in the infected animals. PMID:17357424

  11. Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

    PubMed

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L

    2014-01-01

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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-01

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

  16. Canine Leishmania vaccines: still a long way to go.

    PubMed

    Gradoni, Luigi

    2015-02-28

    Dogs are the main reservoir host for zoonotic visceral leishmaniasis, a sand fly-borne disease caused by Leishmania infantum. In endemic areas, "susceptible" dogs suffer from a severe disease characterized by chronic polymorphic viscerocutaneous signs that manifest several months from the exposure, whereas "resistant" dogs can remain subclinically infected for years or lifelong. The protective immune response to Leishmania is cell-mediated; for visceralizing Leishmania species a mixed T helper (Th)1/Th2 response with a dominant Th1 profile is required for protection. The activation of the adaptive immune system in naturally resistant dogs is revealed by parasite-specific lymphoproliferation, delayed-type hypersensitivity, the production of interferon-γ and tumour necrosis factor-α cytokines, and enhanced macrophage leishmanicidal activity via nitric oxide. Hence, an effective canine Leishmania vaccine should induce strong and long-lasting Th1-dominated immunity to control both infection progression and the parasite transmissibility via the vector. Preclinical research in rodent models has evaluated the efficacy of several categories of Leishmania antigens including killed parasites, cell purified fractions, parasite protein components or subunits, single or multiple chimeric recombinant proteins, plasmid DNA and viral particles encoding parasite virulence factors. Promising antigen(s)/adjuvant combinations from each of the above categories have also been tested in dogs; they mostly resulted in limited or no protection in Phase I-II studies (designed to test vaccine safety, immunogenicity and laboratory-induced protection) in which vaccinated dogs were challenged by the artificial intravenous injection of high-load L. infantum promastigotes. The recombinant A2 antigen plus saponin conferred about 40% protection against infection by this challenge system and has been registered in Brazil as a canine vaccine (LeishTec(®)). An increasing number of efficacy studies

  17. Immunological consequences of stress-related proteins--cytosolic tryparedoxin peroxidase and chaperonin TCP20--identified in splenic amastigotes of Leishmania donovani as Th1 stimulatory, in experimental visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Rawat, Keerti; Yadav, Narendra; Sundar, Shyam; Dube, Anuradha

    2015-04-01

    In earlier studies, proteomic characterization of splenic amastigote fractions from clinical isolates of Leishmania donovani, exhibiting significant cellular responses in cured Leishmania subjects, led to the identification of cytosolic tryparedoxin peroxidase (LdcTryP) and chaperonin-TCP20 (LdTCP20) as Th1-stimulatory proteins. Both the proteins, particularly LdTCP20 for the first time, were successfully cloned, overexpressed, purified and were found to be localized in the cytosol of purified splenic amastigotes. When evaluated against lymphocytes of cured Leishmania-infected hamsters, the purified recombinant proteins (rLdcTryP and rLdTCP20) induced their proliferations as well as nitric oxide production. Similarly, these proteins also generated Th1-type cytokines (IFN-γ/IL-12) from stimulated PBMCs of cured/endemic Leishmania patients. Further, vaccination with rLdcTryP elicited noticeable delayed-type hypersensitivity response and offered considerably good prophylactic efficacy (~78% inhibition) against L. donovani challenge in hamsters, which was well supported by the increased mRNA expression of Th1 and Th2 cytokines. However, animals vaccinated with rLdTCP20 exhibited comparatively lesser prophylactic efficacy (~55%) with inferior immunological response. The results indicate the potentiality of rLdcTryP protein, between the two, as a suitable anti-leishmanial vaccine. Since, rLdTCP20 is also an important target, for optimization, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up. PMID:25498563

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

    PubMed

    Borborema, Samanta Etel Treiger; Osso Jr, João Alberto; Andrade Jr, Heitor Franco de; Nascimento, Nanci do

    2013-08-01

    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 eluc