Clinical presentations of Leishmania braziliensis braziliensis

Leishmania braziliensis braziliensis (Lbb) is probably the most serious, leishmanial infection of the New World. Although epidemiological information is incomplete, its distribution is believed to extend from Belize in Central America to northern Argentina, involving all countries east of the Andes. Lbb causes a variety of clinical lesions and is one of the most difficult forms of leishmaniasis to treat. We still do not know how many patients suffer from mucosal disease requiring treatment. In this review of 10 years field experience in an endemic area of Brazil, Philip Marsden shows that parasitologists have much to contribute, especially in improving diagnostic methods, developing better animal models, and providing new drugs suitable for routine treatment on a large-scale.     

Experimental canine mucocutaneous leishmaniasis (Leishmania braziliensis braziliensis)

Four mongrel dogs were intradermically inoculated with 3 x 10(6) Leishmania braziliensis braziliensis promastigotes. Three out of the four animals developed cutaneous lesions respectively 4, 7, and 8 months after. The fourth dog did not develop lesion at the inoculation site, but a mucosal ulcer was seen 16 months after the inoculum. Clinical, histopathological, and serological findings were similar to what is found in natural canine infection as well as in the human disease. These results suggest that dogs may be an useful model for L. b. braziliensis infection.     

Experimental infection of Lutzomyia whitmani in dogs infected with Leishmania braziliensis braziliensis

Lutzomyia (N.) whitmani was infected on leishmaniotic lesions of three out of nine dogs infected with Leishmania braziliensis braziliensis. The infectivity rates in these sandflies were 8.3% (1/12), 7.1% (1/14) and 1.8% (3/160), respectively. In addition, 180 Lu. whitmani fed on non-ulcerated regions of one of the infected dogs and none became infected. We emphasize the vector potentiality of Lu. whitmani for L.b. braziliensis in the endemic region of Três Bra?os, Bahia, Brazil.     

Preliminary results in favor of the existence of a major surface antigen, specific for Leishmania braziliensis braziliensis

The comparative surface antigen study of 10 bolivian strains and 1 brazilian reference strain from L. b. braziliensis sub-species shows an important homogeneity within this group. All the strains tested so far present similar antigenic patterns with a major antigen at 72 kD. On the contrary, the comparative analysis of surface antigens of L. b. braziliensis with those of L. b. guyanensis, L. b. panamensis, L. mexicana amazonensis and L. donovani chagasi shows a large antigenic heterogeneity between the Leishmania sub-species and species we studied. The 72 kD antigen was only detected on L. b. braziliensis surface.     

Identification of serine proteases from Leishmania braziliensis

Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nalpha-benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-L-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-L-phenyl-alanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.     

Disseminated American muco-cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis in a patient with AIDS: a case report.

The authors report a case of culture-proven disseminated American muco-cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis in an HIV positive patient. Lesions began in the oropharynx and nasal mucosa eventually spreading to much of the skin surface. The response to a short course of glucantime therapy was good.     

Oxidation of fatty acids by Leishmania braziliensis panamensis

Heating cultures of Leishmania braziliensis panamensis (grown at 26 degrees C) to 34 degrees C for 1.5-12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-14C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26 degrees C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of 14CO2 release from [1-14C]laurate to that from [12-14C]laurate was generally larger than four, whereas this ratio from [1-14C]oleate relative to [10-14C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the omega-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Multilocus Sequence Analysis for Leishmania braziliensis Outbreak Investigation

With the emergence of leishmaniasis in new regions around the world, molecular epidemiological methods with adequate discriminatory power, reproducibility, high throughput and inter-laboratory comparability are needed for outbreak investigation of this complex parasitic disease. As multilocus sequence analysis (MLSA) has been projected as the future gold standard technique for Leishmania species characterization, we propose a MLSA panel of six housekeeping gene loci (6pgd, mpi, icd, hsp70, mdhmt, mdhnc) for investigating intraspecific genetic variation of L. (Viannia) braziliensis strains and compare the resulting genetic clusters with several epidemiological factors relevant to outbreak investigation. The recent outbreak of cutaneous leishmaniasis caused by L. (V.) braziliensis in the southern Brazilian state of Santa Catarina is used to demonstrate the applicability of this technique. Sequenced fragments from six genetic markers from 86 L. (V.) braziliensis strains from twelve Brazilian states, including 33 strains from Santa Catarina, were used to determine clonal complexes, genetic structure, and phylogenic networks. Associations between genetic clusters and networks with epidemiological characteristics of patients were investigated. MLSA revealed epidemiological patterns among L. (V.) braziliensis strains, even identifying strains from imported cases among the Santa Catarina strains that presented extensive homogeneity. Evidence presented here has demonstrated MLSA possesses adequate discriminatory power for outbreak investigation, as well as other potential uses in the molecular epidemiology of leishmaniasis.     

Leishmania braziliensis: localization of glycoproteins in promastigotes

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Enhanced Leishmania braziliensis infection following pre-exposure to sandfly saliva

Background

Sand fly saliva has an array of pharmacological and immunomodulatory components, and immunity to saliva protects against Leishmania infection. In the present study, we have studied the immune response against Lutzomyia intermedia saliva, the main vector of Leishmania braziliensis in Brazil, and the effects of saliva pre-exposure on L. braziliensis infection employing an intradermal experimental model.

Methodology/principal findings

BALB/c mice immunized with L. intermedia salivary gland sonicate (SGS) developed a saliva-specific antibody response and a cellular immune response with presence of both IFN-γ and IL-4. The inflammatory infiltrate observed in SGS-immunized mice was comprised of numerous polymorphonuclear and few mononuclear cells. Mice challenged with live L. braziliensis in the presence of saliva were not protected although lesion development was delayed. The inoculation site and draining lymph node showed continuous parasite replication and low IFN-γ to IL-4 ratio, indicating that pre-exposure to L. intermedia saliva leads to modulation of the immune response. Furthermore, in an endemic area of cutaneous leishmaniasis, patients with active lesions displayed higher levels of anti-L. intermedia saliva antibodies when compared to individuals with a positive skin test result for Leishmania.

Conclusion

These results show that pre-exposure to sand fly saliva plays an important role in the outcome of cutaneous leishmaniasis, in both mice and humans. They emphasize possible hurdles in the development of vaccines based on sand fly saliva and the need to identify and select the individual salivary candidates instead of using whole salivary mixture that may favor a non-protective response.     

Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

BackgroundNeutrophils 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.ConclusionsWe 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.     

Metagenomic Analysis of Healthy and White Plague-Affected Mussismilia braziliensis Corals

Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga–Flavobacterium–Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium.     

Leishmania braziliensis: dissemination of Panamanian strains in golden hamsters

Dissemination of Panamanian strains of Leishmania braziliensis was observed in experimentally infected golden hamsters, Mesocricetus auratus, during the characterization of 164 strains isolated from patients (67), two species of edentates (88), and dogs (9). A total of 614 hamsters was employed in these studies. Hamsters were inoculated intradermally in the nose with 5–10 × 10⁶ promastigotes from cultures of strains in their first to third passage in vitro. Parasites were recovered by culture from skin samples, viscera, blood and bone marrow. All strains studied disseminated to various areas of the skin and to the ear pinnae. Highest incidence of dissemination occurred in the skin from the tip of the tail, feet, and ears. Positive cultures obtained from liver and spleen were not considered as evidence for metastasis since they may have been due to the transitory presence in the blood of rare parasitized macrophages. Dissemination of various areas of the body was directly proportional to the length of the postinoculation period of the sloth strains.     

Ornithine decarboxylase and trypanothione reductase genes in Leishmania braziliensis guyanensis

Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC 1. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania.     

Ornithine Decarboxylase and Trypanothione Reductase Genes in Leishmania braziliensis guyanensis

. Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC I. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania .     

Leishmania braziliensis panamensis: increased infectivity resulting from heat shock

Promastigotes of Leishmania braziliensis panamensis were subjected to a heat shock transformation yielding an amastigote-like stage. During the process of conversion, the heat-induced differentiating form displayed an increase in infectivity (as determined by lesion size) accompanied by a total protein composition unlike that of the promastigote and a morphology resembling that of the amastigote. These biological/functional changes may be related to an involvement of a heat shock response in the differentiation of leishmania, thus having important implications in the development of prevention and treatment stratagems.