Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors.

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.

Sampling Rhodnius neglectus in Mauritia flexuosa palm trees: a field study in the Brazilian savanna.

Two sampling methods (manual capture and live-baited adhesive traps) were compared for collecting the bug Rhodnius neglectus Lent (Hemiptera: Reduviidae: Triatominae) from palm trees, Mauritia flexuosa L. (Arecaceae), in the savanna of Brasília DF. R. neglectus was found in 19/50 (38%) of palm trees sampled. The detection rate was much higher by visual inspection and manual capture (18/50=36%) than by our trapping method (5/50=10%), although one tree was found to be positive by trapping but not by manual capture. Bugs collected manually were mostly (146/154=95%) found among the dead organic material in palm crowns. In combination, these sampling techniques are useful for quick detection of triatomine bug infestation in palm trees, especially in areas of high ecological value where the palms should not be cut and dissected, but arboreal Rhodnius are suspected to transmit enzootic Trypanosoma cruzi that might represent a risk of causing human cases of Chagas disease.

Biogeography of Triatominae (Hemiptera: Reduviidae) in Ecuador: implications for the design of control strategies.

Chagas disease control strategies strongly depend on the triatomine vector species involved in Trypanosoma cruzi transmission within each area. Here we report the results of the identification of specimens belonging to various species of Triatominae captured in Ecuador (15 species from 17 provinces) and deposited in the entomological collections of the Catholic University of Ecuador (Quito), Instituto Oswaldo Cruz (Brazil), the Natural History Museum London (UK), the London School of Hygiene and Tropical Medicine (UK), the National Institute of Hygiene (Quito), and the Vozandes Hospital (Quito). A critical review of published information and new field records are presented. We analysed these data in relation to the life zones where triatomines occur (11 life zones, excluding those over 2,200 m altitude), and provide biogeographical maps for each species. These records are discussed in terms of epidemiological significance and design of control strategies. Findings relevant to the control of the main vector species are emphasised. Different lines of evidence suggest that Triatoma dimidiata is not native to Ecuador-Peru, and that synanthropic populations of Rhodnius ecuadoriensis in southern Ecuador-northern Peru might be isolated from their sylvatic conspecifics. Local eradication of T. dimidiata and these R. ecuadoriensis populations might therefore be attainable. However, the presence of a wide variety of native species indicates the necessity for a strong longitudinal surveillance system.

Immunological selection for Leishmania (Viannia) braziliensis antigens.

Comparative ELISA and selective immunoblotting procedures were used in attempts to identify differential serological indicators of infection with the Leishmania (Viannia) braziliensis complex, infection with the L. braziliensis species, and therapeutic cure of localized or mucocutaneous leishmaniasis (LCL or MCL). Although mean ELISA absorbance values were significantly higher for MCL sera than for LCL sera, absorbance could not be used as a reliable indicator of the clinical form of disease. Immunoblotting profiles were similar with sera from MCL and LCL. Pre-adsorption with heterologous trypanosomatid antigens indicated that recognition of antigens of about 56, 60, 66, 72, 88 and 110 kDa might be specific to the subgenus Viannia. In two-colour, sequential, dual ELISA-based immunoblotting, no antigens recognized only by sera from MCL patients were detected. After glucantime therapy, immunoblotting profiles with LCL sera were reduced both in intensity and in the range of antigens detected; a 104-kDa antigen was newly detected with post-treatment LCL sera. Overall, the results show the value of differential immunological detection strategies and support the close relationship between species of the subgenus Viannia but fail to indicate a prognostic antigen for MCL.

Molecular and biologic characterization of Leishmania parasites implicated in an epidemic outbreak in northwestern Argentina.

Leishmania (Viannia) braziliensis and its variants were implicated in the epidemic outbreak of mucocutaneous leishmaniasis that occurred in Salta, northwestern Argentina, in 1985. A total of 24 suspected, untreated cases were evaluated clinically and parasitologically. Four of five stable isolates were consistent with the reference strain of L. (V.) braziliensis as determined by monoclonal antibodies and indirect immunofluorescence or radioimmunobinding assays. Zymodeme analysis in agarose gels showed a close relationship with L. (V.) guyanensis and L. (V.) panamensis. All zymograms obtained with polyacrylamide gels belonged to the subgenus Viannia; the patterns were different from, but very closely related to, the reference strains of L. (V.) braziliensis as determined by dendrogram analysis. Hamsters infected with two isolates showed a pattern consistent with L. (V.) braziliensis. The pattern of development in the gut of Lutzomyia longipalpis was consistent with members of Viannia.

Phylogenetic taxonomy of Leishmania (Viannia) braziliensis based on isoenzymatic study of 137 isolates.

Biochemical characterization of 137 Leishmania braziliensis isolates from South and Central America, and from selected endemic foci in Bolivia, Brazil and Colombia, performed by isoenzymatic electrophoresis using 10 enzymatic systems, showed a high enzymatic polymorphism (44 zymodemes obtained) based on the variation of a small number of enzymes. Cladistic analysis showed close links between the zymodemes within the L. braziliensis s.s. cluster. The position of 2 Colombian zymodemes obtained (MON*204 and MON*205) justify the inclusion of L. peruviana within the L. braziliensis cluster.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Estudos de diagnostico, caracterizacao parasitaria e identificacao foram conduzidos em pacientes humanos com lesoes cutaneas de leishmaniose na Provincia de Santiago del Estero, no norte da Argentina. Os procedimentos de diagnostico foram: biopsias de lesoes para utilizacao em esfregacos e inoculacao em hamster; aspiracao (com agulha) de ulceras, para cultura "in vitro". As tecnicas a IFAT-IgG e o teste intradermico de Montenegro. Oito cepas de parasitas foram isoladas, sendo estas obtidas de pacientes com lesoes ativas...

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes.

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and species-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly species-specific Monoclonal Antibody (Clone: B-18, Leishmania-(Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosine as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Dissemination of Leishmania (Viannia) braziliensis.

Destructive human mucocutaneous leishmaniasis may appear many years after the primary cutaneous infection with Leishmania (Viannia) braziliensis. Hamsters (Mesocricetus auratus) were infected with metacyclic L. braziliensis promastigotes. It was found that secondary metastatic visceral lesions could arise from a primary cutaneous lesion, or secondary cutaneous lesions from a primary visceral lesion. Parasites in the viscera were shown to be viable, multiplying and capable of metastasis to either secondary visceral or cutaneous sites. The finding of an early metastasis in the wall of a small cutaneous vessel indicates that dissemination can occur by the haematogenous route. Slow growing organisms in viscera may thus be a source for late metastasis to mucocutaneous sites or for systemic relapse after immunosuppression.

Marmosets in New World leishmaniasis research

En leismaniasis, la búsqueda de un modelo animal experimental apropiado es muy recomendable, especialmente en las investigaciones de laboratorio con la especie Leishmania (Viannia) braziliensis, L. (V) b. Diversas especies de primates neotropicales han sido usados con frecuencia como huéspedes experimentales de Leishmania. Este artículo descrive aspectos de la interacción del parásito, L. (V) b. y los micos "estrella" o "titles" (primates, Callithricidae) como huéspedes experimentales y resume datos sobre la biologia del parasitismo en este modelo primate. Se comentan sumariamente los resultados de estudios recientes sobre infecciones primarias con varias cepas de L. (V) b y sobre la evolución del parasitismo, los patrones clínicos resultantes y los experimentos de immunoprotección. Se discute la relevancia de los hallazgos en los experimentos de reinfección homóloga en el desarrollo de una vacuna para leishmaniasis

Marmosets in New World leishmaniasis research

En leismaniasis, la búsqueda de un modelo animal experimental apropiado es muy recomendable, especialmente en las investigaciones de laboratorio con la especie Leishmania (Viannia) braziliensis, L. (V) b. Diversas especies de primates neotropicales han sido usados con frecuencia como huéspedes experimentales de Leishmania. Este artículo descrive aspectos de la interacción del parásito, L. (V) b. y los micos "estrella" o "titles" (primates, Callithricidae) como huéspedes experimentales y resume datos sobre la biologia del parasitismo en este modelo primate. Se comentan sumariamente los resultados de estudios recientes sobre infecciones primarias con varias cepas de L. (V) b y sobre la evolución del parasitismo, los patrones clínicos resultantes y los experimentos de immunoprotección. Se discute la relevancia de los hallazgos en los experimentos de reinfección homóloga en el desarrollo de una vacuna para leishmaniasis (AU)

[An evaluation of the effect of a bark extract from the cashew (Anacardium occidentale L.) on infection by Leishmania (Viannia) braziliensis]. / Avaliação do efeito do extrato de casca de cajueiro-branco (Anacardium occidentale L.) sobre a infecção por Leishmania (Viannia) brasiliensis.

This paper presents an evaluation of the therapeutic effect of an hydroalcoholic extract of A. occidentale L. bark against Leishmania (Viannia) brasiliensis. The extract showed high activity in the in vitro model against the promastigotes of this species. However in the in vivo model no curative activity was observed.

Metacyclogenesis of Leishmania (Viannia) braziliensis in vitro: evidence that lentil lectin is a marker of complement resistance and enhanced infectivity.

Axenic culture in modified Grace's medium was used to induce metacyclogenesis of Leishmania (Viannia) braziliensis in vitro. Morphological characteristics, lectin agglutination profiles, susceptibility to complement lysis, and infectivity in vivo were compared between metacyclic promastigotes and promastigotes in mid-log phase growth. Short, arrow-like promastigotes and round, oval promastigotes were defined as putative metacyclic forms on the basis of being highly motile and free swimming, with a small cell body and long flagellum. These forms increased during metacyclogenesis to > 80% whereas long-bodied, slender promastigotes and intermediate slender promastigotes declined progressively. Lentil lectin selectively agglutinated L. braziliensis after the induction of metacyclogenesis, whereas concanavalin A, wheat germ agglutinin and peanut agglutinin similarly agglutinated metacyclic promastigotes and mid-log phase promastigotes. Metacyclic promastigotes survived in 7.5%-20% human serum whereas mid-log phase promastigotes did not. Five hundred metacyclic promastigotes were highly infective to hamsters whereas 500 mid-log phase promastigotes rarely caused any lesion. Specific agglutination by lentil lectin should allow purification of metacyclic organisms for standardization of immunoprotection and challenge experiments.

Marmosets in New World leishmaniasis research.

In leishmaniasis, the search for a suitable experimental animal model is highly recommended, especially in Leishmania (Viannia) braziliensis (L. (V) b) research. Diverse species of neotropical primates have increasingly been used as experimental hosts of Leishmania. This article describes aspects of parasite L(V)b and marmoset, Callithrix penicillata (Primates, Callithricidae), interaction and summarizes the biology of parasitism in this primate model. Results of recent studies on primary infections with several strains of L(V)b as well as parasitism evolution, clinical outcome patterns and immunoprotection experiments, are summarily discussed. The relevance of homologous reinfection experiments for vaccine development in leishmaniasis is discussed.

Marmosets in New World leishmaniasis research.

In leishmaniasis, the search for a suitable experimental animal model is highly recommended, especially in Leishmania (Viannia) braziliensis (L. (V) b) research. Diverse species of neotropical primates have increasingly been used as experimental hosts of Leishmania. This article describes aspects of parasite L(V)b and marmoset, Callithrix penicillata (Primates, Callithricidae), interaction and summarizes the biology of parasitism in this primate model. Results of recent studies on primary infections with several strains of L(V)b as well as parasitism evolution, clinical outcome patterns and immunoprotection experiments, are summarily discussed. The relevance of homologous reinfection experiments for vaccine development in leishmaniasis is discussed.

Clonal variation within a mucosal isolate derived from a patient with Leishmania (Viannia) braziliensis infection.

Three isolates over 5 years from a patient with persistent relapsing mucosal leishmaniasis due to Leishmania (Viannia) braziliensis and 7 clones from one of these isolates were studied by zymodemes and serodemes analysis. Results showed evidences of clonal phenotypic variation. Eight isoenzymes markers demonstrated clear differences on Cellulose Acetate (CA) and thin starch gel electrophoresis. Also a panel of specific monoclonal antibodies showed such differences. Our observations provide additional evidence that Leishmania (Viannia) braziliensis is composed by subpopulations of parasites with peculiar biochemical and antigenic characteristics.

Experimental infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates:Callithricidae).

Fourteen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) braziliensis (L. (V) b.) strains MHOM/BR/83/LTB-300 MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but 3 of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. Parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm2 to 107 mm2. Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the end of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed but with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L. (L) a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesion developed in a similar manner to L. (V) b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species.(ABSTRACT TRUNCATED AT 250 WORDS)