Splenectomy increases mortality in murine Trypanosoma cruzi infection.

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-γ and TNF-α production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Endothelin-1 receptors play a minor role in the protection against acute Trypanosoma cruzi infection in mice.

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is a major cause of cardiovascular disability in countries where it is endemic. Damage to the heart microvasculature has been proposed to be an important factor in the pathogenesis of heart dysfunction. Endothelin-1 (ET-1) is a potent vasoconstrictor and exerts its effects via specific ET A and ET B receptors. A few studies have suggested a role for ET-1 and its receptors in the pathogenesis of Chagas' disease. We investigated the effects of treatment with bosentan, an ET A/ET B receptor antagonist, on the course of T. cruzi infection (Y strain) in C57Bl/6 mice. Treatment with bosentan (100 mg kg-1 day-1) was given per os starting day 0 after infection until sacrifice. Bosentan significantly increased myocardial inflammation, with no effects on parasitemia. Although the total number of nests was similar, a lower number of intact amastigote nests was found in the heart of bosentan-treated animals. Bosentan failed to affect the infection-associated increase in the cardiac levels of the cytokines IFN-g and TNF-a and the chemokines CCL2/MCP-1, CCL3/MIP-1a and CCL5/RANTES. In vitro, pre-incubation with ET-1 (0.1 microM) 4 h before infection enhanced the uptake of the parasites by peritoneal macrophages, and this effect was abrogated when macrophages were pre-treated with bosentan (1 microM) 15 min before incubation with ET-1. However, ET-1 did not alter killing of intracellular parasites after 48 h of in vitro infection. Our data suggest that bosentan-treated mice have a delay in controlling parasitism which is compensated for exacerbated inflammation. Infection is eventually controlled in these animals and lethality is unchanged, demonstrating that ET-1 plays a minor role in the protection against acute murine T. cruzi infection.

Constituents from stem barks of Luehea ochrophylla Mart and evaluation of their antiparasitic, antimicrobial, and antioxidant activities.

Luehea species are found in almost all Central and South American countries. The present work describes the phytochemical study, isolation, and structural characterisation of friedelin, ß-friedelinol, lupeol, pseudotaraxasterol, ß-sitosterol, betulinic acid, taraxasterol, (-)-epicatechin, ß-sitosterol-3-O-ß-d-glucopyranoside, and (+)-epicatechin-(4ß→8)-epicatechin from stem barks of Luehea ochrophylla Mart. The structural identification of the isolated compounds was mainly performed by NMR analyses and comparison with the data from literature. These compounds were isolated for the first time in the genus Luehea, except ß-sitosterol glucopyranoside, (-)-epicatechin, and lupeol. Hexane extract (HE) and dichloromethane (DF) and ethyl acetate (AF) fractions exhibited antiparasitic activity against amastigote (intracellular) and trypomastigote culture forms of Trypanosoma cruzi. The ethanol extract (EE), DF, and ethanol fraction (EF) exhibited considerable antifungal activity against Candida albicans. Moreover, extracts and fractions exhibited significant percentage of capture free radicals of 2,2-diphenyl-picrylhydrazyl (DPPH) when compared to the standard of ascorbic acid.

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years.

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-gamma as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi.

We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.

Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox.

Twenty-seven Trypanosoma cruzi strains, susceptible or naturally resistant to the nitroderivatives benznidazole and nifurtimox, were analyzed using the following molecular markers: (i) isoenzyme patterns of six enzymes; (ii) genetic variability assayed by randomly amplified polymorphic DNA (RAPD) with two different primers; and (iii) gene probes for P-glycoprotein (TcPGP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the ribosomal RNA gene (rDNA) and the mini-exon gene (MEX), RAPD and isoenzyme profiles divided the T. cruzi strains into three groups, whereas the gene probes divided the T. cruzi strains in two groups. Strains classified as group I or II by RAPD or zymodemes Z1 or Z2 by isoenzyme analysis were either susceptible or naturally resistant to the nitroderivatives. In contrast, strains classified as group III by RAPD and zymodeme ZB by isoenzyme analysis were only drug susceptible and showed polymorphisms for HGPRT and TcPGP. No correlation was observed between drug susceptibility and polymorphisms of rDNA and MEX. Eighteen T. cruzi strains isolated from different geographic regions were included in this study. Thus, from a total of 45 T. cruzi strains analyzed, all 19 of zymodeme B were susceptible to the experimental treatment independent of their geographic origin.

Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains.

DNA extracted from 32 isolates of Trypanosoma cruzi was subjected to polymerase chain reaction amplification using 4 arbitrary primers resulting in relatively complex DNA profiles that include polymorphic markers known as random amplified polymorphic DNAs (RAPDs). The RAPD profiles of 18 strains belonging to zymodeme 1 (Z1) collected from various regions of South America exhibited a consistant pattern and 59 (59%) of the bands produced were present in all Z1 strains. A similar level of consistency was seen in the number of bands shared between 5 Z2 strains, 4 ZB strains and 2 ZC strains. A phenetic analysis of the 5 most different Z1 strains based on band sharing showed that their interrelationships mirrored their geographical origin. Comparison of the RAPD profiles of strains from different zymodemes showed that less than 7% of bands of strains in one zymodeme are present in strains of another zymodeme. Analysis of band sharing using bands present in all strains of a given zymodeme showed ZB and ZC to be closely related and Z1 and Z2 to form distinct groups.

The use of RAPDs for the study of the genetic diversity of Schistosoma mansoni and Trypanosoma cruzi.

Arbitrary primers have been used for the production of complex, PCR generated DNA profiles in order to undertake a preliminary random amplified polymorphic DNA (RAPD) analysis of strains (and related species) of two parasitic organisms that are responsible for important diseases endemic in Brazil: Schistosoma mansoni that causes schistosomiasis, and Trypanosoma cruzi that causes Chagas' disease. A relatively low level of polymorphism was found in S. mansoni when strains isolated from different regions of Brazil were compared, with less than 10% of bands exhibiting polymorphism. Comparison of different schistosome species, on the other hand, showed them to be distantly related with very few bands shared by even the more closely related species. Trypanosome strains were found to be much more variable. When strains were compared between zymodemes (groups of parasite strains with the same isoenzyme profiles), a maximum of 7% of bands were found to be common whereas among strains in the same zymodeme a clear characteristic pattern was observed. In the zymodeme most thoroughly studied, it was found that 59% of bands were shared. Band sharing analysis showed that the relationships of strains within a zymodeme correlate with their geographical origin and that the relationship between zymodemes correlates closely with that previously determined by isoenzyme analysis. These preliminary data indicate the ready applicability of RAPD analysis to the study of parasites where largely unexplored genetic variations may have an important bearing on the complexity and diversity of diseases.

Trypanosoma cruzi: parasitaemia produced in mice does not seem to be related to in vitro parasite-cell interaction.

The interaction of Trypanosoma cruzi strains producing subpatent or high parasitaemia in mice with mouse macrophages, Vero and L929 cells was evaluated using tissue culture trypomastigotes. Macrophages were the cells most readily infected while Vero cells presented the highest parasite intracellular multiplication rates. Subpatent strains were equal or more infective than the high parasitaemia. Due to the small number of strains, no correlation could be established between the zymodemes and parasitaemia or parasite-cell interaction in vitro. However parasitaemia in mice does not seem to be related to in vitro parasite-cell interaction.

Populational structure of Schistosoma mansoni assessed by DNA microsatellites.

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.

Trypanosomatid isolates from Honduras: differentiation between Trypanosoma cruzi and Trypanosoma rangeli.

With the aim of identifying and differentiating Trypanosoma cruzi from Trypanosoma rangeli, culture epimastigotes from 30 Honduran trypanosomatid isolates were analyzed by susceptibility to complement lysis, reactivity to lectins, reactivity to monoclonal antibodies specific for T. cruzi, and isoenzymatic electrophoretic patterns. Using these four methodologies, 27 of the 30 trypanosomatid isolates, as well as 5 clones, were identified as T. cruzi, whereas the remaining three isolates were classified as T. rangeli. None of the isolates presented mixed trypanosome species. Results indicate that both trypanosomatid species circulate in Honduras and that any of the four methods employed may be used to reliably differentiate T. cruzi from T. rangeli.

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

Identification of morphologically similar Rhodnius species (Hemiptera: Reduviidae: Triatominae) by electrophoresis of salivary heme proteins.

We examined intraspecific variability in the genus Rhodnius using starch gel electrophoresis of salivary heme proteins. Salivary protein profiles of 8 Rhodnius species (R. prolixus, R. robustus, R. neglectus, R. nasutus, R. ecuadoriensis, R. pallescens, R. pictipes, and R. domesticus) were compared. All species could be distinguished by this technique. The greatest protein polymorphism was found in R. ecuadoriensis, R. nasutus, R. robustus, and R. pictipes, followed by R. prolixus, R. neglectus, R. pallescens, and R. domesticus. This approach was able to distinguish R. prolixus from R. robustus and R. neglectus from R. nasutus, species with extreme phenotypical similarity.

Early, intermediate, and late acute stages in Chagas' disease: a study combining anti-galactose IgG, specific serodiagnosis, and polymerase chain reaction analysis.

The acute phase of Chagas' disease was classified as early, intermediate, and late based on the levels of anti-Galalpha, 3Gal IgG (Gal) and specific IgM (M) and IgG (G) anti-T. cruzi reactivity. While the early phase was M+G-Gal-, the intermediate phase was M+G-Gal+, M+G+Gal-, or M+G+Gal+, and the late phase was M-G+Gal+. This sequence of stages was consistent with our previous studies on acute-phase proteins. Analysis by the polymerase chain reaction (PCR) of parasite DNA in 65 blood samples of children living in Cochabamba, Bolivia showed a significant correlation (90.8%) between ELISA and PCR positivity. A lower correlation was observed between indirect hemagglutination, PCR (58%), and ELISA. Electrocardiographic analysis of 43 children studied by the PCR did not show any alteration typical of acute chagasic myocarditis. The PCR positivity was observed in eight samples where only Gal was increased, suggesting a very early T. cruzi infection, when specific antibodies were not yet present. By associating anti-Gal IgG with specific serology, early T. cruzi infection can be detected with greater precision. We suggest the use of anti-Gal antibody reactivity as an aid for the detection of recent T. cruzi infections, at least in endemic areas where diseases caused by other trypanosomatids do not overlap.

A study of experimental reinfection by Trypanosoma cruzi in dogs.

The role of reinfection in the evolution of Chagas' disease was evaluated in dogs alternately infected with the 147 and SC-1 strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain 147, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas' disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas' disease. All infected dogs developed this form of the disease, which was independent of the number of infections.

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Surface antigens of stocks and clones of Trypanosoma cruzi isolated from humans.

The total surface polypeptide pattern was analyzed after radioiodination of 12 different stocks and clones of Trypanosoma cruzi belonging to four different zymodemes isolated from humans of the region of Bambuí (M.G., Brazil). Although some minor differences were encountered, it is concluded that this pattern cannot be used as a method for classification of strains. Sera obtained from chagasic patients harboring parasites typed according to each zymodeme and from rabbits immunized with either epimastigotes or trypomastigotes from the Y strain immunoprecipitated surface antigens of apparent Mr 55 kDa, 80 kDa and 95 kDa in all the stocks (epimastigotes) tested. These antigens thus appear to be conserved among stocks of T. cruzi and to be common to epimastigotes and trypomastigotes. Immunoprecipitation of antigens of surface radioiodinated trypomastigotes (Y strain) with 16 different chagasic sera indicates a remarkable identity among the observed patterns, suggesting that the antigenic characteristics of the surface of T. cruzi infective forms are highly conserved and insensitive to the zymodeme type.

Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae).

Rhodnius prolixus interpopulation variability was studied based on a new approach using salivary heme proteins (nitrophorins) electrophoresis in starch gel. We compared salivary proteins profiles of R. prolixus from three different laboratory colonies from Honduras, Venezuela, Brazil and Rhodnius robustus from Venezuela, constructing a UPGMA. The Honduran and Venezuelan populations could not be distinguished from each other, but the Brazilian population was well separated from the others. The high similarity between Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus may have been introduced from a Venezuelan origin. The low polymorphism shown by the Honduran specimens is in agreement with a possible founder effect. This new approach also distinguished R. prolixus populations from R. robustus, species with extreme phenotypical similarity.

Genetic variability of the main intermediate host of the Schistosoma mansoni in Brazil, Biomphalaria glabrata (Gastropoda: Planorbidae) assessed by SSR-PCR.

The genetic variability of Brazilian Biomphalaria glabrata populations was studied using SSR-PCR. This technique is a variant of the polymerase chain reaction (PCR), which consists of using a single primer directed towards microsatellite regions under high stringency reaction conditions. Twenty snails of each population from eight distant Brazilian localities were analyzed. Morphology and PCR-RFLP were used for previous specific identification of the snails. Bands generated after gel electrophoresis of the SSR-PCR products of each snail were used to study intra- and interpopulation genetic variability. Fifty-five prominent bands were considered in a pairwise band comparison for the determination of genetic variability. Genetic variability was greater between populations than within populations. Snail populations from the field and the laboratory presented almost no genetic differences. No relationship between genetic variability and geographic distance was found. SSR-PCR proved to be a good alternative molecular tool for the population study of B. glabrata.