Role of two Triatoma (Hemiptera: Reduviidae: Triatominae) species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) to man in the West Coast of Mexico

From August 1997 to August 1998, 334 specimens of Triatoma longipennis and 62 of T. picturata were collected in four groups of localities placed in the zone from Guadalajara, Jalisco to Tepic, Nayarit, in the West Coast of Mexico. Most T. longipennis were collected outdoors (69.2%) while most T. picturata (58.1%) were collected indoors. All collected specimens were examined for Trypanosoma cruzi infection, which was detected on 98 (29.3%) T. longipennis and 17 (27.4%) T. picturata. This study confirms the role of T. longipennis and T. picturata as some of the main T. cruzi vectors to humans in Mexico. Habitation Infestation Rate with T. longipennis was of 0.09 and with T. picturata was of 0.03 and the predominating ecotopes were pile of blocks, chicken coops, pigsties, wall crawls and beds.

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus

PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard´s distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.

A α-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

α-glycerophosphate dehydrogenase (α-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

Triatomines Involved in Domestic and Wild Trypanosoma cruzi Transmission in Concepción, Corrientes, Argentina

An entomological and serological survey was performed in three localities of the Department of Concepción, Province of Corrientes, Argentina in 1998 and 1999, to identify triatomines species involved in domestic and wild transmission of Chagas disease. Triatomines were collected by man/hour capture in 32 houses randomly selected and 44 nearby outdoor ecotopes. Trypanosoma cruzi infection in triatomines was assessed by direct microscopic observation (400x) of feces and polymerase chain reaction. Serological techniques used for people were Indirect Hemagglutination Test and Indirect Fluorescent Test. Triatomines were collected in 28.1% of the houses and 31.8% of the wild biotopes. Triatoma infestans (Klug 1834) was exclusively found indoors and T. cruzi infected 60% of them. Triatoma sordida (Stål 1859) was mainly found in extradomestic ecotopes where trypanosome infection rate reached 12.7%. Serological study of 98 local people showed that 29.6% were seroreactive; most of their houses were closed to wild biotopes colonized by T. sordida. Results indicate that there is an active T. infestans mediated transmission of Chagas disease in this zone that yields important human prevalence and that the populations of T. sordida in wild biotopes not only sustain the wild T. cruzi cycle but also represent an actual risk for people living in the area.

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.

Fibrogenesis and collagen resorption in the heart and skeletal muscle of Calomys callosus experimentally infected with Trypanosoma cruzi: immunohistochemical identification of extracellular matrix components

Intense inflammatory lesions and early development of interstitial fibrosis of the myocardium and skeletal muscle with spontaneous regression, have been described in Calomys callosus infected with Trypanosoma cruzi. The genetic types of collagen present in this model were investigated through immunohistochemistry using specific antibodies, combined with histopathology and Picro-Sirius staining of collagen. Thirty-five calomys were infected with the Colombian strain of T. cruzi and sacrificed at 24, 30, 40, 60 and 90 days post-infection. Inflammatory lesions and fibrogenesis were prominent at the early phase of infection and significantly decreased during late infection. Immunoisotyping of the matrix components was performed by indirect immunofluorescence on 5 µm thick cryostat sections using specific antibodies against laminin, fibronectin and isotypes I, III and IV of collagen. In the early phase, positive deposits of all the matrix components were present, with predominance of fibronectin, laminin and collagens types I and III in the myocardium and of types III and IV in the skeletal muscles. From the 40th day, type IV collagen predominates in the heart. At the late phase of infection (60th to 90th day), a clear fragmentation and decrease of all the matrix components were detected. Findings of the present study indicate that a modulation of the inflammatory process occurs in the model of C. callosus, leading to spontaneous regression of fibrosis independent of the genetic types of collagen involved in this process.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

Biological characterization of a beta-galactosidase expressing clone of Trypanosoma cruzi CL strain

Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37°C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.