Search

Several groups have recently developed molecular tests for the detection of Trypanosoma cruzi, the causative agent of Chagas' disease. Polymerase chain reaction (PCR) amplification of kinetoplast minicircle DNA sequences appears to be the most sensitive method. However, the specificity of PCR-based diagnostic methods was challenged when the complete sequence of Trypanosoma rangeli DNA minicircles was discovered. In the present study, we conducted. PCR experiments using the S35/S36 primers in Rhodnius prolixus and Balb/c mice with single and mixed infections of T. cruzi and/or T. rangeli. In single infections, the profile of each trypanosome was easily distinguishable in haemolymph, salivary gland and intestinal tissues and faeces of insect vectors. In mixed infections of anterior intestine (where T. rangeli is more predominant than T. cruzi), the DNA amplification profile of both parasites was observed simultaneously. Conversely, only the T. cruzi profile was observed in rectal ampulla (where T. cruzi is more abundant than T. rangeli). In mice with single infections of T. cruzi or T. rangeli, the profiles of amplified DNA were easily distinguishable in each case. The T. cruzi profile was dominant in most mixed infections, probably due to the fact that T. cruzi minicircles are more abundant and consequently compete more eagerly for annealing with the S35/S36 primers. In cases of mixed infections where T. rangeli was initially more abundant than T. cruzi, the specific T. rangeli 760 bp band was present for 7 days after infection and then this band and others ranging from 300 to 450 bp disappeared and only the typical T. cruzi 330 bp band remained. The S35/S36 primers used in polyacrylamide gel electrophoresis (PAGE) detected T. cruzi specifically, and prevented misdiagnosis due to the presence of T. rangeli. This technique can also be used to identify parasites in different stages of the infection (acute or chronic) in vertebrate hosts and to localize the parasites in the insect vector.

This short communication informs the discovery of Trypanosoma rangeli for the first time at Triângulo Mineiro region, South-east of Brazil, a highly endemic area of Chagas' disease and also the natural infection of Didelphis albiventris with the same trypanosome. Both the findings were demonstrated through blood smears, xenodiagnosis, microhematocrit technics and PCR. The last one was realized in faeces and hemolymph of Triatoma infestans utilizing as controls strains of T. rangeli from Colombia.

Trypanosomatids are characterized by the presence of kinetoplast DNA (kDNA), a peculiar form of mitochondrial DNA that consists of several thousand minicircles and a few dozen maxicircles catenated in a network. Within a species, the minicircles are known to differ in nucleotide sequence, but are homogeneous in size and always cross-hybridize. In all species of trypanosomatids, kDNA minicircles have at least one copy of a conserved 100-200 nucleotide region containing an almost invariant 'universal' 12-mer sequence (5'-GGGGTTGGTGTA-3'). We here report that Trypanosoma rangeli, a non-pathogenic parasite of man, contains two distinct classes of kDNA, minicircles called KP1 and KP2, which differ in size and molecular organization. Both were cloned and sequenced in both directions. KP2 was 1587 bases along and contained two copies of the conserved region as direct repeats 180 degrees apart. In contrast, KP1 had 1764 bases and showed a single conserved region. Moreover, KP1 differed further from KP2 and from most other previously sequenced trypanosomatid minicircles by containing a nucleotide substitution (5'-GGGGTTAGTGTA-3') in the 12-mer universal sequence tag. Polymerase chain reaction and hybridization studies suggest that the sequence of KP1 is very conserved in several other T. rangeli strains from Honduras, Colombia and Venezuela. It thus could provide a good target for the molecular diagnosis of infection with this parasite.

Very little is known about the structure and sequence of the genomic DNA and kDNA of T. rangeli and no highly polymorphic markers are known. In this paper, we show that the Jeffreys' multilocal probe 33.15 produces characteristic DNA fingerprints with these trypanosomes. The multiband patterns can be used to differentiate T. cruzi from T. rangeli and for recognizing relationships between strains of the latter from widely different geographic areas and different hosts. The topology of a UPGMA phenetic tree constructed from band-sharing data suggests the existence of two groups of T. rangeli: one encompassing parasites from Central America and the northern part of South America and another with the parasites from southern Brazil. This splitting was confirmed by the use of both nuclear and kinetoplast unique sequence probes. Among strains of T. rangeli, band sharing was generally negatively correlated with geographical distance. This work confirms the usefulness of DNA fingerprints as a potent technique for the analysis of relationships in trypanosomatid populations.

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.

Abstract: Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the β-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease. [Copyright &y& Elsevier]

Abstract: We describe here an extension of a previous genetic characterization of Trypanosoma cruzi strains (Be-62 and Be-78) isolated from the patient Berenice, the first human case of Chagas disease [Chagas, C., 1909. Nova Tripanomíase humana. Estudos sobre morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., n. sp., agente etiolójico da nova entidade morbida do homem. Mem. Inst. Oswaldo Cruz 1, 159–218]. We wanted to verify the composition of T. cruzi populations originated from these two isolates. In the present work, 22 enzymatic loci (MLEE), nine RAPD primers and 7 microsatellite loci were analyzed. Clones from both strains were also characterized to verify whether these strains are mono or polyclonal. Be-62 and Be-78 strains were different in 3 out of 22 enzymatic systems, in 3 out of 9 RAPD primers tested and in all microsatellite loci investigated. However, our data suggests that both strains are phylogenetically closely related, belonging to genetic group 32 from Tibayrenc and Ayala [Tibayrenc, M., Ayala, F.J., 1988. Isoenzime variability in Trypanosoma cruzi, the agent of Chagas’ disease: genetical, taxonomical, and epidemiological significance. Evolution 42, 277–292], equivalent to zymodeme 2 and T. cruzi II major lineage which, in Brazil, comprises parasites from the domestic cycle of the disease. Microsatellite analyses showed differences between the parental strains but suggested that both populations are monoclonal since each strain and their respective clones showed the same amplification products. [Copyright &y& Elsevier]