Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.     

Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene

306-314. Three synthetic oligonucleotides corresponding to sequences within the D7a divergent domain of the large subunit ribosomal RNA gene have been used to amplify the total DNA of Trypanosoma rangeli and Trypanosoma cruzi, two morphologically similar protozoa with overlapping geographical distribution and hosts. The two organisms may be distinguished by the electrophoretic mobilities of their respective amplification products. For T. rangeli a 210-bp product was obtained. The presence of this fragment was confirmed in 14 T. rangeli strains. For T. cruzi two possible amplification products were originated: a 265-bp DNA fragment for strains typed as lineage 1 and a 250-bp fragment for lineage 2 strains. Eleven unidentified trypanosome stocks, recently isolated from Amazonian vectors, could be discriminated using the proposed assay. The potential field application of multiplex PCR was further demonstrated by identification of the two parasite species in samples containing intestinal tract and feces of triatomines. In the present study we have also amplified the D7a domain of several trypanosomatids employing primers complementary to the conserved flanking regions. Size and sequence polymorphisms were observed, indicating that this region could also be explored as a target for specific detection of other members of the Trypanosomatidae family.     

First report on the occurrence of Trypanosoma rangeli Tejera, 1920 in the state of Ceará, Brazil, in naturally infected triatomine Rhodnius nasutus Stål, 1859 (Hemiptera, Reduviidae, Triatominae)

The aim of this work was to identify and report the occurrence of Trypanosoma rangeli and Trypanosoma cruzi in naturally infected Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) in the state of Ceará, Brazil. Triatomines feces, salivary glands, and hemolymph were collected for fresh examination, and specific detection of T. rangeli and T. cruzi DNA by polymerase chain reaction was carried out. The specific characterization of these two parasites showed the simultaneous presence of both parasites in two (7.7%) of the 26 positive insects. Our results provide further knowledge on the geographical distribution of T. rangeli in Brazil.     

Genes for cysteine proteinases from Trypanosoma rangeli

Abstract PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli , using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi , allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.     

Characterization of Trypanosoma rangeli strains isolated in Central and South America: an overview

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.     

Molecular characterization of the short interspersed repetitive element SIRE in the six discrete typing units (DTUs) of Trypanosoma cruzi

Repetitive sequences constitute an important proportion of the Trypanosoma cruzi genome; hence, they have been used as molecular markers and as amplification targets to identify the parasite presence via PCR. In this study, a molecular characterization of the SIRE repetitive element was performed in the six discrete typing units (DTUs) of T. cruzi. The results evidenced that this element, located in multiple chromosomes, was interspersed in the genome of all DTUs of the parasite. The presence of several motifs implicated in element insertion, duplication, and functionality suggests that SIRE could be an active element in the parasite genome. Of interest, there were SIRE specific Alu I fragments that allowed to discriminate DTU I from the others DTUs. Moreover, an UPGMA phenetic tree constructed from fragment sharing Southern blot data showed that T. cruzi I isolates conform a cluster separated from the T. cruzi II-VI isolates. When the relative number of SIRE copies was determined, a variation from 105 to 2,000 copies per haploid genome was observed among the different isolates without kept a DTU-relationship. In all, these findings suggest that SIRE sequence is a good target for parasite DNA amplification.     

Amplification of Trypanosoma cruzi-specific DNA sequences in formalin-fixed raccoon tissues using polymerase chain reaction

This investigation applied polymerase chain reaction (PCR) using 3 sets of Trypanosoma cruzi-specific primers to amplify DNA from 31 archived formalin-fixed and fresh-frozen raccoon hearts. PCR successfully amplified T. cruzi-specific sequences, with at least 1 primer set, from multiple sites within the myocardium of formalin-fixed and fresh-frozen raccoon hearts that had previously tested positive using enzyme-linked immunosorbent assay and indirect immunofluorescent antibody titer in the absence of positive hemoculture results. Trypanosoma cruzi DNA was most frequently amplified from the interventricular septum, right ventricle, and left atrium. In addition, T. cruzi DNA was amplified with all 3 primers in at least I raccoon that was hemoculture positive and 2 animals that were borderline negative for the T. cruzi antibody and hemoculture negative. The amplification of T. cruzi-specific DNA sequences in the presence of an elevated antibody titer and negative culture results suggests good sensitivity of this method for detecting the presence of the parasite in archival tissues.     

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.     

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed.     

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.     

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.     

Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids

Trypanosoma cruzi lipids contain a high content of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2). Previous data suggest that this parasite is able to convert oleic acid into linoleic acid; humans are not able to do this. Presently, we show that T. cruzi has a gene with high similarity to the delta12 (omega6)-oleate desaturase from plants. Northern blot analysis of the oleate desaturase gene from T. cruzi (OD(Tc)) indicated that this gene is transcribed in epimastigote, amastigote, and trypomastigote forms. Pulsed-field analysis showed that OD(Tc) is located at distinct chromosomal bands on distinct T. cruzi phylogenetic groups. In addition, the chromoblot analysis demonstrated the presence of homologous OD(Tc) genes in several trypanosomatids; namely, Crithidia fasciculata, Herpetomonas megaseliae, Leptomonas seymouri, Trypanosoma freitasi, Trypanosoma rangeli, Trypanosoma lewisi, Blastocrithidia sp., Leishmania amazonensis, Endotrypanum schaudinni, and Trypanosoma conorhini. The native OD(Tc) activity was detected by metabolic labeling and analysis of total fatty acids from epimastigotes and trypomastigotes of T. cruzi, coanomastigotes of C. fasciculata, and promastigotes of L. amazonensis, H. megaseliae, and L. seymouri. The fact that the enzyme oleate desaturase is not present in humans makes it an ideal molecular target for the development of new chemotherapeutic approaches against Chagas disease.     

Prevalence and molecular phylogenetic characterization of Trypanosoma (Megatrypanum) minasense in the peripheral blood of small neotropical primates after a quarantine period

Neotropical primates of the Cebidae and Callitrichidae, in their natural habitats, are frequently infected with a variety of trypanosomes including Trypanosoma cruzi, which causes a serious zoonosis, Chagas' disease. The state of trypanosome infection after a 30-day quarantine period was assessed in 85 squirrel monkeys (Saimiri sciureus) and 15 red-handed tamarins (Saguinus midas), that were wild-caught and exported to Japan as companion animals or laboratory animals, for biomedical research, respectively. In addition to many microfilariae of Mansonella (Tetrapetalonema) mariae at a prevalence of 25.9%, and Dipetalonema caudispina at a prevalence of 3.5%, a few trypomastigotes of Trypanosoma (Megatrypanum) minasense were detected in Giemsa-stained thin films of blood from 20 squirrel monkeys at a prevalence of 23.5%. Although few T. minasense trypomastigotes were found in Giemsa-stained blood films from tamarins, a buffy-coat examination detected trypanosomes in 12 red-handed tamarins (80.0%), and PCR amplification of a highly variable region of the small subunit ribosomal RNA genes (SSU rDNA) for Trypanosoma spp. detected the infection in 14 of the 15 tamarins (93.3%). Nucleotide sequences of the amplicons were identical for trypanosomes from tamarins and squirrel monkeys, indicating a high prevalence but low parasitemia of T. minasense in imported Neotropical nonhuman primates. Based on the SSU rDNA and 5.8S rDNA, the molecular phylogenetic characterization of T. minasense indicated that T. minasense is closely related to trypanosomes with Trypanosoma theileri-like morphology and is distinct from Trypanosoma (Tejeraia) rangeli, as well as from T. cruzi. Using some blood samples from these monkeys, amplification and subsequent sequencing of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene fragments detected 4 trypanosome genotypes, including 2 types of T. cruzi clade, 1 type of T. rangeli clade, and 1 T. rangeli-related type, but failed to indicate its phylogenetic position based on the gGAPDH gene. Furthermore, species ordinarily classified in the Megatrypanum by morphological criteria do not form a clade in any molecular phylogenetic trees based on rDNA or gGAPDH genes.     

Trypanosoma rangeli expresses a β-galactofuranosyl transferase

Glycoconjugates play essential roles in cell recognition, infectivity and survival of protozoan parasites within their insect vectors and mammalian hosts. β-galactofuranose is a component of several glycoconjugates in many organisms, including a variety of trypanosomatids, but is absent in mammalian and African trypanosomes. Herein, we describe the presence of a β(1-3) galactofuranosyl transferase (GALFT), an important enzyme of the galactofuranose biosynthetic pathway, in Trypanosoma rangeli. The T. rangeli GALFT gene (TrGALFT) has an ORF of 1.2 Kb and is organized in two copies in the T. rangeli genome. Antibodies raised against an internal fragment of the transferase demonstrated a 45 kDa protein coded by TrGALFT was localized in the whole cytoplasm, mainly in the Golgi apparatus and equally expressed in epimastigotes and trypomastigotes from T. rangeli. Despite the high sequence similarity with Trypanosoma cruzi and Leishmania spp. orthologous TrGALFT showed a substitution of the metal-binding DXD motif, conserved amongst glycosyltransferases, for a DXE functionally analogous motif. Moreover, a reduced number of GALFT genes were present in T. rangeli when compared with other pathogenic kinetoplastid species.     

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%). The immunological and clinical implications of these findings are discussed.     

Antigenic make-up of Trypanosoma cruzi culture forms: identification of a specific component.

A comparison of Trypanosoma cruzi water soluble antigens with those of stercorarian and salivarian trypanosomes, and Leishmania using immunoprecipitation in gels and immunoelectrophoresis, with the aid of hyperimmune rabbit serum and heterologous adsorptions showed the following. 1) There is a high complexity of soluble antigens of T. cruzi and T. rangeli. 2) At the intraspecific level our results demonstrated the antigenic stability of T. cruzi when maintained in vitro, and that there was quantitative antigenic consistency of the culture forms of different strains of T. cruzi from diverse geographic and parasite sources. At the interspecific level, the antigenic relationships between T. cruzi and the other Trypanosomatidae were established, as follows: 6/10ths of the antigens are shared by stercorarian species (T. dionisii, T. rangeli); 4/10ths by a salivarian trypanosome (T. brucei); and 3/10ths by Leishmania (L. donovani, L. mexicana). 3) Among the 4/10ths of antigenic components specific to T. cruzi, one component was characterized by its antigenicity and immunogenicity in natural and experimental infections, and in immunization experiments; this component was specific to T. cruzi when compared to the other Trypanosomatidae antigens.     

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.     

Evolutionary relationships in Trypanosoma cruzi: molecular phylogenetics supports the existence of a new major lineage of strains

For the purpose of investigating the evolutionary relationships among strains of the human parasite Trypanosoma cruzi, we have determined the nucleotide sequence, in 16 T. cruzi stocks, of a DNA fragment having approximately 1030 nucleotides in length. Phylogenetic analyses show the presence of at least three major groups of T. cruzi strains, a result that contradicts previous phylogenetic inferences based on polymorphism data. We also performed an analysis of the relative extent of nucleotide divergence among T. cruzi strains compared to the divergence between Leishmania species, using the gene encoding pteridine reductase. The results presented in this work show that the divergence among the most distant T. cruzi strains is at least as high as the divergence between two different species complexes of Leishmania, those containing L. major and L. mexicana.     

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.