Triatominae-Trypanosoma cruzi/T. rangeli: Vector-parasite interactions.

Of the currently known 140 species in the family Reduviidae, subfamily Triatominae, those which are most important as vectors of the aetiologic agent of Chagas disease, Trypanosoma cruzi, belong to the tribes Triatomini and Rhodniini. The latter not only transmit T. cruzi but also Trypanosoma rangeli, which is considered apathogenic for the mammalian host but can be pathogenic for the vectors. Using different molecular methods, two main lineages of T. cruzi have been classified, T. cruzi I and T. cruzi II. Within T. cruzi II, five subdivisions are recognized, T. cruzi IIa-IIe, according to the variability of the ribosomal subunits 24Salpha rRNA and 18S rRNA. In T. rangeli, differences in the organization of the kinetoplast DNA separate two forms denoted T. rangeli KP1+ and KP1-, although differences in the intergenic mini-exon gene and of the small subunit rRNA (SSU rRNA) suggest four subpopulations denoted T. rangeli A, B, C and D. The interactions of these subpopulations of the trypanosomes with different species and populations of Triatominae determine the epidemiology of the human-infecting trypanosomes in Latin America. Often, specific subpopulations of the trypanosomes are transmitted by specific vectors in a particular geographic area. Studies centered on trypanosome-triatomine interaction may allow identification of co-evolutionary processes, which, in turn, could consolidate hypotheses of the evolution and the distribution of T. cruzi/T. rangeli-vectors in America, and they may help to identify the mechanisms that either facilitate or impede the transmission of the parasites in different vector species. Such mechanisms seem to involve intestinal bacteria, especially the symbionts which are needed by the triatomines to complete nymphal development and to produce eggs. Development of the symbionts is regulated by the vector. T. cruzi and T. rangeli interfere with this system and induce the production of antibacterial substances. Whereas T. cruzi is only subpathogenic for the insect host, T. rangeli strongly affects species of the genus Rhodnius and this pathogenicity seems based on a reduction of the number of symbionts.

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.

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease.

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification.

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.

Chagas disease and human migration.

Human Chagas disease is a purely accidental occurrence. As humans came into contact with the natural foci of infection might then have become infected as a single addition to the already extensive host range of Trypanosoma cruzi that includes other primates. Thus began a process of adaptation and domiciliation to human habitations through which the vectors had direct access to abundant food as well as protection from climatic changes and predators. Our work deals with the extraction and specific amplification by polymerase chain reaction of T. cruzi DNA obtained from mummified human tissues and the positive diagnosis of Chagas disease in a series of 4, 000-year-old Pre-Hispanic human mummies from the northern coast of Chile. The area has been inhabited at least for 7,000 years, first by hunters, fishers and gatherers, and then gradually by more permanent settlements. The studied specimens belonged to the Chinchorro culture, a people inhabiting the area now occupied by the modern city of Arica. These were essentially fishers with a complex religious ideology, which accounts for the preservation of their dead in the way of mummified bodies, further enhanced by the extremely dry conditions of the desert. Chinchorro mummies are, perhaps, the oldest preserved bodies known to date.

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.

Isolation of Trypanosoma cruzi DNA in 4,000-year-old mummified human tissue from northern Chile.

A segment of DNA unique to the kinetoplast of Trypanosoma cruzi was isolated from spontaneously mummified human remains from the coastal area of northern Chile at sites dated from 2000 BC to about AD 1400. Following rehydration of the desiccated human tissue samples of heart, esophagus, or colon, the samples were extracted and primers employed to bind to a 330 bp kinetoplast minicircle DNA sequence present in T. cruzi. This segment was then amplified using the polymerase chain reaction (PCR), and the target segment was visualized by gel electrophoresis. This method enables the identification of Chagas' disease in an ancient body in the absence of recognizable anatomic pathological changes.

Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA.

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.

[New sources of lectins to differentiate between Trypanosoma cruzi y T. rangeli]. / Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli.

Extracts of 176 species of Colombian plant seeds, corresponding to 49 families and 147 genera, were tested for detecting agglutinins against human red blood cells from A+, B+ and O+ groups, dog, horse and rabbit. Extracts with haemagglutination activity were used for agglutination of Trypanosoma cruzi and T. rangeli. In addition the hemolymph of 16 native species of invertebrates were tested in the same conditions. Serial dilution of extracts were used for agglutination reactions. Both T. cruzi and T. rangeli epimastigotes showed agglutination with the extract of seven different species of plant seeds and with two types of haemolymph of invertebrates. The seeds of five plants exclusively agglutinated the epimastigotes of T. cruzi and thus can be used for the differentiation between culture forms of the trypanosomes. The secretion of the lung of a snail (Bulimus sp.) lysed entirely the epimastigotes of T. cruzi but did not affect T. rangeli forms. No extracts were found which agglutinated or lysed exclusively the epimastigotes of T. rangeli.

Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli / Further sources of lectins for differentiation between Trypanosoma cruzi y T. rangeli

Os extratos de 176 espécies de sementes de plantas colombianas, correspondentes a 49 famílias e 147 gêneros foram testados para detectar a presença de aglutininas frente a hemácias humanas dos grupos A+, B+, O+ e de cachorro, cavalo e coelho. Os extratos que apresentaram alguma atividade hemaglutinante, foram usados para testar a aglutinaçao de Trypanosoma cruzi e T. rangeli. Além disso, a hemolinfa de 16 espécies nativas de invertebrados foram testadas nas mesmas condiçoes. Diluiçoes seriadas dos extratos foram usadas para as aglutinaçoes. Ambas epimastigotas de T. cruzi e T. rangeli foram aglutinadas com os extratos de sete sementes de plantas diferentes e com dois tipos de hemolinfa de invertebrados. As sementes de cinco plantas aglutinaram exclusivamente as epimastigotas de T. cruzi podendo assim ser usadas para a diferenciaçao entre as formas de cultura desses tripanossomos. A secreçao do pulmao do caramujo (Bulimus sp.) lisou completamente as epimastigotas de T. cruzi mas nao afetou as formas de T. rangeli. Nao foram encontrados extratos que aglutinaram ou lisaram exclusivamente as epimastigotas de T. rangeli.

Kinetoplast DNA from Trypanosoma rangeli contains two distinct classes of minicircles with different size and molecular organization.

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.

DNA fingerprinting reveals relationships between strains of Trypanosoma rangeli and Trypanosoma cruzi.

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.

[Presence of neuraminidase in Rhodnius prolixus infected with Trypanosoma rangeli]. / Estudio sobre la presencia de neuraminidasa en Rhodnius prolixus infectado con Trypanosoma rangeli.

Using three different methods, the activity of neuraminidase was studied in the promesenteron, postmesenteron, rectal ampulla, haemolymph and salivary glands in 600 Rhodnius prolixus experimentally infected with Trypanosoma rangeli stock San Agustín. The haemagglutination method with peanut lectin, and the fluorescence test with peanut lectin conjugated with fluorescein isothiocyanate, and the fluorescence emitted by 4-methylumbelliferone showed in all cases the presence of neuraminidase in the supernatant culture of T. rangeli in Tobie's medium between 8 to 15 days growth. None of the three methods was able to detect the presence of neuraminidase in R. prolixus infected with T. Rangeli, thus suggesting that this enzyme is not produced in vivo, and consecutively is not implicated in the pathogenicity that this trypanosome has to its vector.

The affinity of the lectins Ricinus communis and Glycine maxima to carbohydrates on the cell surface of various forms of Trypanosoma cruzi and Trypanosoma rangeli, and the application of these lectins for the identification of T. cruzi in the feces of Rhodnius prolixus.

Flagellates of Trypanosoma cruzi (stock Molino 1), obtained from the intestine of experimentally infected Rhodnius prolixus, grown in cellular or acellular culture, as well as from the blood of infected mice, were examined by a direct fluorescence test using the lectins RCA (Ricinus communis-120) and SBA (soy bean agglutinin; Glycine maxima), conjugated with fluorescein isothiocyanate, for the detection of beta-D-galactose and alpha,beta-N-acetyl-D-galactosamine on the membranes of the flagellates. The same reactions were carried out using Trypanosoma rangeli (stock San Agustin), obtained from the intestine, hemo-lymph or salivary glands of experimentally infected R. prolixus, as well as from cultures and from the blood of experimentally infected CFW mice. The results indicate that the membrane of T. rangeli in the salivary glands of the vector contains beta-D-galactose, but that this sugar is absent from all other developmental stages of this trypanosome. All stages of intestinal and cultured. T. cruzi presented positive reactions with RCA-FITC and SBA-FITC. The high specificity of this technique makes it useful for the examination of R. prolixus, previously used in xenodiagnosis of Chagas' disease and for the examination of intradomiciliary or sylvatic vectors in epidemiological surveys in areas where T. cruzi and T. rangeli coexist. Formaldehyde fixed samples can be examined months later and false reports due to T. rangeli can be avoided.

[Laboratory maintenance of Trypanosoma (Herpetosoma) rangeli Tejera, 1920]. / Mantenimiento en el laboratorio de Trypanosoma (Herpetosoma) rangeli Tejera, 1920.

Two laboratory maintenance systems of Trypanosoma rangeli were compared. The maintenance by weekly subinoculations in Tobie's culture medium and the intrafemoral inoculation of Rhodnius prolixus with cultured flagellates, resulted in loss of infectivity of the metacyclic salivarian trypomastigotes for mice, ten months after maintenance in culture. With the system of cyclical passes through culture-Rhodnius-mouse-culture-Rhodnius, the infectivity of the metacyclic trypomastigotes for mice, was maintained during the three years of the experiment. The number and percentage of metacyclic trypomastigotes formed in the salivary glands of R. prolixus, previously inoculated intrafemorally or intracoelomically with culture forms of T. rangeli, did not show correlation with the inoculated dose, however the inoculated quantity demonstrated a direct relation with the mortality rate of the insects. The results indicate that T. rangeli requires an adequate maintenance system, so that under experimental condition the biological characteristics, normally expressed under natural conditions, are conserved.