Molecular detection of trypanosomatids in neotropical primates in the state of Mato Grosso, Midwest, Brazil / Detecção molecular de tripanossomatídeos em primatas neotropicais no estado de Mato Grosso, Centro-Oeste, Brasil

Abstract Trypanosomatids are uniflagellate protozoa belonging to the Trypanosomatidae family. The genera Trypanosoma and Leishmania are of paramount importance as they contain species that cause serious diseases, such as Chagas disease and Leishmaniasis, respectively. The objective of the present study was to identify trypanosomatids present in the whole blood of free-living and captive neotropical primates in Mato Grosso State, Midwest Brazil. Between 2017 and 2019, 38 blood samples were collected from seven different neotropical primate species in seven cities in the state. Through molecular techniques, including polymerase chain reaction (PCR) to amplify a fragment of the kinetoplast DNA (kDNA) and 18S ribosomal RNA (18S rRNA) gene, sequencing, and phylogenetic analysis, nine Leishmania spp. [seven L. infantum and two L. (Leishmania) amazonensis] and two Trypanosoma spp. (T. minasense and T. rangeli) were identified. This study contributes to understanding the occurrence and epidemiology of trypanosomatids in Mato Grosso State and the importance of neotropical primates as trypanosome hosts and possible infection sources for other animals and humans. Future identification of other blood pathogens in neotropical primates will assist in disease control and prevention strategies.

Resumo Tripanossomatídeos são protozoários uniflagelados pertencentes à família Trypanosomatidae. Os gêneros Trypanosoma e Leishmania são de suma importância por conterem espécies causadoras de doenças graves, como doença de Chagas e Leishmaniose, respectivamente. O objetivo do presente estudo foi identificar tripanossomatídeos presentes no sangue total de primatas neotropicais de vida livre e cativos no Estado de Mato Grosso, Centro-Oeste do Brasil. Entre 2017 e 2019, foram coletadas 38 amostras de sangue de sete diferentes espécies de primatas neotropicais em sete cidades do Estado. Foram identificados por meio de técnicas moleculares, incluindo reação em cadeia da polimerase (PCR), para amplificar um fragmento do DNA do cinetoplasto (kDNA) e do gene do RNA ribossômico 18S (rRNA 18S), sequenciamento e análise filogenética, nove Leishmania spp. [sete L. infantum e dois L. (Leishmania) amazonensis] e dois Trypanosoma spp. (T. minasense e T. rangeli). Este estudo contribui para o entendimento da ocorrência e epidemiologia dos tripanossomatídeos no Estado de Mato Grosso e a importância dos primatas neotropicais como hospedeiros tripanossômicos e possíveis fontes de infecção para outros animais e humanos. A identificação futura de outros patógenos do sangue em primatas neotropicais ajudará no controle de doenças e em estratégias de prevenção.

Clinical-dermatological, histological abnormalities and prevalence of Trypanosoma caninum and Leishmania infantum in dogs from Midwest region of Brazil / Anormalidades clínico-dermatológicas, histológicas e prevalência de Trypanosoma caninum e Leishmania infantum em cães da região Centro-Oeste do Brasil

Abstract Leishmania infantum is a trypanosomatid that causes parasitic dermatopathy in dogs. Trypanosoma caninum is another trypanosomatid, which infects the skin of dogs, although cutaneous abnormalities are absent. This study aimed to investigate the occurrence of T. caninum infection and its associated cutaneous and histological changes and compare it with the occurrence of L. infantum infection in dogs. The study included 150 dogs, of which T. caninum infection was identified in 3 (2%) and L. infantum infection in 15 (10%) of them, with no association (p>0.05) of these infections with the breed, gender, age, or cutaneous abnormalities. The cutaneous abnormalities were based on 1 (4.8%) and 12 (57.1%) dogs infected by T. caninum and L. infantum, respectively. The dermatohistopathological abnormalities in the dogs infected with T. caninum included mild perivascular lymphohistioplasmacytic infiltrates in the clinically asymptomatic ones, while in those with dermatological abnormalities, acanthosis, epidermal orthokeratotic hyperkeratosis, melanomacrophages, and co-infection with Microsporum sp. and Trichophyton sp. were observed. InL. infantum infected, the histopathological findings included chronic granulomatous inflammatory infiltrates and structures compatible with amastigotes. Despite the low frequency of T. caninum infection, our findings suggest that this trypanosomatid, unlike L. infantum, does not cause any macroscopic skin abnormalities.

Resumo Leishmania infantum é um tripanosomatídeo que causa dermatopatia parasitária em cães. Trypanosoma caninum é outro tripanosomatídeo, que infecta a pele de cães, embora anormalidades cutâneas sejam ausentes. Este estudo teve como objetivo investigar a ocorrência da infecção por T. caninum e suas alterações cutâneas e histológicas associadas e compará-las com a ocorrência da infecção por L. infantum em cães. O estudo incluiu 150 cães, dos quais a infecção por T. caninum foi identificada em 3 (2%) e a infecção por L. infantum em 15 (10%) deles, sem associação (p>0,05) dessas infecções com a raça, sexo, idade ou anormalidades cutâneas. As alterações cutâneas foram observadas em 1 (4,8%) e 12 (57,1%) cães infectados por T. caninum e L. infantum, respectivamente. As anormalidades dermato-histopatológicas nos cães infectados por T. caninum incluíram infiltrados linfo-histioplasmocitários perivasculares leves nos clinicamente assintomáticos, enquanto naqueles com anormalidades dermatológicas, foram observados acantose, hiperqueratose ortoqueratótica epidermal e melanomacrófagos e co-infecção por Microsporum sp. e Trichophyton sp. Nos cães infectados por L. infantum, os achados histopatológicos incluíram infiltrados inflamatórios granulomatosos crônicos e estruturas compatíveis com amastigotas. A despeito da baixa frequência da infecção por T. caninum, nossos achados sugerem que esse tripanosomatídeo, diferentemente de L. infantum, não causa anormalidades macroscópicas na pele.

Genetic diversity and molecular survey of Trypanosoma (Megatrypanum ) theileri in cattle in Brazil's western Amazon region / Diversidade genética e levantamento molecular de Trypanosoma (Megatrypanum) theileri em bovinos na região da Amazônia Ocidental do Brasil

Abstract Trypanosoma (Megatrypanum) theileri is a flagellated protozoan that infects ruminants and it displays high genetic diversity. In this study, we investigated the prevalence rates of this protozoan based on hemoculture and molecular diagnosis. The isolates of T. theileri thus obtained were characterized by molecular markers SSU rDNA and gGAPDH and molecular diagnosis based on Cathepsin L-like gene (PCR-TthCATL). The PCR-TthCATL and hemoculture indicated an overall prevalence rate of 8.13%, and the CATL derived sequence named IB was identified for the first time in cattle in the western Amazon region, as well as IF in Brazil. We also describe a possible new PCR-TthCATL derived sequence in cattle, designated IL.

Resumo Trypanosoma (Megatrypanum) theileri é um protozoário flagelado que infecta ruminantes e apresenta alta diversidade genética. Neste estudo, investigamos as taxas de prevalência deste protozoário com base na hemocultura e no diagnóstico molecular. Os isolados de T . theileri obtidos foram caracterizados pelos marcadores moleculares SSU rDNA e gGAPDH e o diagnóstico molecular foi baseado no gene do tipo Catepsina L (PCR-TthCATL). O PCR-TthCATL e a hemocultura indicaram uma taxa de prevalência total de 8,13% e a sequência derivada do gene Catepsina L denominada IB de T. theileri foi identificada pela primeira vez em bovinos da Amazônia Ocidental, bem como a IF no Brasil. Também descrevemos uma possível nova sequência derivada da PCR-TthCATL em bovinos, designada IL.

Risk factors associated with Trypanosoma cruziexposure in domestic dogs from a rural community in Panama

Chagas disease, caused by Trypanosoma cruziinfection, is a zoonosis of humans, wild and domestic mammals, including dogs. In Panama, the main T. cruzivector is Rhodnius pallescens, a triatomine bug whose main natural habitat is the royal palm, Attalea butyracea. In this paper, we present results from three T. cruziserological tests (immunochromatographic dipstick, indirect immunofluorescence and ELISA) performed in 51 dogs from 24 houses in Trinidad de Las Minas, western Panama. We found that nine dogs were seropositive (17.6% prevalence). Dogs were 1.6 times more likely to become T. cruziseropositive with each year of age and 11.6 times if royal palms where present in the peridomiciliary area of the dog’s household or its two nearest neighbours. Mouse-baited-adhesive traps were employed to evaluate 12 peridomestic royal palms. All palms were found infested with R. pallescenswith an average of 25.50 triatomines captured per palm. Of 35 adult bugs analysed, 88.6% showed protozoa flagellates in their intestinal contents. In addition, dogs were five times more likely to be infected by the presence of an additional domestic animal species in the dog’s peridomiciliary environment. Our results suggest that interventions focused on royal palms might reduce the exposure to T. cruzi infection.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

La ADN topoisomerasa tipo I de protozoos patógenos como Diana terapéutica de fármacos antitumorales / Type I DNA topoisomerase from protozoan pathogens as a potential target for anti-tumoral drugs

La utilización intensiva de fármacos antiparasitarios es la causa principal de la aparición de microorganismos parásitos multirresistentes en las regiones del planeta donde son precisamente endémicos. Los agentes etiológicos de las denominadas enfermedades tropicales -malaria, criptosporiodiosis, enfermedad del sueño, enfermedad de Chagas o los distintos tipos de leishmaniosis- son protozoos unicelulares sobre los que no se ha desarrollado en la actualidad ninguna vacuna eficaz y cuyo tratamiento se basa en medidas sanitarias preventivas y en el uso de medicamentos. La quimioterapia antiparasitaria actual es cara, no está ausente de efectos adversos y no supone beneficios a las empresas que la comercializan, por lo que la inversión en I & D es marginal comparada con la llevada a cabo para otros procesos patológicos de menor relevancia médica. La identificación de las ADN topoisomerasas como dianas farmacológicas se basa en los excelentes resultados obtenidos en los ensayos clínicos llevados a cabo con los derivados de la camptotecina en la terapia antitumoral. Las importantes diferencias estructurales entre las ADN topoisomerasas de tipo I de tripanosomas y leishmanias con respecto a sus homólogas de mamífero ha abierto un nuevo campo de investigación que combina las técnicas de biología molecular con la cristalización de proteínas para poder diseñar nuevos fármacos dirigidos específicamente a su inhibición. Revisamos aquí las características de estas nuevas dianas farmacológicas, así como los compuestos que en el momento están siendo utilizados para su inhibición en los agentes parasitarios que causan las principales enfermedades tropicales.

The intensive use of antiparasitic drugs is the main cause of the emergence of multiresistant parasite strains on those regions where these parasites are endemic. The aetiological agents of the so-called tropical diseases viz. malaria, cryptosporidiosis, sleeping sickness, Chagas disease or leishmaniasis, among others, are unicellular protozoan parasites with no immune-prophylactic treatment and where the chemotherapeutical treatment is still under controversy. At present, the chemotherapeutic approach to these diseases is expensive, has side or toxic effects and it does not provide economic profits to the Pharmaceuticals which then have no or scarce enthusiasm in R & D investments in this field. The identification of type I DNAtopoisomerases as promising drug targets is based on the excellent results obtained with camptothecin derivatives in anticancer therapy. The recent finding of significant structural differences between human type I DNAtopoisomerase and their counterparts in trypanosomatids has open a new field in drug discovery, the aim is to find structural insights to be targeted by new drugs. This review is an update of DNA-topoisomerases as potential chemotherapeutic targets against the most important protozoan agents of medical interest.

La región intergénica del gen H2A apoya las subpoblaciones KP1(-) y KP1(+) de Trypanosoma rangeli / The intergenic region of the histone h2a gene supports two major lineages of Trypanosoma rangeli

Introducción. Con base en la amplificación del ADN de los minicírculos del cinetoplasto y de los genes miniexón, Trypanosoma rangeli ha sido clasificado en las subpoblaciones KP1(-) y KP1(+). Objetivo. Comparar la región intergénica de los genes H2A entre cepas KP1(+) y KP1(-) de T. rangeli, con el fin de aportar evidencias a dicha división. Materiales y métodos. Se amplificó, clonó y determinó la secuencia de la región intergénica de los genes h2a de las cepas KP1(-) Tre y 5048 y de la cepa Choachí [KP1(+)]. Dichas secuencias, junto con las reportadas para las cepas C23 [KP1(-)] y H14 [KP1(+)], fueron utilizadas para la reconstrucción de árboles filogenéticos basados en los métodos de neighbor-joining, máxima parsimonia y máxima verosimilitud, utilizando la cepa Y de Trypanosoma cruzi como grupo raíz externo. Resultados. Se evidenció heterogeneidad intraespecífica en el tamaño de la región estudiada, soportados por valores bootstrap de 85 por ciento (neighbor-joining), 66 por ciento (máxima parsimonia) y 57 por ciento (máxima verosimilitud), los resultados indican que las cepas KP1(-) se agrupan aparte, claramente diferenciadas de las cepas KP1(+), las cuales presentan una mayor heterogeneidad intraespecífica en tamaño y secuencia. Además, se encontró mayor proximidad filogenética entre T. rangeli y T. cruzi que entre T. rangeli y Trypanosoma brucei. Conclusiones. Los análisis filogenéticos basados en la región intergénica de los genes h2a de las cepas estudiadas apoyan la división de T. rangeli en las subpoblaciones KP1(-) y KP1(+). Sin embargo, se requiere estudiar un mayor número de cepas para confirmar estos hallazgos.

Introduction. Trypanosoma rangeli has been classified in the KP1(+) and KP1(-) subpopulations, based on the mini-exon gene and kinetoplast DNA minicircle amplification profiles. Objective. The intergenic region of the histone h2a gene was compared between KP1(+) and KP1(-) strains of T. rangeli to substantiate this classification. Materials and methods. The amplification, cloning and sequencing of the h2a gene intergenic region was undertaken for the Tre and 5048 [KP1(-)] strains for comparison with the Choachí [KP1(+)] strain. These sequences, along with those previously reported for the KP1 (+) and KP1 (-) H14 and C23 strains, were used to reconstruct phylogenetic trees based on the “neighborjoining”, maximum parsimony and maximum likelihood methods. The Y strain of Trypanosoma cruzi was chosen as the outgroup. Results. Intra-specific heterogeneity was observed in the size of the gene region under study, supported by bootstarp values of 85% (neighbor-joining), 66% (maximum parsimony) and 57% (maximum likelihood). The KP1(-) strains were grouped apart, clearly differentiated from the KP1(+) strains. The latter demonstrated a higher intra-specific heterogeneity, both in sequence length and composition. In addition, a closer phylogenetic relationship between T. rangeli and T. cruzi was found to be more closely related to one another than to T. rangeli and Trypanosoma brucei. Conclusion. Phylogenetic analyses of analyzed strains based on the intergenic region of the h2a genes supported the T. rangeli grouping in two major subpopulations known as KP1(+) and KP1(-) strains. However, a higher number of strains are needed to confirm this finding.

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 percent) of the 26 positive insects. Our results provide further knowledge on the geographical distribution of T. rangeli in Brazil.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100 percent of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71 percent with TrF/R2 and in 6 percent with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100 percent with both PCRs and T. rangeli in 14 percent with TrF/R2 and 10 percent with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Embora o Trypanosoma rangeli não seja patogênico para o homem, sua importância médica e epidemiológica reside no fato de compartilhar vetores, reservatórios e áreas geográficas com o Trypanosoma cruzi, agente causal da Doença de Chagas. Neste estudo, para distinguir T. cruzi de T. rangeli em vetores com infecções mistas, se utilizaram duas amplificações de PCR; TcH2AF/R para o gen da histona H2A/SIRE e TrFR2, para um gen repetitivo de ARN nucleolar Cl1 (sno-RNA-Cl1). Assim como a PCR S35/S36, ambas as reações foram capazes de detectar corretamente a presença de T. cruzi ou T. rangeli em triatomíneos infectados experimentalmente. Nas infecções mistas, o ADN de T. cruzi foi amplificado em 100 por cento das amostras quando se utilizaram TcH2AF/R e S35/S36, enquanto T. rangeli foi detectado em 71 por cento delas com os iniciadores TrF/R2 e em 6 por cento, com S35/S36. Adicionalmente, em um grupo de Rhodnius colombiensis coletados na região de Coyaima (Tolima), T. cruzi foi identificado em 100 por cento com ambas PCRs e T. rangeli em 14 por cento delas com os iniciadores TrF/R2 e em 10 por cento, com S35/S36. Estes resultados mostram que as reações de PCR TcH2AF/R e TrF/R2, capazes de reconhecer todas as cepas e linhagens de T. cruzi e T. rangeli, podem ser úteis no diagnóstico e também nos estudos epidemiológicos do campo com vetores infectados pelo T. cruzi e T. rangeli.

Molecular trees of trypanosomes incongruent with fossil records of hosts

Molecular trees of trypanosomes have confirmed conventionally accepted genera, but often produce topologies that are incongruent with knowledge of the evolution, systematics, and biogeography of hosts and vectors. These distorted topologies result largely from incorrect assumptions about molecular clocks. A host-based phylogenetic tree could serve as a broad outline against which the reasonability of molecular phylogenies could be evaluated. The host-based tree of trypanosomes presented here supports the " invertebrate first " hypothesis of trypansosome evolution, supports the monophyly of Trypanosomatidae, and indicates the digenetic lifestyle arose three times. An area cladogram of Leishmania supports origination in the Palaearctic during the Palaeocene.

Identificación de una agrupación génica que codifica para ARN pequeños de nucleólo en Trypanosoma Rangeli / Identification of a Genetic Cluster Codingfor Six Small Nucleolar RNA in Trypanosoma rangeli

Objetivo. Realizar un análisis in silico de un fragmento de 801 pb de Trypanosoma rangeli, previamente reportado como codificante para el ARN pequeño de nucléolo C1, perteneciente a la familia de los C/D snoARN. Materiales y métodos. El tamizaje de las secuencias se realizó en las bases de datos de los diferentes proyectos de genomas de los parásitos usando el programa BLASTN. Las alineaciones de las secuencias se hicieron con los programas L-ALIGN y Clustal W. Resultados. La secuencia C1 constituye una agrupación génica que codifica para seis ARN pequeños de nucléolo, tres pertenecientes a la familia C/D snoARN y tres a la famila H/ACA snoARN. Conclusión. Los ARN pequeños de nucléolo de Trypanosomatidae presentan una organización genómica similar y elevados porcentajes de identidad en las secuencias consenso, lo cual unido a las funciones de estos ARN en el procesamiento y modificación posteriores a la transcripción del ARN ribosómico y, también, en el proceso de trans splicing, hace de estas moléculas un blanco atractivo para el control de estos parásitos

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 percent for trypanosome infections, a seroprevalence of 2.9 percent against T. cruzi and a predominance of T. rangeli infection (6.8 percent). The immunological and clinical implications of these findings are discussed.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Caracterización parcial de los genes codificantes para la proteína de choque térmico HSP70 de Trypanosoma rangeli / Partial characterization of coding genes for Trypanosoma rangeli HSP70

Objetivo: dada la relevancia inmunológica de la región amino terminal de la proteína HSP70 de la Tripanosoma cruzi, así como el hecho de que la inmunización de ratones con Tripanosoma rangeli protege a los animales contra la infección por T. cruzi, el presente trabajo se centró en el aislamiento y caracterización molecular del fragmento homólogo en T. rangeli. Materiales y métodos: la región amino terminal del gen codificante para la HSP70 de T. rangeli fue amplificada mediante PCR utilizando los oligonucleotidos TrHSP70f/R2, diseñados con base en la secuencia homóloga de T. cruzi. El fragmento amplificado fue purificado, clonado en el vector pGEM® –Teasy (Promega) y secuenciado en un 373 Automatic DNA sequencer (Applied Biosystems). La organización genómica de los genes HSP70 se determino mediante ensayos de “Southern blot” y PFGE. Resultados: los genes HSP70 de T. rangeli tienen un tamaño aproximado de 2.400 pb, se encuentran repetidos en tandem y se localizan en un cromosoma de 1.030 y 1.090 Kb en la cepa H14, KP1(+) y de 1.100 KB en la cepa Tre, KP1 (-). La secuencia de nucleótidos correspondiente a la región amino terminal de la proteína tiene una identidad del 99 por ciento entre las cepas H14 y Tre de T. rangeli y del 94 por ciento entre éstas y T. cruzi, preséntando además polimorfismos para diversas enzimas de restricción. La secuencia de amiácidos de dicha región entre ambos parásitos tiene una identidad del 95 por ciento. Conclusiones: el extremo amino terminal de las proteínas HSP70 de T. cruzi y T. rangeli guardan una elevada identidad de secuencia lo que abre un camino a la posible utilización de la HSP70 de T. rangeli en inmunoterapia. Asimismo y dado que en esta región se localizan epítopes B y T inductores de respuesta inmune en pacientes chagásicos, la HSP70 puede estar implicada en la reacción inmunológica cruzada entre estos parásitos

Mobile genetic elements in protozoan parasites.

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtleomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain

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

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.

Genetic variability of Colombian populations of Trypanosoma cruzi and Trypanosoma rangeli

This paper presents our study of genetic variability of Trypanosoma cruzi and Trypanosoma rangeli strains isolated from different Colombian biological hosts, using multilocus enzyme electrophoresis for 15 enzyme systems and electrophoretic analysis of kinetoplast DNA (kDNA) digested with EcoRI and MspI endonucleases. Isoenzyme profiles were used to determine genotypes for each of the strains. populations of T. cruzi and T. rangeli were found to have a polymorphic average of 86,7 per cent and 26,7 per cent, respectively. Schyzodeme analysis showed high variability for T. cruzi, since its genetic distance values were found to be greater than 50 per cent, considerably higher than those previously reported for several T. cruzi strains from other countries. these results suggest that Colombian strains should be considered as genetically independent entities and are worth studying independently from each other to clearly establish their biological and clinical characteristics.