Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia + Tc Id, Tc Ia + Tc Ie and Tc Id + Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.     

Contemporary cryptic sexuality in Trypanosoma cruzi

Clonal propagation is considered to be the predominant mode of reproduction among many parasitic protozoa. However, this assumption may overlook unorthodox, infrequent or cryptic sexuality. Trypanosoma cruzi, which causes Chagas disease, is known to undergo non‐Mendelian genetic exchange in the laboratory. In the field, evidence of extant genetic exchange is limited. In this study, we undertook intensive sampling of T. cruzi Discrete Typing Unit I in endemic eastern Colombia. Using Fluorescence‐activated cell sorting, we generated 269 biological clones from 67 strains. Each clone was genotyped across 24 microsatellite loci. Subsequently, 100 representative clones were typed using 10 mitochondrial sequence targets (3.76 Kbp total). Clonal diversity among humans, reservoir hosts and vectors suggested complex patterns of superinfection and/or coinfection in oral and vector‐borne Chagas disease cases. Clonal diversity between mother and foetus in a congenital case demonstrates that domestic TcI genotypes are infective in utero. Importantly, gross incongruence between nuclear and mitochondrial markers is strong evidence for widespread genetic exchange throughout the data set. Furthermore, a confirmed mosaic maxicircle sequence suggests intermolecular recombination between individuals as a further mechanism of genetic reassortment. Finally, robust dating based on mitochondrial DNA indicates that the emergence of a widespread domestic TcI clade that we now name TcI_DOM (formerly TcIa/VEN_Dom) occurred 23 000 ± 12 000 years ago and was followed by population expansion, broadly corresponding with the earliest human migration into the Americas.     

Proteomic map of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> CL Brener: the reference strain of the genome project

In this work two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of soluble proteins from epimastigote form of Trypanosoma cruzi CL Brener. This strain is a hybrid organism derived from two genotypes, T. cruzi I and T. cruzi II and was chosen for genome sequencing. The two-dimensional gel electrophoresis showed that most of proteins focused at 4–7 pH range. The identification demonstrated that several proteins were in multiple isoforms, such as tubulin and heat shock proteins. Potential targets for development of chemotherapeutic agents like arginine kinase, an enzyme absent from mammalian tissues that is involved in the energy supply of the parasite, were also detected.     

Identification by Q-PCR of Trypanosoma cruzi lineage and determination of blood meal sources in triatomine gut samples in México

Triatomine vectors were collected on human dwellings in Michoacán México. Blood meal sources were identified by real time polymerase chain reaction (Q-PCR) using DNA extracted from triatomine guts. The assay was performed with one only specific primer set to amplify a fragment of the mitochondrial 12S ribosomal gene from vertebrate species. Also Trypanosoma cruzi parasites were detected in triatomine gut samples by microscopy and the positive infection was tested in mice. In addition T. cruzi discrete taxonomic units (DTUs) were identified by Q-PCR with two sets of primers that amplify the mini-circle region (miniexon) and 18S ribosomal mitochondrial gene. The sequences obtained from 18S ribosomal gene amplifications confirmed the presence of T. cruzi I and II lineages, and provide evidence of the presence of lineage TcIII and TcIV.     

The identification of two Trypanosoma cruzi I genotypes from domestic and sylvatic transmission cycles in Colombia based on a single polymerase chain reaction amplification of the spliced-leader intergenic region

A single polymerase chain reaction (PCR) reaction targeting the spliced-leader intergenic region of Trypanosoma cruzi I was standardised by amplifying a 231 bp fragment in domestic (TcI_DOM) strains or clones and 450 and 550 bp fragments in sylvatic strains or clones. This reaction was validated using 44 blind coded samples and 184 non-coded T. cruzi I clones isolated from sylvatic triatomines and the correspondence between the amplified fragments and their domestic or sylvatic origin was determined. Six of the nine strains isolated from acute cases suspected of oral infection had the sylvatic T. cruzi I profile. These results confirmed that the sylvatic T. cruzi I genotype is linked to cases of oral Chagas disease in Colombia. We therefore propose the use of this novel PCR reaction in strains or clones previously characterised as T. cruzi I to distinguish TcI_DOMfrom sylvatic genotypes in studies of transmission dynamics, including the verification of population selection within hosts or detection of the frequency of mixed infections by both T. cruzi I genotypes in Colombia.     

Proton nuclear magnetic resonance analysis of metabolic end products of the Bolivia strain of Trypanosoma cruzi and three of its clones

Proton nuclear magnetic resonance (¹H NMR) was used to study the in vivo metabolism of Trypanosoma cruzi, the pathogen causing American trypanosomiasis (Chagas' disease). Three clones were isolated from a strain of T. cruzi (Bolivia strain), The clones I, II and III and the original strain were characterized according to the spectra of their metabolic pathways to test the hypothesis that clonal evolution of T. cruzi has a major impact on biologically relevant properties of this parasite. T. cruzi (Bolivia strain) excreted acetate, alanine, glycerol, and succinate as major end products, in the proportion 6:4:2:2. Comparing the spectra of T. cruzi clones with the original Bolivia strain revealed both quantitative, as well as qualitative differences in the metabolites excreted: the clones I and II, as opposed to the Bolivia strain and clone III, excreted significant quantities of ethanol.     

Trypanosoma cruzi: Genetic diversity influences the profile of immunoglobulins during experimental infection

The clonal evolution model postulated for Trypanosoma cruzi predicts a correlation between the phylogenetic divergence of T. cruzi clonal genotypes and their biological properties. In the present study, the linkage between phylogenetic divergence of the parasite and IgG, IgG1, IgG2a and IgG2b response has been evaluated during the acute and chronic phases of the experimental infection. Eight laboratory-cloned stocks representative of this phylogenetic diversity and including the lineages T. cruzi I (genotypes 19 and 20), T. cruzi II (genotype 32) and T. cruzi (genotype 39) have been studied. The results showed that the pattern of humoral immune response was correlated with T. cruzi genotype, and that stocks included in genotype 20 were responsible for the high IgG response in the acute and chronic phases. Moreover, T. cruzi I lineage was more efficient in over-expressing all subclasses of specific anti-parasite IgG than either T. cruzi II or T. cruzi lineages. Curiously, the alteration in the pattern of antibodies induced by Benznidazole treatment was related to the phase of the infection but not to the genotype of the parasite. The data suggest that genotypes of T. cruzi are able to drive levels/subclasses of specific IgG, hence giving rise to further concerns about the sensitivity of serological assays in the diagnosis of human Chagas disease.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole

The capacity of the polymerase chain reaction (PCR) to detect the DNA of Trypanosoma cruzi was evaluated in 90 blood samples from BALB/c mice infected with T. cruzi cloned stocks of genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II), and treated with benznidazole. The results from the fresh blood examination, hemoculture, and ELISA allowed to group the treated animals into: cured (TC), dissociated (DIS) and non-cured (NC). The PCR detected T. cruzi DNA in 50.9%, 58.3% and 100.0% of the samples from TC, DIS and NC mice, respectively. These DNA possibly derives from live T. cruzi or from recently lysed parasites, suggests that these animals are in fact not cured. The difference between the PCR results and results obtained using other techniques was statistically significant and independent of the parasite genotype. The PCR described has therefore potential to be used in cure control of treated patients.     

Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0–72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2–48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1β, TNF-α, and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.     

Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12–14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD₅₀ values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.     

Phylogeny of tall fescue and related species using RFLPs

The wild species of tall fescue (Festuca arundinacea var.genuina Schreb.) represent a wide range of genetic variation and constitute potential germplasm for tall fescue improvement. Our objective was to evaluate genome specificity of the previously-identified DNA probes and to examine the phylogenetic relationship of tall fescue with six related species by using RFLP data. A total of 29 DNA probes from aPstI-genomic library of tall fescue were hybridized toEcoRI-orHindIII-digested DNA of 32 plants from sixFestuca species and fromLolium perenne L. Fifteen probes hybridized to all seven species. The remaining 14 probes showed differential hybridization patterns (i.e., ±), especially at the diploid and tetraploid levels. This hybridization pattern reflected genome divergence in these species. The DNA probes will be useful markers in breeding programs involving interspecific and intergeneric hybridization. Cluster analyses were performed using the average genetic distances calculated with the RFLP data from 53 probe-enzyme combinations. Generally, genotypes from the same species were grouped in the same cluster. These data indicated that tall fescue has a close relationship withF. pratensis Huds. (diploid),F. arundinacea var.glaucescens Boiss. (tetraploid), andL. perenne L. (diploid) and thatFestuca pratensis andL. perenne had the closest degree of relationship.This paper is a contribution of the Missouri Agricultural Experimental Station, Journal Series no. 11,798     

Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana)

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n = 2 or 3) of each species were inoculated with 1 × 10⁶ culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) – raccoon), TcI (NA – opossum), TcIIb (South American – human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5 weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7 days post inoculation) than opossums (10 days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts.     

Trypanosoma cruzi: Biological characterization of lineages I and II supports the predominance of lineage I in Colombia

The causes of the particular distribution of both Trypanosoma cruzi lineages throughout the American continent remain unknown. In Colombia, T. cruzi I is the predominant group in both domestic and sylvatic cycles. Here, we present the biological characterization of T. cruzi parasites belonging to both T. cruzi I and T. cruzi IIb groups. Our results show the inability of the T. cruzi IIb clones to infect mammalian cells, produce trypomastigotes and replicate in Rhodnius prolixus, the main vector species in this country. Moreover, this result was confirmed when other species from the same genus, such as R. pallescens and R. robustus, were infected with the same TcIIb clone and its parental strain, while the infection in other genera such as Triatoma and Panstrongylus was successful. Furthermore, the growth kinetics and duplication time in vitro suggest that the high prevalence of T. cruzi I in Colombia results from more successful interactions between parasite lineage, vector, and host species. This type of study may help to understand the factors influencing the particular epidemiological patterns of Chagas disease transmission in different endemic regions.     

Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.     

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.     

Production and cytogenetics of Triticum aestivum L. hybrids with some rhizomatous Agropyron species

Summary Intergeneric hybrids between Triticum aestivum L. and conventional rhizomatous Agropyron species were produced in variable frequencies. They were recovered in high percentage frequencies for T. aestivum cultivars with A. acutum (14.6%), A. intermedium (48.0%), A. pulcherrimum (53.3%), and A. trichophorum (46.6%). The crossability percentages with the highly crossable cultivar Chinese Spring for these Agropyron species accessions were 33.12%, 65.0%, 53.3%, and 65.4%, respectively. Autosyndetic associations of two of their three genomes gave mean meiotic chromosome association data of 17.0 I (univalents) +1.53 II (ring bivalents) + 7.04 II (rod bivalents) +1.43 III (trivalents) +0.05 IV (quadrivalents) +0.01 IV (pentavalents) for A. acutum and of 21.8 I + 1.56 II (rings) +7.22 II (rods) +0.84 III + 0.04 IV for A. intermedium. Chromosome pairing at metaphase I was comparatively lower for A. pulcherrimum (34.4 I + 0.2 II (rings) +3.4 II (rods) +0.14 III) and A. trichophorum (36.7 I + 0.35 II (rings) +2.26 II (rods) + 0.04 III) hybrids with T. aestivum. Hybrids of wheat with A. campestre and A. repens were obtained in low frequency. Direct crossing did not permit T. aestivum/ A. desertorum hybridization. However, by utilizing the 2n=10x=70 A. repens/A. desertorum amphiploid as the pollen source, hybridization with T. aestivum did indeed occur. Aneuploidy was prevalent in this hybrid combination while all other hybrid combinations were apparently normal.