Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis

Genetic characterisation of Trypanosoma cruzi variants is of foremost importance, due to considerable genetic and biological heterogeneity in the parasite populations. Two major phylogenetic lineages, each highly heterogeneous, have been previously described within this species. Here we characterised a geographically and ecologically diverse sample of stocks representative of the breadth of the known clonal diversity of each major lineage, using random amplified polymorphic DNA with 20 primers and multilocus enzyme electrophoresis at 22 loci. Molecular hybridisation experiments were performed to control the homology of randomly amplified DNA markers. Both sets of data were highly consistent and supported the existence of two major lineages. Additionally, we found that lineage 2 appeared further partitioned into five sharply delineated phylogenetic clusters, each comprising one of the following reference strains: CanIII cl1 (Z3 reference), M5631 cl5, Esmeraldo cl3 (Z2 reference), CL Brener, and MN cl2. The two first clusters were found mainly in sylvatic environments, whereas the three latter were restricted to domestic transmission cycles and were only collected South to the Amazon Basin. In contrast, lineage 1, which included Miles' Z1 reference strain X10 cl1, was not further subdivided and was encountered across the entire endemic area, in both domestic and sylvatic cycles. Thus, T. cruzi appeared to be subdivided into six discrete typing units, or DTUs, exhibiting distinct geographic and ecological ranges. Reliable diagnostic markers for the two major lineages and the five smaller DTUs of lineage 2 are described, and correspondence with previous classifications of T. cruzi genotypes is given in order to help communication on T. cruzi phylogenetic diversity.     

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.     

Lack of correlation between in vitro susceptibility to Benznidazole and phylogenetic diversity of Trypanosoma cruzi, the agent of Chagas disease

Chagas disease remains an important health problem in Central and South America. Nitroimidazole derivative drugs like Benznidazole are commonly used to treat Trypanosoma cruzi infection. Natural variation of drug susceptibility between various T. cruzi stocks has been proposed as a possible explanation of treatment failure. Thus, the aim of this work was to determine potential correlations between in vitro Benznidazole susceptibility of different T. cruzi stocks and their genetic diversity. For this purpose, 16 natural stocks representing the overall genetic diversity of the parasite were analysed. Genetic characterisation was assessed by both random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) analyses. Drug activity was determined by two complementary methods, the MTT-PMS micro-method and FACs analysis. The 50% inhibitory concentrations (IC(50)s) were determined. Important variation of IC(50) values (7.3-16.9 microM) among stocks belonging to different discrete typing units (DTUs) was recorded. Further, correlation analysis showed that natural susceptibility to Benznidazole in T. cruzi expressed as IC(50) level was not related with its genetic structure represented by the different DTUs. These results are discussed in relation with the proposed hypothesis establishing a link between genetic diversity and biological behaviour in T. cruzi.     

Genetic characterization of Trypanosoma cruzi natural clones from the state of Paraíba, Brazil

Eighteen Trypanosoma cruzi stocks from the state of Paraíba, Brazil, isolated from man, wild mammals, and triatomine bugs were studied by multilocus enzyme electrophoresis and random primed amplified polymorphic DNA. Despite the low number of stocks, a notable genetic, genotypic, and phylogenetic diversity was recorded. The presence of the two main phylogenetic subdivisions, T. cruzi I and II, was recorded. The strong linkage disequilibrium observed in the population under survey suggests that T. cruzi undergoes predominant clonal evolution in this area too, although this result should be confirmed by a broader sample. The pattern of clonal variation does not suggests a recent origin by founder effect with a limited number of different genotypes.     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

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.     

Trypanosoma cruzi: infectivity of clonal genotype infections in acute and chronic phases in mice

Eight Trypanosoma cruzi stocks pertaining to the clonal genotypes 19/20, 32, and 39 have been characterized for three experimental parameters of infectivity in Balb/c mice: (i) percentage of mice with a patent parasitemia (% MPP), (ii) maximum parasitemia (MP), and (iii) percentage of mice with positive hemoculture (% MPH). By order of decreasing values, the values recorded for the clonal genotypes ranked as follows: 19/20, 32, and 39, except for the % MPP parameter, for which 19/20 and 32 were not statistically different. The rate of successful reisolation after infection in mice, analyzed by multilocus enzyme electrophoresis and random amplified polymorphic DNA typing, was statistically different according to the clonal genotype and was different for uniclonal infections and for mixed infections by two different clonal genotypes. These results confirm that T. cruzi clonal genotypes differ significantly in their infectivity in mice.     

Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from South Carolina

Biological and molecular characteristics of a raccoon isolate of Trypanosoma cruzi (R36) were compared with those of a known virulent strain (Brazil). Included in the characterization were growth rate in liver infusion tryptose medium, infectivity for murine fibroblasts, intracellular amastigote replication and trypomastigote release rates, polymerase chain reaction (PCR) profiling of the mini-exon gene, isoenzyme and random amplified polymorphic DNA (RAPD) profiles, and in vivo virulence for C3H/HeJ mice. Similar growth curves were noted for both strains; however, infectivity and rates of intracellular amastigote replication and trypomastigote release were significantly lower for the R36 isolate than for the Brazil strain. To determine virulence, C3H/ HeJ mice were exposed intraperitoneally to the R36 isolate. No parasite was observed in blood by direct examination or in tissues by histology; however, T. cruzi was detected by PCR in tissues (quadriceps and spleen) at 21 days postinfection. Analyses of the mini-exon gene, isoenzyme, and RAPD profiles indicate that R36 is in the T. cruzi II group and the Brazil strain is in the T. cruzi I group. Although infectivity and virulence of the raccoon isolate were lower than those for the Brazil strain, autochthonous infections in the United States have been reported, which suggests the need for further study of local T. cruzi isolates.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

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.     

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization.     

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States.

Thirty U.S. Trypanosoma cruzi stocks isolated mainly from wild mammals were characterized by multilocus enzyme electrophoresis at 22 genetic loci and random amplification of polymorphic DNA for 10 primers. Two main phylogenetic clusters, separated by large genetic distances, were discriminated by both methods, corresponding, respectively, to the formerly described zymodemes I and III. Two stocks isolated from indigenous human cases were identified as zymodeme I. Genetic diversity of the U.S. T. cruzi isolates was considerable, comparable to that scored in similarly sized samples from South America. These results favor the hypothesis that T. cruzi U.S. stocks were not imported at a historical time and are indigenous to the native fauna of the United States. The population structure of these stocks appeared to be basically clonal, as previously reported in South America, and no evidence of hybrid genotypes was found in the United States.     

Trypanosoma cruzi 5S rRNA arrays define five groups and indicate the geographic origins of an ancestor of the heterozygous hybrids

Isolates of the etiological agent of Chagas disease, Trypanosoma cruzi, have been subdivided into six subgroups referred to as discrete typing units. The subgroups are related through two distinct hybridisation events: representatives of homozygous discrete typing units I and IIb fused to form discrete typing units IIa and IIc, whose homozygous genotypes have features of both ancestral types; a second fusion between strains of homozygous discrete typing units IIb and IIc created the heterozygous hybrid strains discrete typing units IId and IIe. The intergenic region of the tandemly repeated 5S rRNA array displays four variant sequence classes, allowing the discrimination of five discrete typing units. The genome project reference strain, CL Brener, is a hybrid discrete typing unit IIe strain that contains both discrete typing unit IIb and IIc classes of 5S rRNA repeats in distinct arrays present on different chromosomes. The CL Brener discrete typing unit IIb-type array contains approximately 193 repeated units, of which about one-third contain a 129 bp sequence that replaces a majority of the 5S rRNA sequence. The 129 bp 'invader' sequence was detected within the arrays of all hybrid discrete typing unit IId and IIe strains and in a subset of discrete typing unit IIb strains. This array invader replaces the internal promoter elements conserved in 5S rRNA. The discrete typing unit IIb Esmeraldo strain contains approximately 135 repeats and shows a region of homology to the array invader in the 5' flank of the array, but no evidence of the invading sequence element within the array. A survey of additional discrete typing unit IIb strains revealed a split within the subgroup, in which some strains contained invaded arrays and others were homogeneous for the 5S rRNA. The putative discrete typing unit IIb ancestor of the hybrid discrete typing units IId and IIe more closely resembles the extant Bolivian/Chilean IIb isolates than the Brazilian IIb isolates based on the correlation with the array invader.     

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.     

Genetic Heterogeneity in Trypanosoma cruzi Strains From Naturally Infected Triatomine Vectors in Northeastern Brazil: Epidemiological Implications

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.     

Minicircle organization and diversity in Trypanosoma cruzi populations

Trypanosoma cruzi strains and isolates can be divided into at least two groups using biochemical and molecular markers such as isoenzymes, ribosomal DNA, mini-exon gene spacers and some maxicircle genes. Despite the accumulating evidence that these major groups are phylogenetically distinct, their kinetoplast minicircle overall organization (i.e. number of conserved regions per length of minicircle molecule) remains conserved in all T. cruzi isolates studied so far, including the two T. cruzi major lineages -T. cruzi I and T. cruzi II - and a third group of uncertain taxonomic status, T. cruzi ZIII. Thus far, despite the extensive intra- and inter-minicircle sequence polymorphism, no group clustering has been observed.