Molecular typing of Trypanosoma cruzi isolates, United States.
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SUBMITTER: Roellig DM
PROVIDER: S-EPMC2600345 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Trypanosoma cruzi, the causative agent of Chagas disease, is a multiclonal parasite with high levels of genetic diversity and broad host and geographic ranges. Molecular characterization of South American isolates of T. cruzi has demonstrated homologous recombination and nuclear hybridization, as well as the presence of 6 main genetic clusters or "discrete typing units" (DTUs). Few studies have extensively investigated such exchange events and genetic diversity in North American isolates. In the current study, we genetically characterized over 50 US isolates from wildlife reservoirs (e.g., raccoons, opossums, armadillos, skunks), domestic dogs, humans, nonhuman primates, and reduviid vectors from nine states (TX, CA, OK, SC, FL, GA, MD, LA, TN) using a multilocus sequencing method. Single nucleotide polymorphisms were identified in sequences of the mismatch-repair class 2 (MSH2) and Tc52 genes. Typing based on the two genes often paralleled genotyping by classic methodologies using mini-exon and 18S and 24S? rRNA genes. Evidence for genetic exchange was obtained by comparing sequence phylogenies of nuclear and mitochondrial gene targets, dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the cytochrome oxidase subunit II- NADH dehydrogenase subunit I region (COII-ND1), respectively. We observed genetic exchange in several US isolates as demonstrated by incongruent mitochondrial and nuclear genes phylogenies, which confirms a previous finding of a single genetic exchange event in a Florida isolate. The presence of SNPs and evidence of genetic exchange illustrates that strains from the US are genetically diverse, even though only two phylogenetic lineages have been identified in this region.
| S-EPMC3572986 | biostudies-literature
Project description:Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century.
| S-EPMC3194829 | biostudies-literature
Project description:BackgroundTrypanosoma cruzi is a protozoan parasite characterized by extensive genetic heterogeneity. There are currently six recognised, genetically distinct, monophyletic clades designated discrete typing units (DTUs). TcI has the broadest geographical range and most genetic diversity evidenced by a wide range of genetic markers applied to isolates spanning a vast geographical range across Latin America. However, little is known of the diversity of TcI that exists within sylvatic mammals across the geographical expanse of Brazil.ResultsTwenty-nine sylvatic TcI isolates spanning multiple ecologically diverse biomes across Brazil were analyzed by the application of multilocus sequence typing (MLST) using four nuclear housekeeping genes. Results revealed extensive genetic diversity and also incongruence among individual gene trees. There was no association of intralineage genotype with geography or with any particular biome, with the exception of isolates from Caatinga that formed a single cluster. However, haplotypic analyses of METIII and LYT1 constitutive markers provided evidence of recombination events in two isolates derived from Didelphis marsupialis and D. albiventris, respectively. For diversity studies all possible combinations of markers were assessed with the objective of selecting the combination of gene targets that are most resolutive using the minimum number of genes. A panel of just three gene fragments (DHFR-TS, LYT1 and METIII) discriminated 26 out of 35 genotypes.ConclusionsThese findings showed geographical association of genotypes clustering in Caatinga but more characteristically TcI genotypes widely distributed without specific association to geographical areas or biomes. Importantly, we detected the signature of recombination events at the nuclear level evidenced by haplotypic analysis and incongruence.
| S-EPMC5824584 | biostudies-literature
Project description:Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
| S-EPMC4148231 | biostudies-literature
Project description:Although it has been known for nearly a century that strains of Trypanosoma cruzi, the etiological agent for Chagas' disease, are enzootic in the southern U.S., much remains unknown about the dynamics of its transmission in the sylvatic cycles that maintain it, including the relative importance of different transmission routes. Mathematical models can fill in gaps where field and lab data are difficult to collect, but they need as inputs the values of certain key demographic and epidemiological quantities which parametrize the models. In particular, they determine whether saturation occurs in the contact processes that communicate the infection between the two populations. Concentrating on raccoons, opossums, and woodrats as hosts in Texas and the southeastern U.S., and the vectors Triatoma sanguisuga and Triatoma gerstaeckeri, we use an exhaustive literature review to derive estimates for fundamental parameters, and use simple mathematical models to illustrate a method for estimating infection rates indirectly based on prevalence data. Results are used to draw conclusions about saturation and which population density drives each of the two contact-based infection processes (stercorarian/bloodborne and oral). Analysis suggests that the vector feeding process associated with stercorarian transmission to hosts and bloodborne transmission to vectors is limited by the population density of vectors when dealing with woodrats, but by that of hosts when dealing with raccoons and opossums, while the predation of hosts on vectors which drives oral transmission to hosts is limited by the population density of hosts. Confidence in these conclusions is limited by a severe paucity of data underlying associated parameter estimates, but the approaches developed here can also be applied to the study of other vector-borne infections.
| S-EPMC2860507 | biostudies-literature
Project description:Oral transmission of Trypanosoma cruzi has gained relevance because of its association with high morbidity and lethality rates. This transmission route is responsible for maintaining the infection of the parasite in sylvatic cycles, and human cases have been associated mainly with the consumption of food contaminated with triatomine feces or didelphid secretions. Several ecological changes allow the intrusion of sylvatic reservoirs and triatomines to the domestic environments with subsequent food contamination. Here, high-resolution molecular tools were used to detect and genotype T. cruzi across humans, reservoirs, and insect vectors in 2 acute outbreaks of presumptive oral transmission in eastern Colombia.
| S-EPMC5034963 | biostudies-literature
Project description:BackgroundThe parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi.MethodsA restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines.ResultsThe COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi.ConclusionsThe COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist.
| S-EPMC3891993 | biostudies-literature
Project description:Chagas disease affects between six and seven million people. Its etiological agent, Trypanosoma cruzi, is classified into six discrete typing units (DTUs). The biological study of 11 T. cruzi strains presented here included four parameters: growth kinetics, parasitemia curves, rate of macrophage infection, and serology to evaluate IgM, total IgG, IgG1, IgG2a, and IgG3. Sequencing of small subunit of ribosomal RNA (SSU rRNA)was performed and the T. cruzi strains were classified into three DTUs. When their growth in liver infusion tryptose medium was represented in curves, differences among the strains could be noted. The parasitemia profile varied among the strains from the TcI, TcII, and TcIII groups, and the 11 T. cruzi strains produced distinct parasitemia levels in infected BALB/c. The TcI group presented the highest rate of macrophage infection by amastigotes, followed by TcII and TcIII. Reactivity to immunoglobulins was observed in the TcI, TcII, and TcIII; all the animals infected with the different strains of T. cruzi showed anti-T. cruzi antibodies. The molecular study presented here resulted in the classification of the T. cruzi strains into the TcI (Bolivia, T lenti, Tm, SC90); TcII (Famema, SC96, SI8, Y); and TcIII (QMM3, QMM5, SI5) groups. These biological and molecular results from 11 T. cruzi strains clarified the factors involved in the biology of the parasite and its hosts. The collection of triatomine (vector) species, and the study of geographic distribution, as well as biological and molecular characterization of the parasite, will contribute to the reporting and surveillance measures in Brazilian states.
| S-EPMC5929169 | biostudies-literature
Project description:BackgroundTrypanosoma cruzi is the etiologic agent of Chagas disease throughout the Americas. Few population-level studies have examined the epidemiology of canine infection and strain types of T. cruzi that infect canines in the USA. We conducted a cross-sectional study of T. cruzi infection in working hound dogs in south central Texas, including analysis of triatomine vectors collected within kennel environments.Methodology/principle findingsPaired IFA and Chagas Stat-Pak serological testing showed an overall seroprevalence of 57.6% (n = 85), with significant variation across kennels. Dog age had a marginally significant effect on seropositivity, with one year of age increase associated with a 19.6% increase in odds of being seropositive (odds ratio 95% CI 0.996-1.435; p = 0.055). PCR analyses of blood revealed 17.4% of dogs harbored parasite DNA in their blood, including both seronegative and seropositive dogs. Molecular screening of organs from opportunistically sampled seropositive dogs revealed parasite DNA in heart, uterus, and mammary tissues. Strain-typing showed parasite discrete typing units (DTU) TcI and TcIV present in dog samples, including a co-occurrence of both DTUs in two individual dogs. Bloodmeal analysis of Triatoma gerstaeckeri and Triatoma sanguisuga insects collected from the kennels revealed exclusively dog DNA. Vector infection with T. cruzi was 80.6% (n = 36), in which T. gerstaeckeri disproportionately harbored TcI (p = 0.045) and T. sanguisuga disproportionately harbored TcIV (p = 0.029). Tracing infection status across dog litters showed some seropositive offspring of seronegative dams, suggesting infection of pups from local triatomine vectors rather than congenital transmission.Conclusions/significanceCanine kennels are high-risk environments for T. cruzi transmission, in which dogs likely serve as the predominant parasite reservoir. Disease and death of working dogs from Chagas disease is associated with unmeasured yet undoubtedly significant financial consequences because working dogs are highly trained and highly valued.
| S-EPMC5287457 | biostudies-literature
Project description:Chagas disease caused by Trypanosoma cruzi is a public health issue in Latin America. This highly diverse parasite is divided into at least seven discrete typing units (DTUs) TcI-TcVI and Tcbat. Some DTUs have been associated with geographical distribution in epidemiological scenarios and clinical manifestations, but these aspects remain poorly understood. Many studies have focused on studying the parasite and its vectors/hosts, using a wide variety of genetic markers and methods. Here, we performed a systematic review of the literature for the last 20 years to present an update of DTUs distribution in the Americas, collecting ecoepidemiological information. We found that the DTUs are widespread across the continent and that there is a whole gamma of genetic markers used for the identification and genotyping of the parasite. The data obtained in this descriptor could improve the molecular epidemiology studies of Chagas disease in endemic regions.
| S-EPMC9232490 | biostudies-literature