Trypanosoma cruzi: new insights on ecophylogeny and hybridization by multigene sequencing of three nuclear and one maxicircle genes.

Natural populations of Trypanosoma cruzi are structured into five genetic lineages, T. cruzi I and T. cruzi II a to e, as the result of clonal evolution with rare genetic recombination events. To explore more in depth these phenomenons, a multigene sequencing approach was used, for the first time in the case of T. cruzi. Three nuclear loci and a maxicircle locus were sequenced on 18 T. cruzi stocks. Sequences were used to build phylogenetic trees from each locus and from concatenated sequences of all loci. The data confirmed the hybrid origin of DTUs IId and IIe, as the result of an ancient genetic recombination between strains pertaining to IIb and IIc. The data confirmed also a hybrid origin of DTUs IIa and IIc. Contrary to previous reports, we failed to detect mosaic genes. The phylogenetic relationship between DTUs and the respective roles of recombination and selection were tested.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Is Predominant Clonal Evolution a Common Evolutionary Adaptation to Parasitism in Pathogenic Parasitic Protozoa, Fungi, Bacteria, and Viruses?

We propose that predominant clonal evolution (PCE) in microbial pathogens be defined as restrained recombination on an evolutionary scale, with genetic exchange scarce enough to not break the prevalent pattern of clonal population structure. The main features of PCE are (1) strong linkage disequilibrium, (2) the widespread occurrence of stable genetic clusters blurred by occasional bouts of genetic exchange ('near-clades'), (3) the existence of a "clonality threshold", beyond which recombination is efficiently countered by PCE, and near-clades irreversibly diverge. We hypothesize that the PCE features are not mainly due to natural selection but also chiefly originate from in-built genetic properties of pathogens. We show that the PCE model obtains even in microbes that have been considered as 'highly recombining', such as Neisseria meningitidis, and that some clonality features are observed even in Plasmodium, which has been long described as panmictic. Lastly, we provide evidence that PCE features are also observed in viruses, taking into account their extremely fast genetic turnover. The PCE model provides a convenient population genetic framework for any kind of micropathogen. It makes it possible to describe convenient units of analysis (clones and near-clades) for all applied studies. Due to PCE features, these units of analysis are stable in space and time, and clearly delimited. The PCE model opens up the possibility of revisiting the problem of species definition in these organisms. We hypothesize that PCE constitutes a major evolutionary strategy for protozoa, fungi, bacteria, and viruses to adapt to parasitism.

Population structure of malaria parasites: the driving epidemiological forces.

The population structure of Plasmodial parasites, especially Plasmodium falciparum, has received much attention in the recent years. Like many other micropathogens, the debate has focused on the clonality/sexuality question. Considered a panmictic species for very long, P. falciparum actually exhibits strong departures from panmictic expectations in many of its populations, which corroborates the proposal that it is able to undergo uniparental propagation.(1) The currently accepted idea to account for this surprising result is kind of "mechanical" self-fertilization due to the lack of availability of gametes with different genetic make-ups in low transmission areas. However, it could be misleading to make this simple working hypothesis a dogma, for many other explanations are possible (unknown cycles, sibling species, mating types) that deserve to be explored as well. The consequences of this combination of uniparental(1) and sexual propagation on the circulation of genes of interest (drug resistance, antigenic variability, pathogenicity) are discussed, together with the need to use more sophisticated technologies, analysing much broader samples and considering better the host and vector factors in P. falciparum population dynamics.

Leishmania infantum is clonal in AIDS patients too: epidemiological implications.

OBJECTIVE: To test, in AIDS patients, a previously proposed hypothesis of clonal population structure in Leishmania infantum, the agent of visceral leishmaniasis. DESIGN: Forty-three stocks of L. infantum isolated from AIDS patients in Spain were analysed by multilocus enzyme electrophoresis. METHODS: The results were analysed in terms of population genetics according to previously described statistical methods. Departures from panmixia were examined by linkage disequilibrium analysis. RESULTS: As previously shown in HIV-negative patients, classical manifestations of clonality were shown, namely strong linkage disequilibrium, over-representation of genotypes and overall lack of genotype diversity. The same dominant clonal genotype (MON1) was recorded in both HIV-positive and HIV-negative patients. Frequency of this dominant genotype was not statistically different in HIV-positive and HIV-negative patients. CONCLUSIONS: The parasite population under survey appears to be clonal; parasite genotypes can therefore be equated to natural clones, stable in space and time, which can be used as multilocus epidemiological markers. Nevertheless, additional studies are required to better estimate the long-term stability of these clonal genotypes and the possible interference of gene exchange at an evolutionary scale.

Evolutionary genetics of Trypanosoma and Leishmania.

We review recent advances in the study of population structure and phylogenetic diversity of parasites belonging to the genera Trypanosoma and Leishmania. In all species properly analyzed, these parasites exhibit a basically clonal population structure, with occasional bouts of genetic exchange or hybridization, and a strong structuration of their populations into discrete evolutionary lineages. On an evolutionary scale, the impact of sex appears to be greater in African than in American trypanosomes. The taxonomic status of some Leishmania 'species' is questionable.

Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers.

Six discrete phylogenetic lineages were recently identified in Trypanosoma cruzi, on the basis of multilocus enzyme electrophoresis and random amplified polymorphic DNA (RAPD) characterisation. The objective of the present study was to develop specific PCR-based markers for the identification of each of the six lineages. Eighty-seven T. cruzi stocks representative of all the lineages were characterised by RAPD with three primers, resulting in the identification of three fragments that were specifically amplified in the given sets of lineages. After cloning and sequencing these fragments, three pairs of sequence-characterised amplified region (SCAR) primers were designed. After PCR amplification using the SCAR primers, the initial polymorphism was retained either as the presence or absence of amplification, or as size variation between the PCR products. Although most PCR products, taken individually, were distributed across several lineages, the combination of the three SCAR markers resulted in characteristic patterns that were distinct in the six lineages. Furthermore, T. cruzi lineages were distinguished from Trypanosoma rangeli, T. cruzi marinkellei and T. cruzi-like organisms. The excellent correspondence of these new PCR markers with the phylogenetic lineages, allied with their sensitivity, makes them reliable tools for lineage identification and strain characterisation in T. cruzi. The approach described here could be generalised to any species of microorganism harbouring clear-cut phylogenetic subdivisions.

Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality.

Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. There was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.

A phylogenetic analysis of the Trypanosoma cruzi genome project CL Brener reference strain by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting.

We have assessed the phylogenetic status of the Trypanosoma cruzi Genome Project CL Brener reference strain by multilocus enzyme electrophoresis (MLEE) and multiprimer random amplified polymorphic DNA (RAPD) including a set of cloned stocks representative of the whole genetic diversity of T. cruzi. MLEE and RAPD data gave congruent phylogenetic results. The CL Brener reference strain fell into the second major phylogenetic subdivision of T. cruzi, and was genetically very close to the Tulahuen reference strain. No reliable RAPD character and only one MLEE character permitted us to distinguish between the CL Brener and Tulahuen reference strains. In contrast, many RAPD and MLEE characters were able to distinguish between the CL Brener reference strain and the other T. cruzi genotypes analyzed here, in particular the formerly described principal zymodemes I, II and III. It is suspected that both CL Brener and Tulahuen are hybrid genotypes, a fact that should be taken into account when interpreting sequence data. Moreover, our study confirms that the species T. cruzi is genetically very heterogeneous. We recommend future comparison of sequencing data from the CL Brener reference strain with those of at least one radically distinct T. cruzi genotype, belonging to the other major phylogenetic subdivision of this species.

Predicted and observed alleles of Plasmodium falciparum merozoite surface protein-1 (MSP-1), a potential malaria vaccine antigen.

The 19-kDa antigenic domain of Plasmodium falciparum merozoite surface protein (MSP)-1 is a potential malaria vaccine candidate. Based on the amino acid substitution, four known alleles, E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type) of this domain have been identified. Using single or double crossover recombinational events, we predicted the existence of additional alleles of this antigen. The presence of the predicted alleles was determined in parasite isolates from western Kenya, by undertaking a cross-sectional and a longitudinal study. Of the ten predicted alleles, we have revealed the presence of three new alleles: E-KSG-L (Kenya-1 type); E-KSR-L (Kenya-2 type); and E-KNG-F (Kenya-3 type). The results of this study suggest that it may be possible to predict the complexity of the genetic makeup of natural parasite populations.

Genetic epidemiology of parasitic protozoa and other infectious agents: the need for an integrated approach.

This paper emphasises the relevance of the concepts and methods of evolutionary genetics for studying the epidemiology of parasitic protozoa and other pathogenic agents. Population genetics and phylogenetic analysis both contribute to identifying the relevant evolutionary and epidemiologically discrete units of research (Discrete typing units = DTUs), that can be equated to distinct phylogenetic lines. It is necessary (i) to establish that a given species represents a reliable DTU; (ii) to see whether a given species is further structured into lower DTUs that correspond to either clonal lineages or to cryptic species, and could exhibit distinct biomedical properties (virulence, resistance to drugs, etc). DTUs at the species and subspecies level can be conveniently identified by specific genetic markers or sets of genetic markers ("tags") for epidemiological follow-up. For any kind of pathogen (protozoa, fungi, bacteria, viruses), DTUs represent the relevant units of research, not only for epidemiology, but also, for other applied researches (clinical study, pathogenicity, vaccine and drug design, immunology, etc). The development of an "integrated genetic epidemiology of infectious diseases", that would explore the respective role of, and the interactions between, the genetic diversity (and its biological consequences) of the pathogen, the host and the vector (in the case of vector-borne diseases) is called for.

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.

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages.

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.

Molecular epidemiology and evolutionary genetics of Leischmania parasites.

In order to illustrate the relevance of the concepts and methods of evolutionary genetics in the understanding of the epidemiology of pathogenic agents, we develop in this paper the case of the Leishmania, a genus of parasitic protozoa. An extensive study of various natural populations of Leishmania in different countries (Old and New World) was carried out by using Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA fingerprinting (RAPD) as genetic markers. The data have been interpreted in evolutionary genetic terms. The main benefit of this approach has been to better define the concept of species in the genus Leishmnania, on rigorous phylogenetic bases. As a matter of fact, a sound taxonomical background is a prerequisite for any epidemiological approach. Since the biological concept of species is difficult or impossible to apply for most pathogenic microorganisms, we recommend relying on criteria of both phylogenetic discreteness and of epidemiological/medical relevance to describe new species of Leishmania. Through this approach, for example, we have shown that the species status of L. ( V.) perzzl.ianza can be supported. On the contrary, we have been unable to clearly distinguish L. (V.) panamensis from L. (V.) guyanensis with genetic tools. Additionally, we have shown that the epidemiological inferences based on a limited set of genetic markers can be misleading. As a matter of fact, we have demonstrated that a collection of L. (L.) infantum stocks identified as zymodeme 'MON 1' by other authors present additional genetic heterogeneity and do not correspond to a distinct 'Discrete Typing Unit' DTU, and are actually polyphyletic. Lastly, in the samples that were conveniently designed, we have confirmed that Leishmania parasites have a basically clonal population structure. As the clonal model specifies it, occasional bouts of genetic exchange remain nevertheless possible. Telling comparisons are drawn with the evolutionary genetics of other pathogens Trypanosoma cruzi and Trypanosoma congolense.

Experimental Trypanosoma cruzi biclonal infection in Triatoma infestans: detection of distinct clonal genotypes using kinetoplast DNA probes.

Monitored biclonal densities of parasites were offered to third-stage larvae of Triatoma infestans via an artificial feeding device and 30 days later, the gut contents of the insects were processed for microscopic examination and polymerase chain reaction (PCR) detection of Trypanosoma cruzi kinetoplast DNA [kDNA]). A total of 15 mixtures involving nine different stocks attributed to the 19/20, 32 and 39 major clonal genotypes of Trypanosoma cruzi were used. The presence of each T. cruzi clonal genotype after completion of the cycle through the insects was investigated by hybridising the PCR amplification products with genotype-specific minicircle kDNA probes. Sixty-five out of 90 examined insects (72.2%) were positive for parasites by microscopic examination and 85 (94.4%) were positive by PCR. The results show that almost half of the biclonal infections are not detectable after completion of the cycle, and that there are important differences in detection of such biclonal infections according to the clonal genotypes considered. Moreover, elimination of a clonal genotype by another is a frequent, but not constant, pattern in biclonal infections of T. infestans. The use of PCR and kDNA probes makes it possible to avoid the culture phase, which makes detection of mixed infections much easier in epidemiological surveys. Moreover, the fact that T. infestansdoes not transmit different T. cruzi clonal genotypes with the same efficiency has strong implications for the reliability of xenogiagnosis.

Phylogenetic diversity of bat trypanosomes of subgenus Schizotrypanum based on multilocus enzyme electrophoresis, random amplified polymorphic DNA, and cytochrome b nucleotide sequence analyses.

Trypanosome stocks isolated from bats (Chiroptera) and belonging to the subgenus Schizotrypanum were analyzed by multilocus enzyme electrophoresis (MLEE) at 22 loci, random amplified polymorphic DNA (RAPD) with 14 primers and/or cytochrome b nucleotide sequence. Bat trypanosomes belonged to the species Trypanosoma cruzi marinkellei (10 stocks), Trypanosoma dionisii (four stocks) and Trypanosoma vespertilionis (three stocks). One T. rangeli stock and seven stocks of T. cruzi sensu stricto, the agent of Chagas disease, were included for comparison. The homology of several RAPD fragments shared by distinct species was verified by hybridization. The sequence of a 516-nucleotide portion of the maxicircle-encoded cytochrome b (CYb) coding region was determined in representative stocks of the species under study. Phylogenetic analysis of the data confirmed the previous taxonomic attribution of these bat trypanosomes based on biological, epidemiological and ecological features. However, a new finding was that within T. cruzi marinkellei two major subdivisions could be distinguished, T.c.m. I, found in the spear-nose bats Phyllostomus discolor and Phyllostomus hastatus, and T.c.m. II, from P. discolor. In addition, the T. c. marinkellei 'Z' stock from a short-tailed bat (Carollia perspicillata) was distantly related to these two subdivisions, and the monophyly of T. c. marinkellei is unclear based on the present data. Based on the present sample, the European species T. dionisii and T. vespertilionis appeared to be more homogeneous. RAPD and CYb data both suggested the monophyly of a group composed of T. cruzi and the two major subdivisions of T. cruzi marinkellei. This study shows that MLEE, RAPD and CYb can be used for taxonomic assignment and provide valuable phylogenetic information for strains and taxa within the subgenus Schizotrypanum. An evolutionary scenario in which the broad host-range parasite T. cruzi would be derived from a bat-restricted trypanosome ancestor is discussed.

The population structure of Neisseria meningitidis serogroup A fits the predictions for clonality.

The population structure of Neisseria meningitidis is supposedly epidemic according to. The model predicts that linkage disequilibrium in N. meningitidis populations is only temporary and arises due to the outgrowth of highly successful clonal genotypes from an essentially sexual population. These clones should disappear after a few years because of frequent recombination. In contrast, multilocus enzyme electrophoresis (MLEE) data had previously been interpreted as showing that serogroup A meningococci are truly clonal and possess only limited genetic variability (Wang et al., 1992). The two interpretations are contradictory. In order to elucidate the true population structure of serogroup A meningococci, we analyzed data for a representative group of 84 serogroup A isolates obtained by MLEE, random amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). Analysis of linkage disequilibrium and bootstrap analyses of cluster analysis showed a strongly structured population with highly significant linkage disequilibrium. This was not due to the overrepresentation of certain genotypes, in contrast to the expectations for an epidemic population. The analyses identify two main clades, within each of which linkage disequilibrium was also highly significant, thus, excluding a cryptic speciation model. These observations support a population structure based on clonal evolution, in which clones are much more stable than expected for epidemic clonality. We propose that serogroup A meningococci may possess a different population structure from other serogroups of Neisseria meningitidis.

Methicillin-resistant Staphylococcus aureus: phylogenetic relatedness between European epidemic clones and Swiss sporadic strains.

We have compared the phylogenetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) strains from Switzerland and their phylogenetic relationships with European epidemic clones, using multiprimer random amplification polymorphic DNA (RAPD). Strains included 24 European epidemic clones (59 strains), 66 sporadic strains isolated in Switzerland in 1996-1997, and 15 reference strains of five other Staphylococcus species. Similarity and clustering analysis with the Jaccard's coefficient showed that the maximum genetic distance between MRSA strains was 0.43, whereas the minimum genetic distance between the six Staphylococcus species was 0.97, indicating that the method permits phylogenetic hierarchization. The 24 MRSA clones reported to be epidemic in European countries during the 1990s were distributed into seven different genetic clusters with a maximum distance of 0.29 among them. This clustering pattern was confirmed by the analysis of a subset of MRSA strains by multilocus enzyme electrophoresis at 12 loci. Most of the sporadic Swiss strains were distributed into these seven different genetic clusters, together with the epidemic MRSA clones. This suggests that there is no phylogenetic cluster specific to epidemic clones of MRSA.

Exotic stock of Trypanosoma cruzi (Schizotrypanum) capable of development in and transmission by Triatoma protracta protracta from California: public health implications.

A stock of Trypanosoma cruzi was recovered from a Triatoma dimidiata from Tegucigalpa, Honduras. This stock was shown to be capable of development and transmission by native California Triatoma protracta protracta. Isozyme analysis indicated that this T. cruzi is closely related to the Tehuantepec strain and to a lesser extent the Miles' zymodeme 1 strain. The potential public health significance of development and transmission of exotic stocks of T. cruzi by native reduviids is discussed.

Chagas' disease in Bolivia: clinical and epidemiological features and zymodeme variability of Trypanosoma cruzi strains isolated from patients.

We performed serological and pathological studies on 495 patients with Chagas' disease from different areas of Bolivia. Eighty-nine Trypanosoma cruzi strains, isolated by xenodiagnosis, were characterized by 12 isoenzyme loci and were related to the presence of cardiac changes and enteric disease with megacolon. There was a high heterogeneity of human zymodemes, presenting evidence of 2 predominant zymodemes genetically dissimilar from each other and ubiquitous in Bolivia. The frequencies of these predominant zymodemes among strains from patients were compared to strains from triatomine bugs previously studied. We observed mixtures of different zymodemes within the same patient, a phenomenon seen previously in Bolivian patients. There was no apparent difference of pathogenicity between the 2 more frequent zymodemes isolated from humans.