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.

Phylogenetic character mapping of RADES Probing, a new marker for exploring the clonal evolution of expressed coding sequences in Trypanosoma cruzi, the agent of Chagas disease.

We have tested a new genetic marker, RADES Probing (RADES-P), on a standard sample of 19 laboratory-cloned stocks of Trypanosoma cruzi, the agent of Chagas disease. This set of stocks, fully characterized using multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD), is representative of this parasite's main genetic subdivisions. RADES-P consists in hybridizing RAPD profiles with probes composed of the products of random amplified differentially expressed sequences (RADES). The profiles thus obtained uncover only expressed coding sequences that are as well present on RAPD gels. Direct visual examination and the banding record show that these RADES-P profiles are different of, and not redundant with, both RAPD and RADES patterns obtained on the same set of stocks with the same primers. Phylogenetic character mapping (PCM) of the RADES-P polymorphism fairly confirms the known population structure and phylogenetic diversity of T. cruzi. This suggests that the impact of clonal evolution on T. cruzi has been predominant enough over the long term to carve the polymorphism of all types of DNA sequences, including polymorphisms of expressed coding sequences, although these sequences are subject to natural selection.

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.

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.

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.

Clonal propagation and the fast generation of karyotype diversity: An in vitro Leishmania model.

In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology.

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.

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.

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.

An isoenzyme survey of Trypanosoma brucei s.l. from the Central African subregion: population structure, taxonomic and epidemiological considerations.

In order to improve our knowledge about the taxonomic status and the population structure of the causative agent of Human African Trypanosomiasis in the Central African subregion, 169 newly isolated stocks, of which 16 came from pigs, and 5 reference stocks, were characterized by multilocus enzyme electrophoresis, for 17 genetic loci. We identified 22 different isoenzyme profiles or zymodemes, many of which showed limited differences between them. These zymodemes were equated to multilocus genotypes. UPGMA dendrograms revealed one main group: Trypanosoma brucei gambiense group I and 3 T. brucei 'non-gambiense' stocks. T. b. gambiense group I zymodemes were very homogenous, grouping all the human stocks and 31% of the pig stocks. Two main zymodemes (Z1 and Z3) grouping 74% of the stocks were found in different remote countries. The genetic distances were relatively high in T. brucei 'non-gambiense' zymodemes, regrouping 69% of pig stocks. The analysis of linkage disequilibrium was in favour of a predominantly clonal population structure. This was supported by the ubiquitous occurrence of the main zymodemes, suggesting genetic stability in time and space of this parasite's natural clones. However, in some cases an epidemic population structure could not be ruled out. Our study also suggested that the domestic pig was a probable reservoir host for T. b. gambiense group I in Cameroon.

Impact of number of isoenzyme loci on the robustness of intraspecific phylogenies using multilocus enzyme electrophoresis: consequences for typing of Trypanosoma cruzi.

Thirty-one stocks of Trypanosoma cruzi, the agent of Chagas disease, representative of the genetic variability of the 2 principal lineages, that subdivide T. cruzi, were selected on the basis of previous multilocus enzyme electrophoresis analysis using 21 loci. Analyses were performed with lower numbers of loci to explore the impact of the number of loci on the robustness of the phylogenies obtained, and to identify the loci that have more impact on the phylogeny. Analyses were performed with numerical (UPGMA) and cladistical (Wagner parsimony analysis) methods for all sets of loci. Robustness of the phylogenies obtained was estimated by bootstrap analysis. Low numbers of randomly selected loci (6) were sufficient to demonstrate genetic heterogeneity among the stocks studied. However, they were unable to give reliable phylogenetic information. A higher number of randomly selected loci (15 and more) were required to reach this goal. All loci did not convey equivalent information. The more variable loci detected a greater genetic heterogeneity among the stocks, whereas the least variable loci were better for robust clustering. Finally, analysis was performed with only 5 and 9 loci bearing synapomorphic allozyme characters previously identified among larger samples of stocks. A set of 9 such loci was able to uncover both genetic heterogeneity among the stocks and to build robust phylogenies. It can therefore be recommended as a minimum set of isoenzyme loci that bring maximal information for all studies aiming to explore the phylogenetic diversity of a new set of T. cruzi stocks and for any preliminary genetic typing. Moreover, our results show that bootstrap analysis, like any statistics, is highly dependent upon the information available and that absolute bootstrap figures should be cautiously interpreted.

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.

Significant linkage disequilibrium and high genetic diversity in a population of Plasmodium falciparum from an area (Republic of the Congo) highly endemic for malaria.

A study based on 28 microsatellite loci was performed on 32 isolates of Plasmodium falciparum from Pointe Noire (Republic of the Congo) and compared with a cosmopolitan sample of 21 isolates collected from different countries in Africa, Latin America, and Asia. The Pointe Noire population exhibited very high genetic diversity (A = 7.8 +/- 2.6, He = 0.79 +/- 0.11). Significant linkage disequilibria were observed in 28 of 378 pairs of microsatellite loci. This result could be explained by two non-exclusive hypotheses: 1) uniparental propagation (i.e., selfing), leading to non-panmictic associations, and/or 2) a Wahlund effect (i.e., spatial population genetic heterogeneity). These observations are in agreement with data previously obtained from isozyme loci of the same isolates, but contrast with other population genetic analyses conducted in other hyperendemic zones.

Trypanosoma cruzi isoenzyme variability in Ecuador: first observation of zymodeme III genotypes in chronic chagasic patients.

We have analysed by multilocus enzyme electrophoresis (MLEE) at 21 genetic loci 10 Trypanosoma cruzi stocks isolated from chronic chagasic patients and 3 stocks isolated from Triatoma dimidiata collected in human habitats from the coastal part of Ecuador (all stocks isolated in August-December 1998). Isoenzyme profiles were compared to those of 4 laboratory-cloned stocks representing the major phylogenetic subdivisions of T. cruzi. This parasite's genetic variability in Ecuador proved to be considerable, even in this limited sample, since all main isoenzyme genotypes were recorded. Four stocks from patients were identical at all loci to the reference stock MNcl2 ('major clonet #39'; T. cruzi II) isolated in Chile. The 3 stocks isolated from T. dimidiata were closely related to the formerly described zymodeme I (T. cruzi I). Finally, 3 stocks from chronic chagasic patients (one with an asymptomatic form, 2 with a cardiac-digestive form) were closely related to the formerly described zymodeme III (presently not classified in either T. cruzi I or T. cruzi II). This is the first observation of this category of T. cruzi genotypes in chronic chagasic patients. In the past it was recorded only in acute patients, wild mammals and wild triatomine bugs. The epidemiological implications of these results are discussed.

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico.

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5 percent and 9 percent with IHA and ELISA tests, respectively, and up to 7.7 percent using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7 percent), Bolivia (13.7 percent) and Argentina (3.5 percent). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1 percent) and AIDS (0.1 percent) in the same region

Impact of Trypanosoma cruzi clonal evolution on its biological properties in mice.

Twenty Trypanosoma cruzi stocks attributed to the 19, 20, 39, and 32 clonal genotypes were comparatively studied in BALB/c mice during the acute and chronic phases of the infection to test the working hypothesis that T. cruzi clonal structure has a major impact on its biological properties. Fourteen parameters were assayed: (1) infectivity; (2) prepatent period; (3) patent period; (4) maximum of parasitemia; (5) day of maximum of parasitemia; (6) parasitemia; (7) mortality, (8) percentage of positive hemoculture, (9) tissue parasitism; (10) inflammatory process during the acute phase of the infection; (11) mortality, (12) percentage of positive hemoculture; (13) tissue parasitism; and (14) inflammatory process during the chronic phase of the infection. Statistical comparison showed that the results are overall consistent with the working hypothesis that biological differences are proportional to the evolutionary divergence among the genotypes. Thus, closely related genotypes (19 vs 20 and 32 vs 39) show in general fewer differences than distantly related groups (19 or 20 vs 32 or 39) except for the comparison between 19 and 32. The working hypothesis is even more strongly supported by the result of the nonparametric Mantel test, which showed a highly significant correlation (P = 2.3 x 10(-3)) between biological differences and genetic distances among all pairs of stocks. These data taken together emphasize that it is crucial to take into account the phylogenetic diversity of T. cruzi natural clones in all applied studies dealing with diagnosis, drug and vaccine design, epidemiological surveys, and clinical diversity of Chagas' disease. Index Descriptors and Abbreviations: Trypanosoma cruzi; phylogenetic distance; biological properties; clonal theory; multilocus enzyme electrophoresis (MLEE); randomly amplified polymorphic DNA (RAPD); acute phase (AP); chronic phase (CP); days after inoculation (d.a.i.); liver infusion tryptose (LIT); gastrointestinal tract (GIT); genitourinary tract (GUT); percentage of infectivity (%INF); percentage of mortality during the acute phase (%MORT AP); percentage of mortality during the chronic phase (%MORT CP); prepatent period (PPP); patent period (PP); maximum of parasitemia (MP); day of maximum of parasitemia (DMP); parasitemia (PAR); percentage of positive hemoculture during the acute phase (% + HC AP); percentage of positive hemoculture during the chronic acute phase (% + HC CP); tissue parasitism (TP); inflammatory process (IP); tissue parasitism during the acute phase (TP AP); tissue parasitism during chronic phase (TP CP); inflammatory process during acute phase (IP AP); inflammatory process chronic phase (IP CP); Mann-Whitney test (MW); Kruskal-Wallis (KW); Kolmogorow-Smirnov test (KS).

Restricted genetic and antigenic diversity of Plasmodium falciparum under mesoendemic transmission in the Venezuelan Amazon.

The study of genetic diversity in malaria populations is expected to provide new insights for the deployment of control measures. Plasmodium falciparum diversity in Africa and Asia is thought to reflect endemicity. In comprehensive epidemiological surveys reported here the genetic and antigenic structure of P. falciparum in the Venezuelan Amazon were studied over a 2-year period. DNA polymorphisms in glutamate-rich protein (GLURP), merozoite-surface protein 1 (MSP1) and MSP2 genes, in a multicopy element (PfRRM), all showed low diversity, 1 predominant genotype, and virtually no multi-clonal infections. Moreover, linkage disequilibrium was seen between GLURP, MSP1 and MSP2. Specific antibody responses against MSP1 and MSP2 recombinant antigens reflected the low genetic diversity observed in the parasite population. This is unexpected in a mesoendemic area, and suggests that the low diversity here may not only relate to endemicity but to other influences such as a bottleneck effect. Linkage disequilibrium and a predominant genotype may imply that P. falciparum frequently propagates with an epidemic or clonal population structure in the Venezuelan Amazon.