Cruzi Stocks Research Articles

Virulence and pathogenicity associated with diversity of Trypanosoma cruzi stocks and clones derived from Chagas' disease patients.

The intraspecific variation that has been described in Trypanosoma cruzi was examined in recent isolates from Chagas' disease patients, using behavioral and molecular markers for characterization of the parasite stocks and derived clones. We used these parasite populations to determine virulence and pathogenicity in vivo. The T.cruzi stocks mSLU142 (megaesophagus) and hSLU239 (heart disease) and the clones h1 and h2 induced very low parasitemias in BALB/c mice, whereas high parasitemias were obtained with clones m1, m2, m3, and m4. Clones m1-m4 also produced heart lesions of higher intensity than those observed in mice infected with the h1 and h2 clones. Furthermore, the heart lesions produced by all of these clones were significantly more intense than those seen in mice infected with either of the T. cruzi parental stocks. In addition, neither the kinetics of growth, doubling time, differentiation in axenic culture, zymodemes, nor DNA restriction length polymorphisms showed correlations with parasitemias and pathogenicity in mice. This study suggests that multiple biochemical and physiological markers are required to enable an association of clinical and pathologic manifestations of the disease with intrinsic characters of the T. cruzi populations.

Polymorphisms in Trypanosoma cruzi: evidence of genetic recombination

The ploidy of Trypanosoma cruzi is until now undetermined although analysis of isoenzymes, molecular karyotype and DNA content suggest diploidy in a very plastic genome. Also, there has been no convincing demonstration of genetic exchange and it has been proposed that reproduction is clonal. We have compared 18 T. cruzi stocks and clones from the same area or host by means of isoenzyme analysis (12 loci) and restriction site polymorphisms in and around three glycolytic genes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and glucosephosphate isomerase). The analysis demonstrated the presence of homozygotes and heterozygotes and is compatible with diploidy for these housekeeping genes. This strongly supports the hypothesis of genetic exchange in T. cruzi and further elucidates the genetic diversity within natural T. cruzi populations.

Biochemical, Immunological, and Biological Characterization of Trypanosoma cruzi Populations of the Andean North of Chile

Twenty-one Trypanosoma cruzi stocks isolated from Triatoma infestans and humans of the Chilean Andean highlands were studied at the genotypic level by schizodeme and molecular karyotype analyses, which allowed a clear distinction of the parasites from those hosts. A phenotypical characterization was performed by proteolytic activity after electrophoretic fractionation without discrimination among the stocks. Metacyclic trypomastigotes obtained in vitro proved to be infective to Swiss mice and the study of immune response and biological behavior was assessed. Of a total of 21 T. cruzi stocks, only 11 proved to be infective in mice due to difficulties in obtaining metacyclic trypomastigotes with the parasite populations isolated from humans. Western blot analysis revealed a complex immune response even in the first days postinfection with each T. cruzi strain studied. Antigenic recognition by each immune serum is characteristic, although several major and common antigens were detected. Lytic antibodies were studied by the in vitro complement-mediated lytic assay using purified metacyclic trypomastigotes as target cells. All the T. cruzi isolates tested induced lytic antibodies in this experimental model. Parasitemias were moderate and characteristic for each T. cruzi strain. Results are compared with metacyclic forms of the infective and pathogenic Tulahuen strain.

Intraspecific Diversity in the Response of Trypanosoma cruzi to Environmental Stress

Epimastigotes of 5 Trypanosoma cruzi stocks were cultivated in liver infusion tryptose (LIT) medium at 23-35 C or cocultivated with vertebrate cells at 35 C. A temperature decrease from 26 to 23 C resulted in a stable 60% increase in population doubling time. In zymodeme I and II stocks, a temperature increase to 35 C resulted in a transient approximately 25% increase in doubling time during the first month followed by a approximately 30% decrease after 2 mo. A zymodeme III stock did not grow at 35 C. Flow cytometric analyses showed that the total DNA/cell, guanine + cytosine (G-C), and adenine + thymidine content of 2 zymodeme II stocks increased by 3-11% when cultivated in LIT at 35 C, whereas the DNA values of 2 zymodeme I stocks did not change. The increased DNA levels, due predominantly to an increased kinetoplast G-C content, returned to normal levels when the culture temperature was reduced to 26 C. The effects of cocultivation with vertebrate cells at 35 C were identical to cultivation in LIT at 35 C except that the DNA increase in a zymodeme II stock was not stable. Total DNA/cell, nuclear, and kinetoplast DNA decreased by 8-13% upon prolonged cocultivation. No change in total protein, antigen profiles, complement sensitivity, or heat shock protein gene expression was observed as a consequence of culturing the parasites above 26 C.

Molecular biology studies of tubercidin resistance in Trypanosoma cruzi.

Trypanosomatids are incapable of de novo purine synthesis; purines are obtained through the scavenging of exogenous nucleosides. To advance our understanding of purine utilization, we mutagenized a Trypanosoma cruzi stock and selected for resistance to high levels of tubercidin (7-deazaadenosine, TUB), a purine analog. The TUB-resistant stocks were > 100 times more resistant to TUB than was the parental stock. TUB and uridine transport in the TUB-resistant stocks decreased by 50%-90%, whereas thymidine and adenosine transport were unaffected. These data imply that TUB-resistant stocks have defects in the pathways involved in the transport of TUB and uridine but not in the thymidine and adenosine transport pathways. Karyotype analyses using specific probes showed that the deletion of a 950-kb chromosome-size DNA occurred in both of the TUB-resistant stocks. These data suggest that genes involved in nucleoside transport are located in this DNA region. This study will facilitate the identification and characterization of the specific genes involved in nucleoside transport and aid in the elucidation and development of new chemotherapeutics for Chagas' disease.

Characterization of Chilean, Bolivian, and Argentinian Trypanosoma cruzi populations by restriction endonuclease and isoenzyme analysis

Ninety-one Chilean, 15 Bolivian, and 9 Argentinian Trypanosoma cruzi stocks, isolated from various hosts and vectors, were characterized by schizodeme analysis with EcoRI and MspI endonucleases. The three major similar pattern groups that emerged from this sample correlated with results of isoenzyme analysis. This result confirms previous work and supports the hypothesis of the clonal structure of natural populations of T. cruzi, fully defined at the level of isoenzyme analysis, quantitative kinetoplast DNA restriction fragment length polymorphism, and kinetoplast DNA hybridization analysis. In Chile, sylvatic and domestic cycles of T. cruzi transmission appear to be mainly independent: genetically different families of natural clones are specific to these cycles. Nevertheless, the possibility of overlap remains unclear. Results described here indicate that natural clones inhabiting Chilean regions appear genetically related to the natural clones identified in neighboring countries. In Chile the more frequently sampled parasite types are natural clone 39 and a genetically closely related clone NP13. In this work an evaluation of T. cruzi natural clone mixtures in T. cruzi stocks from Chile was performed for the first time by schizodeme analysis before and after serial transfer in mouse maintenance. The results indicate that six of nine stocks are composed of two or more natural clones. This observation raises the relevant question of whether specific T. cruzi natural clones generate different clinical features of Chagas' disease.

Molecular characterization of Mexican stocks of Trypanosoma cruzi using total DNA.

Seventeen Mexican Trypanosoma cruzi stocks and five South American reference strains were analyzed by Hind III restriction fragment length polymorphisms associated with ribosomal RNA gene spacers and by HinfI digestion patterns of total DNA. Our findings demonstrate the occurrence of genetic heterogeneity within these stocks. Hierarchic and non-hierarchic clustering of these molecular characters allowed the formation of groups that correlate with the geographic origin of the stocks. The HinfI digestion pattern permitted the identification of DNA fragments from the kinetoplast maxi-circles, and therefore represents a simple and convenient method for conducting epidemiologic surveys in laboratories in developing countries.

Trypanosoma cruzi: Flow Cytometric Analysis of Developmental Stage Differences in DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2-12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4-8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5-13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.

Isolate-dependent differences in the oxidative metabolism of Trypanosoma cruzi epimastigotes

Epimastigote cultures of the two cloned Trypanosoma cruzi stocks, CA-I/72 and HO-3/15, grown under identical conditions, differ both qualitatively and quantitatively in their cytochrome content. The CA-I/72 stock has a four-fold higher cytochrome b content (19.2 nM (mg protein) −1) than the HO-3/15 stock (4.9 nM (mg protein) −1). Cytochrome o is present at 29 nM (mg protein) −1 in the CA-I/72 stock but is below detectable limits in the HO-3/15 stock. There is no inter-stock difference in oxygen utilization (12–15 nM O 2 min −1 (mg protein) −1) during exponential growth. However, stationary phase CA-I/72 epimastigotes utilize twice as much oxygen as HO-3/15 epimastigotes. Oxygen utilization by HO-3/15 epimastigotes incubated in Dulbecco's phosphate buffer solution (starvation conditions), was stimulated earlier and to higher levels by the addition of glucose than by CA-I/72 epimastigotes under identical conditions. Under starvation conditions and with the cytochrome chain partially inhibited by antimycin A,(anti-A) the addition addition of glucose also increased oxygen utilization by CA-I/72 epimastigotes. In contrast, anti-A did not influence glucose-stimulated oxygen utilization by HO-3/15 epimastigotes. Following partial inhibition with anti-A, salicylhydroxamic acid produced an additional 50% inhibition in oxygen utilizatin in both stocks irrespective of the growth phase of the organisms. These data indicate that marked intra-specific differences in oxidative metabolism exist within the T. cruzi population and that an α-glycerophosphate oxidase or similar salicylhydroxamic acid-inhibitable compound may be present in the organism.

Characterization and grouping ofTrypanosoma cruzi stocks by DNA base-specific fluorochromes and discriminant analysis

Fluorochromes with G-C and A-T specificity were used for a single-cell DNA analysis of the blood-stream forms of 14 Trypanosoma cruzi stocks in a cytofluorometric assay. The kinetoplast contained 22.3%-37.9% of the total DNA G-C base content and 42.7%-63.5% of the total DNA A-T base content. In spite of these differences, the mean base A-T/G-C ratio of the total DNA was 1.11 and was nearly constant in all stocks. The G-C base ratio of kinetoplast/nucleus resulted in a grouping corresponding with the peanut agglutinin (PNA)- and wheat germ agglutinin (WGA)-type characteristics of the T. cruzi stocks. The discriminant analysis revealed relationships, in that each stock contained some trypanosomes with DNA fluorescence characteristics of common to at least one other stock. After chromomycin A3 staining, the mean hit rates for the classification into group 1 PNA and the WGA group were 99% and 96%, respectively, and the respective rates obtained after DAPI application were 84% and 94%.

ISOZYME VARIABILITY IN TRYPANOSOMA CRUZI, THE AGENT OF CHAGAS' DISEASE: GENETICAL, TAXONOMICAL, AND EPIDEMIOLOGICAL SIGNIFICANCE.

A genetic interpretation of the zymograms of 524 Trypanosoma cruzi stocks from various hosts and representing a broad geographical range (United States to Southern Brazil) reveals high genetic variability (only one monomorphic locus out of 15) and suggests that this parasite has a diploid structure. The data do not give any indication of Mendelian sexuality, although many opportunities are present for genetic exchange between extremely different genotypes. The population structure of T. cruzi appears to be multiclonal and complex. The natural clones evidenced by isozyme analysis are numerous (43 different ones are recorded among 121 stocks assayed at 15 gene loci) and exhibit a large range of genotypes, in a nonhierarchical structure; it is not possible to cluster them into a few strictly delimited groups which could represent natural taxa. The available data suggest that the genetic variability of T. cruzi reflects the long separate evolution of multiple clones. It is suggested that long clonal evolution may explain the present biological and medical variability of the causative agent of Chagas' disease.

Lack of enzyme polymorphism in Trypanosoma rangeli stocks from sylvatic and domiciliary transmission cycles in Colombia.

Although Trypanosoma rangeli is biologically and morphologically distinct from Trypanosoma cruzi, these two hemoflagellates are epidemiologically linked. We report the results of enzyme electrophoretic studies of T. rangeli stocks isolated from sylvatic and domiciliary Rhodnius prolixus, and infected humans inhabiting foci in which T. cruzi was sympatrically transmitted. T. rangeli stocks displayed electrophoretically detectable polymorphism for only a single enzyme, isocitrate dehydrogenase (ICD), in contrast with the marked phenotypic heterogeneity previously reported among the T. cruzi stocks. The relatively restricted diversity manifested by stocks from different geographic sites and ecologic habitats may reflect the existence of distinctive biologic or genetic constraints influencing T. cruzi and T. rangeli transmission.

Isoenzyme changes during the life cycle of Trypanosoma cruzi

The isoenzyme profiles, for 14 enzymes, of amastigotes, trypomastigotes and epimastigotes were compared in various cloned and uncloned T. cruzi stocks belonging to different zymodemes. A culture method with a human diploid cell line was developed and produced either pure amastigotes or trypomastigotes in high yields. Trypomastigotes were also isolated from rat blood and from liquid culture. Epimastigotes were harvested from various acellular media and from the overlay of cell monolayers. The isoenzyme patterns of each life-cycle stage showed consistent differences in the number, position and intensity of the electrophoretic bands for certain enzymes. With the single exception of one peptidase, the variable patterns were stage-specific regardless of whether the organisms were harvested from animals or from various cultures at different temperatures.

Isozyme profiles of Trypanosoma cruzi stocks from Colombia and Ecuador.

A total of 74 of 82 domestic Rhodnius prolixus from the same locality in eastern Colombia were found to be infected with Trypanosoma cruzi or T. rangeli. One of three domestic Triatoma dimidiata from Ecuador also showed T. cruzi infection. A total of 59 T. cruzi stocks from these and five other localities in Colombia were isolated from man, marsupials and triatomine bugs. Cellulose-acetate electrophoresis of nine or ten enzymes characterized all T. cruzi stocks as zymodeme 1 (reference clone Silvio X10/1). Differences in electrophoretic patterns between the newly isolated stocks and the zymodeme 1 standard were seen with the enzymes G6PD and HK. These results are in agreement with the previously described geographical distribution of T. cruzi zymodemes. Stocks were isolated from both low and high altitudes and there was no evidence of adaptative significance of T. cruzi enzyme polymorphism.

Isoenzyme characterization of Trypanosoma cruzi from congenital cases of Chagas' disease.

The isoenzyme profiles of a group of five Trypanosoma cruzi stocks obtained from congenital cases and of a second group of seven stocks obtained from chagasic mothers who did not transmit their infection congenitally were studied by electrophoresis of nine enzymes. All the mothers became infected in Bahia State (Brazil). With the exception of a triple 'heterozygous' GPI pattern, all T. cruzi stocks were identified as zymodeme 2 (Z2). The heterozygous pattern has not been recorded previously in Bahia. This study shows that enzymically similar T. cruzi stocks can show different behaviour with respect to transplacental transmission, and also with respect to clinicopathological presentation and therapeutic response of the congenital cases.

Cultivation of Trypanosoma cruzi using human diploid cells.

Interaction between normal diploid cells which had been established from human lung tissue and Trypanosoma cruzi (T. c) stock newly isolated from an Ecuadorian patient with Chagas' disease was studied. Normal diploid cells were found to have susceptibility to invasion by T. c and capacity to support the parasite growth. Intracellular parasites multiplied with a doubling time of 16.9 hours, and after 4 days of infection, trypomastigotes were released into culture supernatant. Development of T. c was relatively synchronized in the infection with parasite : host cell ratio of 20 : 1, providing broad form trypomastigotes with purity of more than 95 per cent in culture supernatant on day 4. Amastigotes were observed during the later stage of cultivation, resulting from degeneration of host cells. Two morphologically distinct forms of trypomastigotes, broad and slender, were obtained. Broad form predominated at early stage of cultivation and then proportion of slender form gradually increased.

Chagas' disease in the Amazon Basin IV

Of 151 Trypanosoma cruzi stocks from 117 different individual hosts collected in the states of Amazonas and Rondonia, 147 from 113 hosts were identified as zymodeme 1 (Z1). These included T. cruzi stocks from three marsupial species, two rodent species and three triatomine species although most were from the common opossum, Didelphis marsupialis. One T. cruzi stock from Rhodnius robustus was identified as Z1 with a Z3 PGM character, one from Sciurus sp. as Z3 and two from Monodelphis brevicaudata and Panstrongylus geniculatus as Z3 with a Z1 ASAT character. The ways in which stocks were isolated and grown up in vitro did not influence isozyme profile. These results support earlier evidence from Pará State that Z2 is absent from the Amazon basin and that the distribution of T. cruzi zymodemes in this region is quite different to that in endemic areas on the south of the continent.

The microdistribution of isoenzymic strains of Trypanosoma cruzi in southern Bolivia; new isoenzyme profiles and further arguments against Mendelian sexuality

132 Trypanosoma cruzi stocks were collected in southern Bolivia (99 stocks in Tupiza, 33 in Tarija), and were characterized using five enzymes (six loci). From these 132 stocks, a sample of 21 was studied using 10 enzymes (12 loci) to establish the genetic distances between them. Only five different isoenzymic strains were registered among the 132 stocks: the taxonomic status of these strains is discussed. The distribution of the strains indicated that a Founder effect was not a constant fact at the level of the house and of the suburb, but that a Founder effect was more apparent for greater geographical distances. All strains were transmitted sympatrically by the same vector Triatoma infestans. Genotype frequencies demonstrated the lack of Mendelian sexuality among stocks of T. cruzi from southern Bolivia, confirming our previous results.

Trypanosoma cruzi: antigenic analysis of cloned stocks.

The antigenic constitution of two Trypanosoma cruzi strains and six cloned stocks was determined immunoelectrophoretically. Five to seven antigens common to all of the T. cruzi stocks were found. However, each stock also contained "strain-specific antigens," and five of the six cloned stocks presented "clone-specific antigens." These data demonstrate that a T. cruzi strain is composed of an antigenically heterogeneous population of organisms.

The characterization of Chilean and Bolivian Trypanosoma cruzi stocks from Triatoma infestans by isoelectrofocusing.

Culture forms of 12 Chilean and 9 Bolivian Trypanosoma cruzi stocks were compared isoenzymatically by the following enzymes: non-specific esterase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and alcohol dehydrogenase. On the basis of the electrophoretic mobility of these enzymes the stocks were classified into two main groups. Ten Chilean stocks were characterized as group II; two stocks showed enzyme patterns of group I. In contrast, five Bolivian stocks were classified as belonging to group I, the other four to group II. The results show that the two groups of T. cruzi overlap in Triatoma infestans suggesting that both groups of T. cruzi are infective for man. The classification of stocks into two groups is discussed in the light of published results of Brazilian T. cruzi stocks. A strong association of groups with the transmission cycles as it seems to be in Brazil does not exist in Chile and Bolivia.