Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain reaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.     

Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients

Parasite DNA amplified by PCR from blood of 73 chagasic children and adults of two endemic areas of Chile were studied by Southern blot and/or dot blot hybridization analysis with a panel of three minicircle probes corresponding to the parasite genotypes (clonets 19, 33 and 39). The hybridization pattern of the PCR positive samples identified clonets 39, 19/20, and 32/33 with frequencies of 0.84, 0.32 and 0.26, respectively. A total of 31 samples corresponded to mixed infections. The most frequently found mixtures were: clonets 39 and 19/20 (14 cases), followed by clonets 39 and 32/33 (8 cases), clonets 39, 32/33 and 19/20 (8 cases), and clonets 32/33 and 19/20 (1 case). Amplified DNA from 9 cases showed no hybridization signal with none of the three studied probes indicating that other genotypes different to the ones mentioned are circulating in humans, but that the clonets used as probes are the most prevalent ones in terms of transmission in the endemic areas studied. A biological characterization of 34 T. cruzi populations isolated from the xenodiagnosis of the patients was performed on an experimental murine model. The biochemical characterization of the parasite populations by molecular karyotype determined that the most frequent parasite isolated from patients belongs to clonet 39.     

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

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

Trypanosoma vivax: adaptation of two East African stocks to laboratory rodents

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10(9)-10(10) trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Trypanosoma vivax: Adaptation of Two East African Stocks to Laboratory Rodents1

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10⁹–10¹⁰ trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi.

A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.     

Integrate study of a Bolivian population infected by Trypanosoma cruzi,the agent of Chagas disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

In Vitro Production of Metacyclic Forms of Trypanosoma musculi and Their Infectivity in CBA Mice1

ABSTRACT. Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Trypanosoma cruzi in wild raccoons and opossums in North Carolina.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.     

Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.     

Plasmodium chabaudi chabaudi: effect of low parasitemias on immunity in CB6F1 mice.

We examined the effect that low parasitemias have on the immune response of CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. Ascending parasitemias were stopped by chloroquine treatment when they were between 1.6 and 9.4%. Mice that suffered low parasitemias developed good immunity to homologous reinfection but, contrary to what happened in mice that suffered full parasitemias, they did not develop immunity to heterologous reinfection with Plasmodium yoelii 17XL. Total IgG antiparasite antibody responses were similar in mice that suffered low or full parasitemia, both in primary infection and after reinfection. At the level of isotypes, IgM, IgG1, IgG2b, and IgG3 responses were similar in mice that suffered low or full parasitemias, but after reinfection, mice that suffered low parasitemias responded with higher levels of IgG2a than mice that suffered full parasitemias. Mice that suffered low parasitemias did not have splenomegaly but their immunity to homologous reinfection was diminished after splenectomy in a manner similar to that of splenectomized mice that suffered full parasitemia. CB6F1 mice can develop homologous immunity even if exposed to low parasitemias but cannot develop heterologous immunity unless exposed to high parasite loads.     

In vitro production of metacyclic forms of Trypanosoma musculi and their infectivity in CBA mice

Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard's distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.     

Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice

The antigenic types in populations of Metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms with heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

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

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

Antigenic Analysis By Immunofluorescence of In Vitro-Produced Metacyclics of Trypanosoma Brucei and Their Infections In Mice*

SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

Behavior of the Y and Peruvian strains of Trypanosoma cruzi in mice, after passage through various media

The behavior of two strains of Trypanosoma cruzi (Y and Peruvian strains) in experimental mouse infection, after being passed through different conditions of maintainance and cultivation was studied. The conditions were: Warren's acellular culture medium, cryopreservation in liquid Nitrogen, passage through the insect vector and direct blood passage from mice to mice. The parameters considered for comparative study were as follows: parasitemia, mortality rate, maximum survival time, morphology of parasites in peripheral blood, tissue tropism and histopathological lesions. Each experimental group consisted of two sub-groups according to the inocula: 10.000 or 50.000 trypomastigotes. The basic characteristics of the strains remained unchanged. These were macrophagotropism, predominance of slender forms of the parasite in early infection and 100 per cent mortality rate in the acute phase of the infection. However, decrease in the virulence was observed when the culture forms were used or when the infection with low inoculum was used (10.00 forms). Therefore the main biological characteristics of the strains tended to remain the same, regardless of the conditions used for maintainance and cultivation.