Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones.

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

A estrutura clonal da cepa Colombiana do Trypanosoma cruzi, biodema Tipo III e zimodema 1, foi analisada com o objetivo de caracterizar as suas populações e estabelecer a homogeneidade ou heterogeneidade das mesmas. Foram isolados sete clones, os quais apresentaram as características básicas do biodema Tipo III, com o mesmo padrão de curvas parasitêmicas, tropismo tecidual para músculo esquelético e miocárdio, alta patogenicidade, com extensas lesões necrótico-inflamatórias, do 20º ao 30º dia de infecção. A cepa parental e os clones C1, C3, C4 e C6 apresentaram os niveis mais elevados de parasitemia entre 20 e 30 dias pós-infecção e alto indice de mortalidade até 30 dias (79 a 100%); os clones C2, C5 e C7 apresentaram niveis mais baixos de parasitemia com baixa mortalidade até 30 dias (7,6 a 23%). Os padrões isoenzimáticos foram característicos do zimodema 1 (Z1) para a cepa parental e os sete clones. Os achados do presente trabalho indicam uma homogeneidade fenotípica entre os clones isolados da cepa Colombiana e sugerem a predominância de um clone principal, responsável pelo padrão de comportamento biológico da cepa parental e dos clones.

Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology.

With the objective of establishing biological and biochemical characteristics of a significant number of Trypanosoma cruzi strains from different geographical areas, 138 strains isolated from naturally infected humans, triatomine or vertebrate hosts were studied; 120 were isolated from different areas of Brazil and 18 from other South and Central American countries. Inocula from triatomine or culture forms were injected into suckling Swiss mice, followed by passages into mice 10 to 12 g. Biological characters and histopathological study permitted the inclusion of the strains into three Types or biodemes: I, II, III. Isoenzymic analysis confirmed a correspondence between the biodemes and zymodemes: Type I and Z2b, Type II and Z2, Type III and Z1. Results showed the ubiquitary distribution of the several types of strains. The predominance of the same Type and zymodeme in one geographical area was confirmed: Type II strains among the human cases from eastern Bahia and east of Goiás; Type III strains from humans of north Brazil and Central America and from silvatic vectors or vertebrates from other geographical areas. The biological types of strains correlate with different histopathological lesions considering cardiac involvement and neuronal lesions. These findings suggest that the biological behavior together with isoenzymes patterns and pathological pictures in the vertebrate host can be an important tool for establishing correlations between strains behavior and clinico-pathological manifestations of Chagas' disease in different geographical areas.

Trypanosoma cruzi strains: behavior after passage into authoctonous or foreign species of traitomine (biological and biochemical patterns).

The behavior of T. cruzi strains from S. Felipe-BA (19 SF, 21 SF and 22 SF) classified as Type II Zymodeme 2, was investigated after passage through the authoctonous (P. megistus) and foreign vectors (T. infestans and R. prolixus). For each strain Swiss mice were infected: I--with blood forms (control); II--with metacyclic forms (MF) from P. megistus; III--with MF from T. infestans; IV--with MF from R. prolixus. Inocula: MF from the three species of triatomine, 60 to 120 days after feeding in infected mice, adjusted to 10(4). Biological behavior in mice (parasitemia, morphology, mortality, virulence and pathogenicity) after passage through triatomine was compared with data from the same strain in control mice. Isoenzymic electrophoresis (ASAT, ALAT, PGM, GPI) were also performed after culture into Warren medium. The three strains maintained the isoenzyme profiles (zymodeme 2), in the control groups and after passages through different species of triatomine. Biological characterization disclosed Type II strains patterns for all groups. An increased virulence was observed with the 22 SF strain isolated from P. megistus and T. infestans and higher levels of parasitemia and predominance of slender forms in mice inoculated with the 19 SF and 21 SF from these same species. Results indicate that the passage through the two species T. infestans and P. megistus had a positive influence on the virulence of the regional strains of S. Felipe, regardless of being autocthonous (P. megistus) or foreign to the area (T. infestans).

Investigation on the possibility of spontaneous cure of mice infected with different strains of Trypanosoma cruzi.

Seventy Swiss mice chronically infected with different strains of Trypanosoma cruzi, with persistently negative parasitemia on routine blood examination were parasitologically investigated to find out whether spontaneous cure occurred. Duration of infection varied from 90 to 250 days in the initial phase of this investigation. Parasitological tests consisted of daily direct blood examination performed during at least 25 days, followed by xenodiagnosis and subinoculation of blood into newborn mice. Mice that persisted negative were treated with Cyclophosphamide with one dose of 250 mg/kg of body weight and then investigated by direct blood examination, xenodiagnosis and subinoculation. A second dose of 250 mg/kg b. w. was given to the persistently negative mice. With one single exception, all mice showed positive parasitological tests in the different stages of the present investigation and we conclude that spontaneous cure did not occur in this group, which is representative of the chronic infection with different strains of T. cruzi.

Specific chemotherapy of Chagas disease: a comparison between the response in patients and experimental animals inoculated with the same strains.

Eleven strains of Trypanosoma cruzi were isolated from patients with Chagas disease in central Brazil by xenodiagnosis and inoculation into newborn mice. Biological characterization and isoenzyme analysis showed that 6 strains were type II (zymodeme 2) and 5 were type III (zymodeme 1). Patients were treated with benznidazole or benznidazole plus nifurtimox. Mice infected with each isolated strain were treated for comparison with the results obtained in the respective patient. Evaluation of cure of the patients was based on the indirect immunofluorescence test, complement fixation reaction and xenodiagnosis. For the mice, haemoculture, indirect immunofluorescence testing, xenodiagnosis and inoculation of blood into newborn mice were used. Tests were performed 3-6 months after the end of treatment. The cure rate was 66-100% in mice infected with type II strains and 0-9% in those infected with type III strains. The correlation between treatment results in patients and mice was 81.8% (9 of 11 cases). Type II strains were more susceptible to treatment, in contrast to type III strains which yielded the majority of therapeutic failures.

[Behavior of Trypanosoma cruzi strains after passage in different triatominae species]. / Estudo do comportamento de cepas de Trypanosoma cruzi após passagem em diferentes espécies de triatomíneos.

To study the influence of the intermediate host stage on the course of mouse infection, Trypanosoma cruzi belonging to the Peruvian (Type I), 12 SF (Type II) and Colombian (Type III) strains were passaged through either Rhodnius prolixus, Panstrongylus megistus or Triatoma infestans. T. cruzi metacyclic forms (dose 10(4)) from the different strains were obtained from each bug and inoculated into 8-10 gm mice. Comparison was made in mice inoculated with blood forms. Parasitaemia curves were plotted in the peripheral blood for each strain, reaching more elevated peaks with Peruvian strain parasites from P. megistus and R. prolixus, 12 SF strain from P. megistus and Colombian strain from R. prolixus. Tissue tropism and histopathological patterns were those usually seen in mice infected with each respective strain type. Peruvian virulence was the same for all groups. Slender forms predominate among mice inoculated with metacyclic forms of Colombian and 12 SF strains, probably an adaptative parasite change related to the intermediate host.

[Therapy of the chronic phase of the experimental infection by Trypanosoma cruzi with benzonidazole and nifurtimox]. / Terapêutica da fase crônica da infeccao experimental pelo Trypanosoma cruzi com o benzonidazol e o nifurtimox.

Fifty-eight mice, chronically infected with different T. cruzi strains (Types II and III) were submitted to chemotherapy either with Nifurtimox (Bay 2502) or Benznidazole (Ro 7-1051). Twenty one mice were not treated and were used as infected controls. The duration of infection was from 90 to 400 days. Inocula varied from 1 x 10(4) to 5 x 10(4) blood forms. Treatment lasted for 90 days, doses being 200mg/kg/day during 4 days, followed by 50mg/kg/day for Nifurtimox and 100mg/kg/day for Benznidazole. Parasitological tests (xenodiagnosis, inoculations into baby mice and hemoculture) showed 85.3% negativation for Type II strains and 43% for Type III in animals treated with Benznidazole. As for Nifurtimox, there were 71.4% of parasitological negativation for the animals infected with Type II strains and 66% for those infected with Type III. IFA tests remained positive in 90% of treated and cured animals. Disappearance or marked regression of myocardial and skeletal muscle lesions was seen in the treated and parasitologically negative animals. The conclusion is that the treatment in the chronic phase of T. cruzi infection can result in parasitological cure in a high percentage of cases with regression of histopathological lesions, although with persistence of positivity of the IFA tests.

Immunological response of Swiss mice to infection with three different strains of Trypanosoma cruzi.

The immunological response of Swiss mice to infection with three strains of Trypanosoma cruzi which differ in their morphobiological, antigenic and isoenzymic characters [Peruvian, 12 SF (São Felipe) and Colombian strains] was investigated. The three strains stimulated an elevation of the immunoglobulin fractions IgG2a, IgG2b and IgM during acute infection, as measured by radial immunodiffusion, and an early drop of IgG1 levels. There were low levels of specific antibodies and a negative cutaneous delayed hypersensitivity test to T. cruzi antigens. Cellular reaction of the spleen was evident, with proliferation of lymphocytes and the presence of blastic lymphoid cells in the red and white pulp, and hyperplasia of germinal centres of the lymphoid follicles. Those aspects were consistent with a depletion of the T-cell zone (periarteriolar lymphocyte sheath). Despite these common features, there were clear differences in the onset, intensity and evolution of the splenic cellular reaction and IgG serum levels and in the relationship between these levels and parasitaemia in the mice infected with the three strains of T. cruzi. A positive correlation was seen between high IgG levels and mortality, corresponding to intense exudative tissue lesions, showing that a raised immunoglobulin level was not associated with protection. It is worth observing that the 12 SF strain, which showed the lowest parasitaemic profile and mortality rate, stimulated the greatest elevation of IgG2b during acute infection; and also that IgG2a and IgG2b were the immunoglobulins which showed the greatest increases following infection by all three strains of T. cruzi.

[Immunological aspects of infection of 6 inbred strains of mice by 3 different strains of Trypanosoma cruzi]. / Aspectos imunológicos da infecção de seis linhagens isogenicas de camundongos por tres diferentes cepas do Trypanosoma cruzi.

We evaluated humoral and cellular immune responses in 6 inbred mouse strains (BALB/c, B-10, C3H, A/J, AKR and DBA) infected with 3 Trypanosoma cruzi strains (Peruvian, 21 SF and Colombian), which are the standards for the 3 strains Types of Andrade's classification. Negative delayed-type hipersensitivity reactions to parasite antigens were evidence of suppressed cell-mediated immunity. An early drop of IgG1 and rise of IgM levels were observed in almost all mouse strains infected by any T. cruzi strain. Elevation of IgG2a and/or IgG2b levels was higher in resistant mouse strains. Anti-T. cruzi antibody levels (Indirect immunofluorescence and ELISA) did not correlate with survival. Despite some differences among mouse strains there was a definition of an overall pattern of host response and the maintenance of biological standards which characterize the basic types of T. cruzi strains.

Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types.

A test was made of the susceptibility of 30 strains of Trypanosoma cruzi to chemotherapy with nifurtimox (Bay 2502) and benznidazole (Ro 7-1051). The strains had previously been classified as type I, II, or III according to their morphobiological and isoenzymic characteristics. Three type I strains, 14 type II strains, and 13 type III strains were studied. Mice were infected with 2 x 10(5) blood forms of these parasites and treated for 90 days with benznidazole or nifurtimox. All the surviving mice were submitted to parasitological tests (direct parasitaemia, xenodiagnosis, inoculation in new-born mice, and haemoculture) and serological tests (indirect immunofluorescence). As the latter remained positive in about 80% of the parasitologically negative animals, the cure rates were based on the more reliable parasitological tests. Type I strains displayed high susceptibility, type II strains showed medium to high susceptibility, and type III strains were highly resistant to both drugs. The fact that a particular strain type, with its own level of susceptibility, usually predominates in a given geographical area may explain the contradictory results after chemotherapy from different endemic areas.

[Behavior of the Y and Peruvian strains of Trypanosoma cruzi in mice, after passage through various media]. / Comportamento das cepas Y e Peruana do Trypanosoma cruzi no camundongo, após passagem em diferentes meios.

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.