Infectivity of amastigotes of Trypanosoma cruzi

The infectivity amastigotes of Trypanosoma cruzi, isolated from the supernatant of the J774G8 macrophage-like cell line infected with trypomastigotes to normal macrophages in vitro was tested. After a period of 1 h of T. cruzi-macrophage interaction about 2% of the mouse peritoneal macrophages had ingested amastigotes. In contrast 12% of the macrophages had ingested epimastigotes. Treatment of the amastigotes with trypsin did not interfere with their ingestion by macrophages. Once inside the macrophages the amastigotes divided and after some days transformed into trypomastigotes. When i.p. inoculated into mice the amastigotes were highly infective, inducing high levels of parasitaemia and tissue parasitism. As previously described for trypomastigotes, amastigotes were not lysed when incubated in the presence of fresh guinea-pig serum. Contrasting with what has been described for trypomastigotes, the resistance of amastigotes to complement-mediated lysis persisted after treatment with trypsin.

Dynamics of transmission of Trypanosoma cruzi in a rural area of Argentina: III. Persistence of T. cruzi parasitemia among canine reservoirs in a two-year follow-up

A new cross-sectional survey of household- associated mongrel dogs as well as follow-up of previously parasitemic individuals was carried out in 1984 toy means of xenodiagnosis and serologic techniques to get a deeper insight into the relationship of T. cruzi parasitemia and age among canine hosts in a rural area of Argentina. Persistence of detectable parasitemia was age-independent, or at most, loosely related to age, confirming the pattern observed in 1982. Similarly no significant age-decreasing effect was recorded among seropositive dogs in: a) the probability of detecting parasites in a 2-year follow-up; b) their intensity of infectiousness (=infective force) for T. infestans 3rd-4th instar nymphs, as measured by the percentage of infected bugs observed in each dog xenodiagnosis. Moreover, not only was the infective force of seropositive dogs for bugs approximately constant through lifetime, but it was significantly higher than the one recorded for children in the present survey, and for human people by other researchers. Therefore, and since T. infestans field populations show high feeding frequencies on dogs, the latter are expected to make the greatest contribution to the pool of infected vectors in the rural household of Argentina. This characteristic should be sufficient to involve canine reservoirs definitely as a risk factor for human people residing in the same house. The increased severity of parasitemia observed among dogs in this survey may be related to the acute undernutrition characteristic of canine populations of poor rural areas in our country, which is expected to affect the ability of the host to manage the infection.

Trypanosoma cruzi: expression of antigenic component 5 among 35 laboratory clones obtained from 18 different isozymic variants

Two monoclonal antibodies anti-component 5 of Trypanosoma cruzi (I-35/115 and II-190/30) were tested in IFA and ELISA respectively against 35 T. cruzi laboratory clones. Among the 35 clones tested, 18 different isozyme patterns were detected. All clones were recognized by both monoclonal antibodies except one clone which did not react with II-190/30. These results support the universal expression of specific component 5 within the taxon T. cruzi.

Avaliação de antígenos do Trypanosoma Cruzi para a reação de hem aglutinação indireta: I. diferentes extratos antigênicos

Alguns procedimentos descritos na literatura (ultrasom, água destilada, NaOH, TRITON x 100 e congelamento-descongelamenao) foram avaliados determinando o melhor extrato antigênico para a reação de hemaglutinação indireta (RHI) no diagnóstico sorológico da doença de Chagas. Para isso, foram ensaiados 30 soros de indivíduos chagásicos e 30 soros de indivíduos não chagásicos. A reação de imuno-fluorescência indireta foi considerada como reação de referência no cálculo dos indices de co-positividade (i.c.p.) e co-negatividade (i.cn.). O valor do i.c.p, para a RHI com antígeno obtido por NaOH foi mais elevado do que para os outros antígenos. Os cinco antígenos apresentaram valores máximos para o i.cn., indicando boa especificdade. Os títulos apresentados pelos soros chagásicos com antígeno obtido por NaOH foram, significativamente, superiores aos demais antígenos. A avaliação de cinco partidas de antígeno extraídas por NaOH em épocas diversas indicam boa sensibilidade, especificidade e reprodutibilidade de resultados, traduzidas pelos valores elevados para os i.c.p. e i.cn., além de títulos próximos entre si.

Avaliação de antígenos do trypanosoma cruzi para a reação de hemaglutinação indireta: II. Antígenos de diferentes amostras e formas evolutivas

Para aprimorar a sensibilidade e a especificidade da reação de hemaglutina-ção indireta no diagnóstico da doença de Chagas, foram utilizados eritrocitos sensibilizados com antígeno de epimastigotas de diferentes amostras (clones Al.7, B12, Cl e cepas D150 e Y), de tripomastigotas metacíclicos de cultura (TMC) e de tri-pomastigotas de cultura de células (TCC) do Trypanosoma cruzi. Foram titulados 132 soros de indivíduos chagásicos e 161 soros de não chagásicos, diagnosticados pela reação de imunofluorescência indireta (RIFI), considerada como reação de referência no cálculo dos índices de co-positividade (i.c.p.), co-negatividade (i.c.n.) e concordância. Utilizando o título discriminante (T.D.) de 1:40, os antígenos da cepa Y e do clone B12 apresentaram valores elevados para os três índices calculados, indicando boa sensibilidade e especificidade em relação à RIFI. Os antígenos dos clones Al .7 e Cl apresentaram baixa especificidade considerando o T.D. de 1:40, mas ocorreu um aumento acentuado para o i.c.n. considerando o T.D. de 1:80, indicando um aumento de especificidade relativa sem alteração significativa da sensibilidade relativa. Com os antígenos T.M.C, e T.C.C, foram observados títulos mais baixos e valores menores para o i.c.p., indicando pouca sensibilidade em relação à RIFI.

Infecção natural de Equus caballus por Leishmania sp - São Paulo, Brasil (Breve comunicação científica)

E assinalado, pela primeira vez, o encontro de um cavalo (Equus caballus) infeclado por Leishmania sp, no Estado de São Paulo.

Lytic antibodies elicited by Trypanosoma cruzi infection recognize epitopes present on both bloodstream trypomastigote and epimastigote forms of parasite

Sera of Chaga's disease patients containing anti-T. cruzi lytic antibodies were submitted to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimasiigote or trypomasiigote forms of the parasite. Epimastigotes were obtained from culture at the exponential growth phase and the trypomastigotes from blood of infected and immunosuppressed mice. Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifugation. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was performed by passing sera from chagasic patients through an immunoadsorbent column containing either epimasiigote or trypomasiigote antigens. Antibodies bound to the column were eluted with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype and lytic activity. The results showed that anti-T. cruzi lytic antibodies present in sera from chagasic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomasiigote and epimastigote forms. A brief report of this work has already been published12.

Effect of scorpion toxin on the enterochromaffin-like cells in normal and Trypanosoma cruzi-infected rats: a morphological study

Intravenous injection of scorpion toxin (Tityus serrulatus) in normal and Trypanosoma cruzi infected rats did not cause ultrastructural morphologic changes on enterochromaffin-like (ECL) cells of the stomach, although it induced a significant increase of the gastric secretion. Our data seem to indicate that gastric ECL cells structure is not affected by stimulation with scorpion toxin or by acute infection with T. cruzi in the rat.

Differences in the antigenic profile of bloodstream and cell culture derived trypomastigotes of Trypanosoma cruzi

A comparative study of the antigenic profile of bloodstream and cell culture derived trypomastigotes showed many differences in their components. Using mouse anti-T. cruzi antibodies the differences were located mostly in the 120 kDa band, whereas using chagasic patient sera the differences were located in the 85 and 52 kDa bands. These findings might explain known physiological differences between trypomatigotes obtained from cell culture and from infected blood. A brief report of this work has already been published9.

Prevention of electrocardiographic and histopathologic alterations in the murine model of Chagas' disease by preinoculation of an attenuated Trypanosoma cruzi strain

The effects of infection with Trypanosoma cruzi on the electrocardiographic tracings of mice were studied in 4.groups of animals: (1) normal; (2) infected with a pathogenic T. cruzi strain (TS COB); (3) immunized with 3 intraperitoneal inocula of 10(6) attenuated T. cruzi epimastigotes (TCC) and (4) immunized-infected, which sequentially received the treatments of groups 3 and 2. Infection and protection were confirmed by xenodiagnosis and histopathology. Isolated alterations such as extrasystolia, 1st degree atrioventricular block, arrhythmia and ST elevation were observed in normal as well as infected mice. However, tracings taken repeatedly on each mouse over a 293 day period revealed a set of alterations which were more frequently seen in infected (14/22) than in normal (4/27) animals (p = 0.00048). These alterations consisted of supraventricular tachycardia, sinus bradycardia and persisting, first degree AV blocks, often associated to pacemaker changes. Inoculation of attenuated T. cruzi (group 3) did not increase these alterations (2/27 mice) but significantly prevented their development after challenge with the pathogenic strain (1/19 versus 14/22 mice, p = 0.000095). Thus, preimmunization reduced not only parasitemia but also a pathogenic consequence of T. cruzi infection. This evidence is relevant for immunoprevention studies against Chagas' disease.

Efecto de la reinfección sobre la evolución de ratas infectadas con Trypanosoma cruzi

El objetivo de este trabajo fue comprobar si una de las variables medio-ambientales, la reinfección, puede modificar el comportamiento observado en un modelo de rata a nivel de parasitemia, anticuerpos séricos, manifestaciones electrocardiográficas y/o lesión miocárdica. Los grupos experimentales fueron: GI: ratas infectadas al destete con 1 x 10(6) T. cruzi; GR: igual a GI más reinfecciones cada 30 días hasta los 150 días post-infección inicial (p.i.i.); GI1 ratas de 51 días infectadas; GT: testigos. Se detectó parasitemia alta en GI y GR hasta los 20 días p.i.i. tendiendo a negativizarse al día 30. En GR no se observaron parásitos despúes del primer reinóculo, resistencia que no es debida sólo a la mayor edad del huésped pués hubo parasitemia en GI1. Los xenodiagnósticos fueron negativos en los tres grupos. Los anticuerpos séricos no se modificaron significativamente en GR respecto de GI, salvo en los anticuerpos 7S, pues los del GR presentaron títulos superiores en algunos de los días estudiados. Los ECG basales no mostraron cambios distintivos en las ratas infectadas. La prueba de ajmalina mostró una disminución de la FC independiente del tratamiento; el PR, QaT y QRS se prolongaron significativamente en todos los grupos respecto del basal (p < 0.05), salvo el QaT en el GT; además, el cambio de PR y QaT fue mayor en los infectados (p < 0.05). En los grupos infectados hubo también una amplia variación en la orientación del eje eléctrico respecto del valor basal, acompañado de cambios morfológicos más manifiestos en GR. La proporción de lesión cardíaca detectada histológicamente en los grupos infectados, fue significativamente superior a la del GT (p < 0.01). Se concluye que la reinfección no reproduce el cuadro agudo inicial y no modifica el tipo y grado de lesión cardíaca observada histológicamete. La prueba de ajmalina muestra cambios electrocardiográficos compatibles con daño miocárdico incipiente en las ratas infectadas y sugieren mayor compromiso en el grupo reinfectado.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Trypanosoma cruzi: modification of alkaline phosphatase activity induced by trypomastigotes in cultured human placental villi

Human term placental villi cultured ''in vitro" were maintained with bloodstream forms of Trypanosoma cruzi during various periods of time. Two different concentrations of the parasite were employed. Controls contained no T. cruzi. The alkaline phosphatase activity was determined in placental villi by electron microscopy and its specific activity in the culture medium by biochemical methods. Results showed that the hemoflagellate produces a significant decrease in enzyme activity as shown by both ultracytochemical and specific activity studies and this activity was lower in cultures with high doses of parasites. The above results indicate that the reduction in enzyme activity coincides with the time of penetration and proliferation of T. cruzi in mammalian cells. These changes may represent an interaction between human trophoblast and T. cruzi.

Epidemiological survey of Trypanosoma cruzi infection in North-Eastern Brazil using different diagnostic methods

A survey of the prevalence of Trypanosoma cruzi infection was carried out in Oitis, a small community in the State of Piaui, Brazil. Two hundred and sixty five individuals were screened by microscopic examination, hémoculture, indirect immunofluorescence (IFA), enzyme-linked immunosorbent assay (ELISA), and competitive enzyme-linked immunosorbent assay (C-ELISA) using the monoclonal antibody TCF87 against to a 25kd T. cruzi antigen. Seropositivity was 14.3% by the IFA test, 14.7% by ELISA, and 13.2% by C-ELISA. The C-ELISA using the TCF87 monoclonal antibody seems to be applicable in serodiagnosis of Chagas' disease.

Neuronal counting and parasympathetic dysfunction in the hearts of chronically Trypanosoma cruzi - infected rats

Ten male Wistar rats, chronically infected with Colombian, São Felipe (12SF) and Y strains of Trypanosoma cruzi and ten non-infected control animals were submitted to the bradycardia responsiveness test, an assessment of heart parasympathetic function, after phenylephrine injection. Six chagasic animals showed heart parasympathetic dysfuntion characterized by reduction in the index of bradycardia baroreflex responsiveness, as compared with the control group. Microscopic examination of the atrial heart ganglia of chagasic rats showed ganglionitis, but no statiscally significant reduction in the number of neurons.