An electron microscopy study was carried out on the salivary glands of the triatomine bug Rhodnius ecuadoriensis experimentally infected with a Peruvian strain of Trypanosoma (Herpetosoma) rangeli. Epimastigotes forms of T. rangeli were found in large numbers in the haemolymph surrounding the salivary gland. Some of them were closely associated with the basal lamina of the gland epithelium, apparently in a process of cell invasion, in which the flagellum breaking through the basal lamina. Within the cytoplasm of the gland epithelial cells, there were rounded forms of T. rangeli, consisting of tightly coiled sphaeromastigotes. The gland lumen was filled with large numbers of mostly epimastigotes forms of the flagellate; only few paramastigotes and trypomastigotes forms could be unequivocally identified. The presence of paramastigotes in the life cycle of T. rangeli has yet to be fully assessed

Fué realizado un estudio ultraestructural de las glándulas salivares del triatomino Rhodnius ecuadoriensis experimentalmente infectado por el tripanosomatídeo, Trypanosoma (Herpetosoma) rangeli, con la finalidad de documentar los aspectos básicos de la infección glandular por este flagelado. Gran número de formas epimastigotes fueron encontradas en la hemolinfa que baña la parte externa de las glándulas en el hemoceloma. Algunos flagelados parecían íntimamente asociados a la membrana basal del epitelio glandular y varios de ellos parecían estar iniciando el proceso de su invasión, rompiendo la lámina basal con el flagelo. En el interior de las células glandulares observamos que, T. rangeli se presentaba en la forma de esferomastigotes, células redondeadas rodeadas externamente por un flagelo, que envolvía apretadamente el parásito. En la luz glandular también observamos gran número de epimastigotes mezclados con los productos de la secreción glandular; el material presentó apenas pocas formas paramastigotes y trypomastigotes identificadas de manera inequívoca. El significado de los paramastigotes en el ciclo evolutivo del T. rangeli aguarda futuros estudios

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, seventy one animals were inoculated with different Trypanosoma cruzi strains and routes. The rabbits were submitted to necropsy in acute (earlier than three months of infection), recent chronic (three to six months) and late chronic (later than six months) phases. Myocarditis, generally focal and endomysial, occurred in 94.1%, 66.7% and 70.8% of the infected rabbits respectively in the acute, recent chronic and late chronic phases. The myocardial inflammatory exudate was composed by mononuclear cells, and also polymorphonuclear cells in the acute phase. In most cases of the late chronic phase, the myocarditis was similar to that described in the indeterminate form of human chagasic patients. Initial fibrosis occurred in the three phases but was more severe and frequent in the early chronic. Advanced fibrosis occurred only in the late chronic phase. Tissue parasites occurred only in the acute phase. The digestive tract and skeletal muscles showed mild and occasional lesions. Our data indicate that experimentally infected chagasic rabbits repeat some lesions similar to that of humans chagasic patients, specially that of the indeterminate form. So, it may be a useful, however not an ideal, model.

Reactivation of chronic chagasic patients may occur upon use of immunosuppressive drugs related to kidney or heart transplantation or when they are affected by concomitant HIV infection. This recrudescence, however, does not occur in all chagasic patients exposed to immunosuppressive agents. We therefore investigated the influence of Trypanosoma cruzi strains in the recrudescence of the parasitism in mice at the chronic phase treated with cyclophosphamide, an immunosuppressor that blocks lymphocytes DNA synthesis and therefore controls B cells response. A large variation was detected in the percentages of newly established acute phases in the groups of mice inoculated with the different strains. We suggest that reactivation of chronic T. cruzi infections is influenced by the parasite intrinsic characteristics, a phenomenon that might occur in the human disease.

Ketoconazole an azole antifungic drug which is already in the market has also been demonstrated to be active against Trypanossoma cruzi experimental infections. In this paper we confirmed the drug effect and investigated its range of activity against different T. cruzi strains naturally resistant or susceptible to both standard drugs Nifurtimox and Benznidazole used clinically in Chagas disease. Moreover, we have shown that the association of Ketoconazole plus Lovastatin (an inhibitor of sterol synthesis), which has an antiproliferative effect against T. cruzi in vitro, failed to enhance the supressive effect of Ketoconazole displayed when administered alone to infected mice. Finally, administration in chronic chagasic patients of Ketoconazole at doses used in the treatment of deep mycosis also failed to induce cure as demonstrated by parasitological and serological tests. The strategy of identify and test drugs which are already in the market and fortuitously are active against T. cruzi has been discussed.

The author investigated the distribution of lectin receptors on Trypanosoma cruzi blood forms collected from mice inoculated with, respectively, the drug-resistant and drug-sensitive strains VL-10 and CL, and treated with the two standard active nitroheterocyclic compounds nifurtimox and benznidazole used for treatment of human Chagas' disease. Blood trypomastigotes purified in Fycoll-Hypaque were incubated with fluorescein-labelled lectins Con A, WGA, EE, WFA, TPA and PNA and then microscopically examined. Neither qualitative or quantitative differences in the fluorescence intensity could be detected between parasites from VL-10 and CL strains submitted or not to treatment. The results suggest that both strains do not differ in their surface membrane carbohydrate moieties. Moreover, the rapid clearance of blood forms the drug-sensitive strain in animals treated with singlo doses of both compounds is not likely to depend on membrane alterations expressed by changes in the carbohydrate components. furthermore, resistance or sensitivity to drugs is not apparently related to carbohydrate distribution on T. cruzi blood forms.

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

Com a finalidade de investigar o uso do coelho como modelo experimental da doença de Chagas, 72 desses animais foram inoculados por via intraperitoneal e conjuntival com formas sanguíneas, tripomastigotas metacíclicos obtidos de triatomíneos e tripomastigotas obtidos de cultura de tecido, usando-se as cepas Y, CL e Ernane do T. cruzi. E, 95,6% dos coelhos o exame de sangue a fresco foi positivo, o que evidencia uma alta suscetibilidade desses animais à injecção pelo T. cruzi. O curso da parasistemia na fase aguda da infecção é intensamente influenciado pela cepa do parasita e via de inoculação usada. Na fase crônica parasitas foram recuperados através de xenodiagnóstico e/ou hemocultura em 40% dos coelhos excaminados. A análise dos resultados dos xenodiagnósticos revelou a existência de interações seletivas entre as cepas do T. cruzi e as quatro espécies de vetores empregados, evidenciadas por significativa variabilidade dos resultados. De um modo geral os resultados obtidos no presente trabalho atendem ao pre-requisito de ordem parasitológica sugerido para um modelo para a doença de Chagas na fase crônica.

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.

A fim de obter metodologias que permitam estabelecer, com segurança, o diagnóstico "post-mortem " da infecção chagásica, adaptou-se o xenodiagnóstico artificial a necropsiados com diferentes tempos de óbito. O testefoi positivo em três (30%) de dez chagásicos autopsiados. O tempo decorrido entre o êxito letal e o início do repasto pelos triatomineos destes chagásicos foi de duas horas, duas horas e quinze minutos e sete horas, respectivamente. Discutem-se os fatores que podem explicara sobrevivência do Trypanosoma cruzi no hospedeiro morto bem como as aplicações práticas do achado.

Reliable post mortem diagnosis of Chagas' disease is highly desirable. Artificial xenodiagnosis was adapted for the human blood collected during autopsies in an attempt to reach thatgoal. The method ivas applied to ten proven chagasics. The test was positive in 3 (30%) of the cases which were autopsied 2, 2 1/4 and 7 hours after death. The survival of Trypanosoma cruzi in a dead host and the practical use of the findings are discussed.

Cães jovens foram infectados com as cepas Y e CL do T. cruzi usando-se como inóculos 107 formas sangüíneas inoculadaspor via intraperitoneal e 2 x 10³ tripomastigotas metacíclicos obtidos do inseto vetor e inoculadospor via conjuntival. As cepas Ye CL induziram nos cães curvas deparasitemia totalmente distintas, confirmando dados parasitológicos obtidos em camundongos e coelhos. Com a cepa CL a parasitemia, com ambos os inóculos, foi gradualmente ascencional ao passo que com Y a parasitemia foi extremamente baixa, irregular e, com freqüência, subpatente. Com ambas as cepas o parasitismo e as lesões predominaram no miocárdio. Entretanto, com a cepa Y a miocardite foi sempre intensa desde as fases mais precoces da infecção, ao passo que com a cepa CL o processo inflamatório tomou-se acentuado somente a partir do 20.° dia. Freqüentemente a intensidade da miocardite observada em alguns animais não guardava relação com a parasitemia; em alguns cães com parasitemia subpatente, nos quais a infecção só foi diagnosticada pelo xenodiagnóstico, a intensidade da miocardite foi comparável àquela observada nos animais com parasitemia patente. Idêntica correlação também não foi assinalada em relação ao parasitismo tissular. Esses achados sugerem a participação de mecanismo imunológicos na gênese das lesões, ainda na fase aguda da infecção.

Young dogs were inoculated with T. cruzi Y and CL strains either by intraperitoneal route with 107 blood forms or by ocular route with 2 x 10³ vector-derived trypomastigotes. With both inocula the patterns of parasitemia were significantly different, confirming data previously obtained in experimentally infected mice and rabbits. CL strain induced a gradual ascencional parasitemia whereas with Y the parasitemia was either subpatent or the number of parasites was very low, rapidly declining. Although myocarditis was present in most animals regardless of the strain used, the inflammatory process was established earlier with the Ystrain. The severity of the myocarditis was not related to parasitemia and in some animals in which parasites could only be detected by xenodiagnosis the lesions were comparable to those detected in animals with high number of blood forms. In a similar way, no correlation was established between tissue parasitism and myocardium inflammatory lesions. Thus, the heart tissue damage in the acute phase was not apparently influenced by the differences inthe course of infection, suggesting a participation of immunological mechanisms even at this early stage of infection.

A pesquisa de anticorpos líticos (AL) através do teste de lise mediada por complemento (LMCo) foi positiva em 11 líquidos pericárdicos obtidos de pacientes chagásicos falecidos subitamente ou por causas não relacionadas à doença de Chagas e que estão incluídos em um estudo da forma indeterminada dessa doença. Em todos esses pacientes a sorologia convencional para doença de Chagas apresentou resultados positivos e o quadro anátomo-patológico mostrou lesões características ou compatíveis com a cardite chagásica crônica. Em 12 líquidos pericárdicos de indivíduos que faleceram por diferentes causas e nos quais a sorologia para doença de Chagas foi negativa, a LMCo também foi negativa. A presença de AL no líquido pericárdico constitui forte evidência da presença de infecção ativa por T. cruzi nos chagásicos necropsiados e abre perspectivas para o estudo e o melhor conhecimento da patologia da doença de Chagas.

Lytic antibodies (LA) investigated by complement-mediated lysis (CoML) test were present in 11 pericardial fluids collected from chagasic patients who died suddenly or from causes not related to the disease and were included in a study of the indeterminate form of Chagas' disease. In all patients diagnostic conventional serology performed with the pericardial fluid was positive and heart lesions were characteristic or strongly suggestive of Chagas' disease myocardiopathy. In 12 pericardial fluids from non-chagasic patients who died from different reasons, the CoML test was negative. The presence of LA in the pericardial fluid is a strong evidence of active T. cruzi infection in the chagasic patients submitted to necropsy and opens perspectives for further studies and better understanding of the pathology of Chagas' disease.

No presente trabalho descreve-se um metodo que permite determinar in vivo e em curto espaço de tempo (4-6 horas) a sensibilidade de cepas de T. cruzi a agentes terapeuticos ativos na doença de Chagas. Usando-se cepas sensíveis e resistentes aos medicamentos foi possível observar uma boa correlação entre os resultados obtidos com o método rápido (que detecta atividade contra as formas circulantes do parasita) e aqueles obtidos com esquema de acao prolongada que envolve a administração da droga por 20 dias e posterior avaliação. Esse metodo pode ser usado para caracterizar a sensibilidade de cepas a drogas ativas usadas clinicamente, fornecer informações específicas sobre a ação medicamentosa em tripomastigotas sanguíneos e, eventualmente, para triagem de novos compostos.

A method is described which permits to determine in vivo an in a short period of time (4-6 hours) the sensitivity of T. cruzo strains to known active chemotherapeutic agents. By using resistant- and sensitive T. cruzi stains a fairly good correlation was observed between the results obtained with this rapid method (which detects activity against the circulating blood forms) and those obtained with long-term schedules which involve drug adminstration for at least 20 consecutive days and a prolonged period of assessment. This method may be used to characterize susceptibility to active drugs used clinically, provide infomation on the specific action against circulating trypomastigotes and screen active compounds. Differences in the natural susceptibility of Trypanosoma cruzi strains to active drugs have been already reported using different criteria, mostly demanding long-term study of the animal (Hauschka, 1949; Bock, Gonnert & Haberkorn, 1969; Brener, Costa & Chiari, 1976; Andrade & Figueira, 1977; Schlemper, 1982). In this paper we report a method which detects in 4-6 hours the effect of drugs on bloodstream forms in mice with established T. cruzi infections. The results obtained with this method show a fairly good correlation with those obtained by prolonged treatment schedules used to assess the action of drugs in experimental Chagas' disease and may be used to study the sensitivity of T. cruzi strains to active drugs.

Um estudo com microscopia eletrônica mostrou que a administração de dose única (500 mg/kg, p.o.) do composto 2-amino-5-(1-metil-5-nitro-2-imidazolil)-1 3, 4-tiadiazole induz, em camundongos inoculados com T. cruzi, lesões degenerativas das formas intracelulares do parasita. Alterações ultra estruturais são observadas 6 horas após a administração da droga e destruição ocorre em 18-36 horas. Tripomastigotas também desaparecem do sangue circulante dos animais 4-6 horas após o tratamento. O efeito em ambos os estágios evolutivos do T. cruzi é aparentemente responsável pelos efeitos in vivo desse derivado que é o mais ativo composto já testado em nosso laboratório na doença de Chagas.

An electron microscopy study shows that the administration of a single dose (500 mg/kg, p.o.) of 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1, 3, 4-thiadiazole induces in mice infected with Trypanosoma cruzi results in degenerative lesions of the intracellular stages. Ultrastructural alterations are detected as early as 6 hours after the drug administration and destruction of the parasites occurs within 18 - 36 hours. Trypomastigotes are cleared from the bloodstream 4 to 6 hours after treatment. The combined effect on both developmental stages is apparently responsible for the in vivo ejfects of this drug which is the most active drug ever tested in our laboratory in experimental Chagas' disease.

Formas de cultura de diferentes cepas do T.cruzi foram submetidas a vários processos de criopreservação. As percentagens de recuperação, avaliadas pela motilidade dos parasitas, foram consideradas como adequadas com algumas das técnicas empregadas, variando entre 60 a 80%. A estabilidade das características biológicas do material criopreservado foi investigada através do estudo das curvas de crescimento e diferenciação em meio acelular, infectividade para celulas de cultura de tecido ("Vero"), diferenciação intracelular em cultura de tecido assim como infectividade e curso da infecção em animais de laboratório. De um modo geral essas características nao foram significativamente alteradas no material congelado e estocado por diferentes períodos de tempo.

A systematic study of the cryopreservation of T. cruzi culture forms was per formed using different parasite strains and freezing methods. The recovery rates with some of the methods as evaluated by motility of the thawed parasites were fairly high (60-80%). The following aspects have been used to investigate the stability of the parasites' biological characteristics atter cryopreservation: growth and differentiation in acelular medium, infectivity to tissue culture "Vero" cells, intracellular differentiation and infectivity to animals. Those characteristics had not been significantly changed by the cryopreservation procedures.