ABSTRACT INTRODUCTION: Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. METHODS: For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). RESULTS: The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). CONCLUSIONS: The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient’ responses to therapeutic treatment or disease clinical manifestations.

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15% fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5%). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occurred at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2%). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process

On few occasions, Phytomonas davidi (McGhee & Postell isolate) cultures in LIT (liver infusion-tryptose) medium around 27oC presented, as seen in Giemsa-stained smears, a set of peculiar morphological features, among them being noticeable the pairs of apposed cells attached by their posterior ends, where occurred a stained line and/or a dilatation, usually bulb-like in shape; sometimes this dilatation could occupy one of the cells or hold both together. In some pairs, the nucleus of each parasite seemed migrating towards the other, entering into such dilatation; in others, both nuclei were inside it, sometimes in close proximity or seeming fused; peculiar chromatin arrangements involving both nuclei were occasionally observed. Several mono or binucleate round forms bearing one or two flagella, as well as flagellate slender cells without nucleus were concomitantly seen there. In some instances, an intriguing small stained body occurred beside a single large nucleus, either in pairs presenting the bulb-like structure or in round cells. These cytological findings seemed steps of a dynamic process suggesting sexuality, since in several of them nuclear interactions following fusion of two parasites appeared to occur

Através deste trabalho fizemos uma revisão do parasitismo tecidual da cepa Y do Trypanosoma cruzi em camundongos albinos (Swiss-Webster). Esta cepa parasitou preferencialmente as fibras musculares lisas, esqueléticas e cardíacas, sendo baixo e transitório seu parasitismo do baço e fígado, conforme já observado por alguns Autores, embora diferindo de outros achados. Estes resultados podem estar relacionados com o padrão genético do hospedeiro e/ou com o grau de virulência da cepa por ocasião deste estudo. Além do mais, discutimos a possibilidade de que o intenso macrofagotropismo descrito para esta cepa em algumas ocasiões possa ser uma condição artificialmente induzida através de sua manutenção seriada em camundongos por tempo prolongado e/ou pelo uso de animais jovens. Também são dignos de nota, o intenso parasitismo e inflamação da bexiga, pâncreas e canal espermático de alguns animais inoculados, assim como, o encontro de ninhos de amastigotas no testículo.

A review of the tissular parasitism of Trypanosoma cruzi Y strain in Swiss mice was carried out. This strain parasitized preferentially smooth, skeletal and cardiac muscle fibers, with low transitory spleen and liver parasitism, as previously found by some Authors, although differing from other reports. These results can be related to the host genetical constitution and/or the degree of the strain virulence at the time of this study. Furthermore, we discuss that the high macrophagotropism reported for this strain in some instances could be an artificially induced condition resulting from its serial maintenance in mice, either for a longer time and/or by using young animals. The heavy parasitism and inflammation observed in the bladder, pancreas and spermatic duct of some inoculated mice, as well as the testis parasitization, were also noteworthy findings.

Tripomastigotas sanguíneos de algumas cepas de Trypanosoma cruzi foram processadas em colunas de DEAE-celulose sob condições padronizadas. Os resultados obtidos sugerem principalmente que estas cepas possuem cargas superficiais diferentes, que em relação a este aspecto existem subpopulações de tripomastigotas e que as formas largas são menos negativas do que as finas.

Bloodstream trypomastigotes of some Trypanosoma cruzi strains were processed through DEAE-cellulose columns under standardized conditions. The results obtained suggest mainly that these strains present different surface charges, that there are subpopulations of bloodstream trypomastigotes as regards electrical charges and that the broad forms are less negative than the slender ones.

Usando colunas de DEAE-cellulose foi padronizado um método para purificação de tripomastigotas de várias cepas de Trypanosoma cruzi (Y, CL, FL, F, "Berenice", "Columbiana" e "São Felipe") a partir do sangue de camundongos. Este método é uma modificação daqueles descritos por Lanham & Godfrey e difere em vários aspectos de outros descritos para purificar as formas sanguíneas deste parasita, particularmente na dispensa de pré-purificações. Por ele foram isolados principalmente os tripomastigotas largos, oque certamente justifica as maiores percentagens de recuperação obtidas com cepas em que predominavam estas formas; por outro lado, o processamento de sangue infectado de camundongos irradiados podia ser vantajoso pela recuperação de maior número e percentagem de parasitas e eluição de mais formas finas. Os tripomastigotas purificados por este método apresentavam morfologia e motilidade normais e continuavam infectantes para barbeiros e camundongos. Nestes últimos apresentaram período pré-patente e curvas parasitêmicas semelhantes aos dos parasitas controles, além de reproduzirem o padrão de polimorfismo típico da cepa. Sua virulência e patogenicidade também manteve-se consideravelmente preservada, sendo que esta última propriedade foi avaliada por testes de DL 50, taxas de mortalidade e tempo médio de sobrevida de camundongos inoculados. Além do masi, os tripomastigotas purificados apresentaram reação de imunofluorescência positiva, nítida e periférica, cujos títulos eram comparáveis aos dos parasitas controles, assim sugerindo uma importante preservação de seus antígenos de superfície.

A method to purify trypanosomastigotes of some strains of Trypanosoma cruzi (Y, CL, FL, F, "Berenice", "Colombiana" and "São Felipe") from mouse blood by using DEAE-cellulose columns was standardized. This procedure is a modification of the Lanham & Godfrey methods and differs in some aspects from others described to purify T. cruzi bloodstream trypomastigotes, mainly by avoidance of prior purifications of parasites. By this method, the broad trypomastigotes were mainly isolated, accounting for higher recoveries obtained with strains having higher percentages of these forms: processing of infected blood from irradiated mice could be advantageous by increasing the recovery of parasites (percentage and/or total number) and elution of more slender trypomastigotes. Trypomastigotes purified by this method presented normal morphology and motility, remained infective to triatomine bugs and mice, showing in the latter prepatent periods and courses parasitemia similar to those of control parasites, and also reproducing the polymorphism pattern of each strain. Their virulence and pathogenicity also remained considerably preserved, the latter property being evaluated by LD 50 tests, mortality rates and mean survival time of inoculated mice. Moreover, these parasites presented positive, clear and peripheral immunofluorescence reaction at titres similar to those of control organisms, thus suggesting important preservation of their surface antigens.

Toddia França, 1912 pela microscopia ótica apresenta-se como corpúsculos de inclusão no citoplasma de hemácias de animais pecilotérmicos, ás vezes acompanhados de corpos cristalóides. Sua relação com os protozoários ou os vírus tem sido discutida por alguns autores, mas não estava ainda esclarecida. Visando a elucidar este problema, fizemos a microscopia eletrônica de cortes ultrafinos do sangue de uma rã (Leptodactylus ocellatus) altamente infectada com Toddia. No citoplasma das células parasitadas não encontramos qualquer protozoário, mas partículas semelhantes a vírus, as quais eram geralmente hexagonais, mediam em média 195 nm de diâmetro não considerando suas duas membranas envolventes, e possuíam uma área central de densidade eletrônica variável. Partículas em diferentes estádios de desenvolvimento foram geralmente encontradas em volta ou sobre uma área menos densa que o citoplasma, a qual parecia um local para formação de vírus. Os cristais nucleares ou citoplasmáticos que acompanhavam os corpúsculos de Toddia pela microscopia eletrônica revelaram estrutura semelhante àquela observada nos corpos de inclusão associados com os vírus da poliedrose e poxvírus de insetos, nas cápsulas dos vírus da granulose e em outros cristais protéicos em cortes ultrafinos. Testes citoquímicos dos corpúsculos de Toddia demonstraram exclusivamente a presença de ácido desoxirribonucléico. Estas observações indicam que Toddia não é um protozoário e demonstram, com toda probabilidade, que ela é um corpúsculo de inclusão virótico. Levando em consideração o tipo de ácido nicléico encontrado em sua estrutura (ADN) e a forma hexagonal geralmente apresentada em cortes ultrafinos pelas suas partículas componentes, as quais, além disto, são formadas e reunidas no citoplasma, relacionadas Toddia com os chamados "Desoxirribovírus Icosaédricos Citoplasmáticas". Acreditamos que o presente trabalho dá a primeira referência a partículas semelhantes a vírus em L. ocellatus.

Toddia França, 1912 under the light microscope occurs as inclusion corpuscles in the cytoplasm of erythrocytes of cold-blooded vertebrates sometimes accompanied by crystalloid bodies. Its position among the protozoans or the viruses has been discussed by some authors, but remained unclear. To elucidate this problem we studied Toddia from a Brazilian frog (Leptodactylus ocellatus) by electron microscopy. In the cytoplasm of the infected cells we found no protozoan, but rather virus-like particles often hexagonal in outline, averaging 195 nm excluding their two involving membranes, and presenting a central area of variable electron density. Particles at different stages of development were generally found around or on area lighter density than the cytoplasm. which resembled a virus synthesis site. At high magnification, the nuclear or cytoplasmic crystals allied to Toddia resembled the crystalline lattice of the inclusion bodies associated with the polyhedrosis viruses and poxviruses from insects, of the capsules of granulosis viruses and of other protein crystals in ultrathin sections. Cytochemical tests in Toddia corpuscles displayed exclusively the presence of deoxyribonucleic acid. These findings indicate that Toddia is not a protozoan and demonstrate that it is in all probability a viral inclusion corpuscle. Taking into account the nucleic acid type found in its structure (DNA) and the hexagonal shape usually shown in ultrathin sections by its component particles, which have a cytoplasmic site of synthesis and assembly, we tentatively relate Toddia with the so-called "Icosahedral Cytoplasmic Deoxyriboviruses". We believe that the present paper gives the first report of virus-like particles in L. ocellatus.

Com o objetivo de fornecer dados para o esclarecimento do controvertido problema da natureza de Cytamoeba bacterifera freqüentemente encontrada nos eritrócitos de Leptodactylus ocellatus, realizamos alguns testes citoquímicos. Demonstramos a presença do ácido ribonucleico e polissacarídeos não digeríveis pela ptialina em sua estrutura. Com o método de Feulgen, teste de referência para a caracterização do ácido desoxirribonucleico, obtivemos principlemnte resultados negativos; porém, um parasito com fraca e difusa positividade e algumas reações duvidosas também foram encontrados. Ao emrpegarmos o Verde Metila-Pironina, mesmo após o tratamento pela ribonuclease, e o Azul de Toluidina, também depois da ação desta enzima, não conseguimos confimar a presença de ADN. Como os elementos constituintes de C. bacterifera são minúsculos e, às vezes, não evidenciáveis, é possível que seu teor de ADN, porventura existente, seja muito pequeno e, conseqüentemente, de difícil demonstração por métodos cujos resultados são observados sob microscopia ótica, além de poder ficar facilmente encoberto por outras substâncias. Não estamos propensos a admitir uma provável natureza virótica para Cytamoeba baseados, principalmente, em alguns de seus aspectos estruturais (figs. 8, 15, 17 e 18) e na ausência de alteração no núcleo das células parasitadas. Apesar de não termos comprovado a presença de ADN, achamos possível que C. bacterifera seja um aglomerado intracitoplasmático de organismos modificados, cujas dimensões situam-se nas proximidades do limite de resolução do microscópio ótico, relacionados com as bactérias, assim como são, por exemplo, os Clamídios e as Riquétsias. Observamos o desenvolvimento de Cytamoeba em rã mantida em cativeiro por três meses e semanalmente examinada; constatamos decréscimo paulatino da parasitemia inicial e também que os seus tipos estruturais e medidas não estavam relacionadas com a etapa da infecção. Não conseguimos transmitir, por inoculação intra-peritoneal, Cytamoeba de L. ocellatus para Bufo crucifer.

In view to furnish data to elucita the controversial problem of the nature of Cytamoeba bacterifera, which it is often found in erythrocytes of the frog Leptodactylus ocellatus, we have done some cytochemical tests. We displayd in its structure the contents of RNA and polysaccharides that do not undergo digestion by ptyalin. With the Feulgen's method we mainly obtained negative results, but one parasite showed a faintly diffused positivity and a few doubtful reaction were also observed. When we employed the Methyl Green-Pyronin, even after the ribonuclease treatment, and the Toluidin Blue, likewise subsequent to the activity of this enzime, we did not evidence the DNA. Taking into account that the components of C. bacterifere are usually very small and sometimes undiscernible, it is probable that their contents of DNA, perhaps existent, would be difficultly demonstrated under the light microscope, and also that it would be easily dissimulated by other substances. We do not believe in a viral nature of Cytamoeba mainly regarding some aspects of its structure (figs. 8, 15, 17 and 18) and the absence of damage on the nucleous of the infected cells. Although we did not prove the presence of DNA, we think that it is possible that C. bacterifera is an intracytoplasmic clump of modified organisms wich sizes are just within the nearness of th limit of visibility with optical microscopes, related with the bacteria, such as are, by exemple, the Chlamydiae and Rickettsiae. The development of Cytamoeba was followed in a frog maintained int he laboratory during three months and weekly examined; we observed progressive decrease of the parasitismo. Its structural types and sizes were not related with the phasis of the infection. We did not get transmission by intraperitonela inoculation of C. bacterifera from L. ocellatus to Bufo crucifer.

A ocorrência de hemagregarinas no sangue dos sapos que examinamos (Bufo crucifer, B. marinus e Melanophryniscus moreirae) revelou-se extremamente rara; apenas um exemplar de B. crucifer procedente de Manguinhos (Rio de Janeiro, Guanabara) apresentou-se parasitado. As formas sanguíneas encontradas eram intra-eritrocitárias, medindo em média 10,8/ 3,6µ; não as identificamos como trofozoítos ou gametócitos e discutimos este fato. As hemácias parasitadas tinham dimensões normais, porém podiam ter o núcleo deslocado. Supomos ser esta a primeira referência a hemogregarinas neste hospedeiro. Não encontramos identidade entre as formas que descrevemos e aquelas referidas em outras espécies de sapos; fornecemos uma lista das hemogregarinas achadas nestes hospedeiros. Denominamos provisoriamente os parasitos que encontramos Haemogregarina "sensu lato", até que novos dados sobre seu ciclo evolutivo sejam conhecidos.

The occurrence of hemogregarines in the blood of Brazilian toads that we have examined (Bufo crucifer, B. Marinus and Melanophryniscus moreirae) has displayed very scarce; only one specimen of B. crucifer from Manguinhos (Rio de Janeiro, Guanabara) was infected. The blood forms that we found were intraerythrocytic, averaging 10,8 by 3,6µ; we did not differenciate them as thophozoites or as gametocytes and we discuss this fact. The infected erythrocytes were not hypertrophied and their nucleus was normal and sometimes displaced. We think that it is the first reference to hemogregarines in this host. We did not evidence identity among the forms that we described and those reported in other species of toads; we give a list of hemogregarines found in those hosts. We provisorily named the parasites that we found Haemogregarina "sensu lato", until new data about their life cycle become known.

Dentre 683 serpentes procedentes de diferentes pontos do Brasil, 16 (2,4%) apresentaram em seu sangue (em glóbulos vermelhos maturos e imaturos) Toddia ou Pirhemocyton. Denominamos provisoriamente Toddia os parasitos encontrados em Bothrops moojeni, B. pradoi, B. jararaca e Chironius flavolineatus e Pirhemocyton os encontrados em B. alternatus; fornecemos a primeira referência nestes hospedeiros. Estes parasitos foram estudados comparativamente com o objetivo de estabelecermos a extensão de suas afinidades. Com Toddia de B. moojeni realizamos técnica citoquímica para caracterização de ácidos nucleicos e encontramos positividade apenas para DNA. As maiores afinidades entre os parasitos estudados residiam nas características dos corpúsculos cromáticos e alterações celulares que determinam; observarmos quase total identidade nas maneiras como os parasitos da mesma espécie de serpente se apresentaram. Constatamos a coexistência de inclusões globóides, cristalóides e formas intermediárias associada aos parasitos de B. pradoi e B. alternatus; em C. flavolineatus encotnramos além dos cristais, raríssimas formas intermediárias entre estes e globóides. Afinidades entre Toddia e Pirhemocyton abrangendo o tipo de inclusão contrariam as descrições originais, tornando pouco válida esta separação genérica. Discutimos também os fundamentos da criação das espécies e a possível natureza virótica destes parasitos.

Among 683 snakes from several localities of Brazil, 16 (2,4%) presented in their blood (in mature or immature red cells) Toddia or Pirhemocyton. We named provisorily Toddia the parasites found in Bothrops moojeni, B. pradoi, B. jararaca and Chironius flavolineatus and Pirhemocyton those found in B. alternatus; we give the first reference in these hosts. These parasites were comparatively studied in view to establish the range of their relationship. With Toddia from B. moojeni we attempted nucleic acids by cytochemical methods and we found only DNA. The most affinities among the parasite that we have studied remained on the characters of the chromatic corpuscles and the cellular changes determined by them; we observed almost total identity among the parasites of same species of snakes. We observed the coexistence of globoides, crystalloids and intermediate forms between them allied to the parasites of B. pradoi and B. alternatus; in C. flavolineatus we found younder cristals, scarce intermediary forms between these and globoids. Affinities between Toddia and Pirhemocyton including the type of inclusion oppose to the original descriptions, turning doubtfull this generic separation. We also discuss the basis of the establishment of the species and the possible viral nature of these parasites.