The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5610 6 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+) CD8(+), CD69(+) CD4(+) and CD25(+) CD122(+) CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Resistance of mice immunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.

Antigen characterization of vector-borne and cultured metacyclic trypomastigotes of Trypanosoma cruzi

Metacyclic trypomastigotes ol the CL strain of Trypanosoma cruzi obtained from triatomid vectors and from axenic cultures were comparatively analysed as to their antigen make-up and immunogenic characteristics. They were found to be similar by the various parameters examined. Thus, sera of mice immunized with either one of the two metacyclic types precipitated a 82Kd surface protein from 131I-labeled culture metacyclics. Sera of mice protected against acute T. cruzi infection by immunization with killed culture metacyclics of a different strain (G) recognized, by immunoblotting, a 77Kd protein in both types of CL strain metacyclics. A monoclonal antibody raised against G strain metacyclics, and specific for metacyclic stages of this strain, reacted with both CL strain metacyclic types. Both metacyclic forms were similarly Iysed by various anti-T. cruzi sera, in a complement-mediated reaction.

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.

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.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Trypanosoma cruzi-like bloodstream trypomastigotes in bats from the State of Piauí, Northeastern Brazil

One-hundred and thirty-five bats from 12 species were examined for thepresence of trypomastigotes by means of direct blood examination, xenodiagnosis, and hemoculture. Of those, 44 specimens (32.6%) from 8 species were infected with trypanosomes. Phyllostomus discolor discolor presented the highest rate of infection, being captured only in one locality, while Phyllostomus hastatus hastatus captured in four localities showed high rates. Two species, Anoura geoffroyii and Pteronotus (Phillodia) pamelli rubiginosa, were found infected by T. cruzi-like trypanosomes, apparently described for thefirst time. Flagellates from Artibeus jamaisensis jamaisensisa and A. geoffroyii were able toproduce detectable parasitaemia in young mice. One triatomine bug was found infected in natural conditions, Triatoma brasiliensis was associated with a P.h. hastatus colony, in which six captured bats were also found infected.

Comparison of Trypanosoma cruzi infection in dogs inoculated with blood or metacyclic trypomastigotes of Berenice-62 and Berenice-78 strains via intraperitoneal and conjunctival routes

This paper aimed to verify the influence of the inoculum source (blood or metacyclic trypomastigote) and the route of inoculation (intraperitoneal or conjunctival) on the course of T. cruzi infection in dogs, using comparatively the T. cruzi strains Berenice-62 and Berenice-78. All dogs inoculated intraperitoneally became infected independently of the T. cruzi strain and source of trypomastigotes used. High level of infectivity was also observed when metacyclic trypomastigotes of both strains were inoculated by conjunctival route. However, when blood trypomastigotes were inoculated by conjunctival route the percentages of infectivity were significantly lower in dogs inoculated with both strains. Parasitaemia was significantly higher in animals infected with metacyclic trypomastigotes via the conjunctival route independently of the T. cruzi strain used. All animals infected with Berenice-78 strain showed severe acute myocarditis. On the other hand, animals infected with Berenice-62 showed severe acute myocarditis only when infected with metacyclic trypomastigote, via the intraperitoneal route. The results suggest that the source of the inoculum and the route of inoculation remarkably influence the evolution of the infection for the T. cruzi in the vertebrate host even when the same strain of the parasite is used.

A simple method to purify biologically and antigenically preserved bloodstream trypomastigotes of Trypanosoma cruzi using Deae-cellulose columns

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.

Surface electrical charge of bloodstream trypomastigotes of Trypanosoma cruzi strains

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.

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi

Polymorphism in Trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense , although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.