Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.     

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.     

Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

Polyclonal B lymphocyte activation during Trypanosoma cruzi infection

Infection of A/J mice with Trypanosoma cruzi results in the polyclonal activation of B lymphocytes in vivo as assessed by the spontaneous plaque-forming cell (PFC) response to trinitrophenyl and to goat, equine, and sheep erythrocytes. The peak response to these antigens is found at 5 to 6 days of infection. Additionally, a polyclonal response to syngeneic erythrocytes can be detected in infected mice by using aged but not fresh indicator cells. Polyclonally stimulated PFC to human gamma-PFC found late in infection during a period of marked splenomegaly and parasitemia. This trypanosoma-induced polyclonal B cell activation may well be responsible for the abnormalities in immunoglobulin synthesis and secretion that have been reported to occur during human infection with T. cruzi.     

Haeme oxygenase activity protects the host against excessive cardiac inflammation during experimental Trypanosoma cruzi infection

The infection with Trypanosoma cruzi induces a robust cardiac inflammation that plays a pathogenic role in the development of Chagas heart disease. In this study, we aimed at investigating the effects of Haem Oxygenase (HO) during experimental infection by T. cruzi in BALB/c and C57BL/6 mice. HO has recently emerged as a key factor modulating the immune response in diverse models of inflammatory diseases. In mice with two different genetic backgrounds, the pharmacologic inhibition of HO activity with zinc-protoporphyrin IX (ZnPPIX) induced enhanced myocarditis and reduced parasitaemia, which was accompanied by an amplified production of nitric oxide and increased influx of CD4⁺, CD8⁺ and IFN-γ⁺ cells to the myocardium in comparison with the control group. Conversely, treatment with haemin (an activator of HO) lead to a decreased number of intracardiac CD4⁺ (but not CD8⁺) cells compared to the control group. The mechanism involved in these observations is a modulation of the induction of regulatory T cells, because the stimulation or inhibition of HO was parallelled by an enhanced or reduced frequency of regulatory T cells, respectively. Hence, HO may be involved in the regulation of heart tissue inflammation and could be a potential target in conceiving future therapeutic approaches for Chagas disease.     

Endogenous IFN-gamma is required for resistance to acute Trypanosoma cruzi infection in mice

In order to study the role of endogenous IFN-gamma in Trypanosoma cruzi infection in mice, a potent murine IFN-gamma-specific mAb was injected i.p. on days -1, 7, and 14, relative to infection. Irrespective of the parasite inocula (100 or 25,000), groups of antibody-treated mice had significantly greater cumulative mortality rates than did appropriate controls. In antibody-treated mice, mean survival times were also significantly shorter, and maximum mean parasitemia levels were significantly higher, than in controls. Moreover, the number of amastigote nests in tissues was higher than in control mice and attained a maximum at the same time as parasitemia. As evident from kinetic studies of neutralizing activity, injected mAb were rapidly consumed in infected, but not in noninfected, mice, which is suggestive of massive IFN-gamma production during the early parasitemic phase of the disease. Nevertheless, IFN-gamma remained undetectable in the sera of infected but untreated mice. Unexpectedly, however, a peak of IFN-like antiviral activity, characterizable as a mixture of IFN-gamma and IFN-beta, appeared in mAb-treated mice that survived to infection at a time when neutralizing activity of injected mAb had drastically decreased in the circulation. We hypothesize that this high level of artificially induced endogenous IFN-gamma, not neutralized by the amounts of injected mAb, was due to the more intense parasite multiplication occurring in mAb-treated mice, which in turn may have induced an increased amount of various cytokines. TNF-alpha was not found in the serum of our mice. The humoral immune response entered its exponential phase at a time point later than that when protection by endogenous IFN-gamma was evident. Treatment with IFN-gamma-specific antibody, as applied in our study, failed to affect the level of different Ig isotypes or of T. cruzi-specific antibodies. Our study clearly indicates that IFN-gamma is produced early in acute T. cruzi infection and exerts a protective effect that is probably independent from the humoral immune response.     

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.     

The innate immune response to Trypanosoma cruzi infection

Trypanosoma cruzi infection is a major public health problem in Latin America. The host innate immune system plays a pivotal role in the recognition of T. cruzi infection and the subsequent development of adaptive immunity. In this review, we focus on the TLR-dependent and -independent innate immune responses to T. cruzi.     

Trypanosoma cruzi: cross-reactive anti-heart autoantibodies produced during infection in mice

Preimmunization with attenuated Corpus Christi stain Trypanosoma cruzi provides survival to C3H mice and enhances resistance of C57 mice to Brazil strain infection. C3H(He) and C57 B1/6 mice surviving acute infection of T. cruzi are shown to have heart specific autoantibodies through acute and chronic infection. ELISA assays were performed using nondenatured extract of hearts from normal syngeneic mice as target antigen reacted with sera from immunized and/or infected mice. Surviving C3H mice developed a specific anti-heart response as early as Day 21 of infection and this response continued at a high level to Day 300. The response in C57 mice, both immunized-infected and infected only, increased to Day 100 followed by a decline in intensity. The heart specificity of the response in mice was suggested by negligible reaction of sera with smooth muscle preparations and a reduced autoreactivity with skeletal muscle. Laminin, a suggested target of autoimmunity in Chagas' disease, was shown not to be the target of the responses in these mice. Immunoaffinity-purified heart specific antibodies show strong cross-reactivity with parasite antigen and like purified parasite specific antibodies, reacted with heart antigen.     

Trypanosoma cruzi: T-cell-dependent mechanisms of resistance during chronic infection

Effector mechanisms of resistance exerted by T cells from BALB/c mice chronically infected with Trypanosoma cruzi, Tulahuén strain, were studied. Spleen cells from chronically infected mice (Chro-SC) prestimulated with heat-killed trypomastigotes (HKT) and/or IL-2 destroyed PHA-labeled p-815 mastocytoma cells, HKT-pulsed macrophages, and normal peritoneal macrophages. However, HKT-stimulated Chro-SC did not affect the infectivity of free bloodstream forms of the parasite. Upon HKT stimulation, Chro-SC or their culture supernatant activated peritoneal macrophages for the destruction of intracellular amastigotes. The effect was abolished after Thy 1.2+ cell depletion. The addition of Cyclosporin A (CyA), which blocks T-cell activation, during HKT-stimulation of Chro-SC, diminished their ability to activate the trypanocidal activity of macrophages. CyA also inhibited the production of both macrophage-activating factors and interferon-gamma by HKT-stimulated Chro-SC. CyA administration to recipients of nylon-wool nonadherent spleen cells from chronically infected mice inhibited their adoptively acquired resistance against T. cruzi, suggesting that the conferred resistance depended on the effect of specifically activated cells. When administered during the chronic stage of the infection, CyA abrogated the antigen-specific delayed type hypersensitivity response but increased the levels of anti-T. cruzi IgG antibodies. Neither parasitemia, tissular parasitism in myocardium or skeletal muscle, nor mortality were detected after CyA treatment, suggesting the presence of a CyA nonsensitive mechanism(s) in the control of T. cruzi during the chronic phase of the infection.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.     

Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes

A rabbit antibody to the neuraminidase of the infective form of Trypanosoma cruzi identifies a subpopulation of trypomastigotes that expresses neuraminidase. Complement-mediated lysis by the antibody selectively destroys 30 to 40% of the trypomastigotes, supporting the conclusion that the immune antibody binds to a subset of parasites. The trypomastigotes that react with the immune antibody are the only ones expressing neuraminidase because the trypomastigotes that survive complement-mediated lysis are depleted of neuraminidase activity. The enzyme seems to negatively modulate infection in vitro, since infection of host cells by trypomastigotes is enhanced when neuraminidase activity is blocked by antineuraminidase antibody; infection is also enhanced when the infecting trypomastigotes have been depleted of parasites that express neuraminidase. Addition of exogenous neuraminidase (from Vibrio cholerae) to trypomastigotes treated with immune antibody, reverts the enhancement observed when infection takes place in the presence of antibody to T. cruzi neuraminidase only. Addition of V. cholerae neuraminidase in the absence of immune antibodies has no effect on infection. These results show that T. cruzi neuraminidase depresses infection and also suggest that sialic acid is involved in the parasite-host cell interaction. The antibody to T. cruzi neuraminidase recognizes on the surface of live trypomastigotes a set of proteins with high m.w. (165,000 to 200,000) and also two antigens of 79,000 to 82,000. The high m.w. proteins appear to be associated with neuraminidase activity as shown by renaturation experiments of released enzyme fractionated on a sodium dodecyl sulfate-polyacrylamide gel.     

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.     

Targeted screening strategies to detect Trypanosoma cruzi infection in children

Background

Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy.

Methods and Findings

We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child''s risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child''s house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children.

Conclusions

We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings.     

Regulation of immunity in Trypanosoma cruzi infection

Immunity to T. cruzi is complex, involving among other components, antibody production, CD4+ helper cells, CD8+ T cells as both regulators and effectors of immunity, and possibly, double-negative T cells. In addition, several of these components have been implicated in pathogenesis in the chronic infection. Although the immunosuppression observed in the infection seems quite severe, it also appears to provide for a sufficient level of immune responsiveness to control the infection in most hosts. At the same time, immunosuppression may provide the regulatory control necessary to prevent massive chronic pathogenesis in all hosts. Continued study of the relative roles of lymphocyte populations and the products they secrete in immunity and pathogenesis may provide the understanding necessary to enhance immunity to T. cruzi without the feared consequence of increased pathogenesis.     

Cell-mediated immunity in experimental Trypanosoma cruzi infection

Infection of humans with the protozoan Trypanosoma cruzi leads to Chagas disease, or American trypanosomiasis, a disease that affects nearly 20 million people, and constitutes one of the largest socioeconomic burdens in Latin America. Much of the present knowledge on pathogenic mechanisms underlying T. cruzi infection comes from experimental murine models. Here, George A. DosReis reviews recent findings about the features of host cell-mediated immunity against the parasite and possible mechanisms leading to chronic infection.     

Trypanosoma cruzi: productive infection is not allowed by chorionic villous explant from normal human placenta in vitro

We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished. Human placental chorionic villi and VERO cells as controls were co-cultured with T. cruzi. Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32%. There were no live parasites in placenta-T. cruzi culture media at 72 h of co-culture. There were significantly increased dead parasites when T. cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed. CONCLUSION: Low productive infection by T. cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances.