Fibronectin enhances macrophage association with invasive forms of Trypanosoma cruzi

Treatment of either mouse peritoneal macrophages (MPH) or invasive blood forms of Trypanosoma cruzi with human plasma fibronectin (FN) significantly enhanced their association (a term to mean surface attachment and parasite internalization) with the untreated counterpart in a dose-dependent manner. This effect involved increases in the percentage of MPH that associated with the parasites and in the number of parasites per MPH. By using indirect immunofluorescence, the percentages of FN-positive MPH and FN-positive parasites found in preparations of these cells were 26 and 13%, respectively, and increased to 70 and 73%, respectively, after incubation with FN for 60 min and multiple washings. These results demonstrated the presence of FN itself and FN-binding sites on the surface of MPH and T. cruzi. Incubation of FN-treated MPH and FN-treated parasites with gelatin, for which FN has a binding site, significantly reduced the stimulatory effect of FN. A reduction was also seen when FN-treated MPH were incubated with anti-FN antibody before adding the parasites. These observations suggested that FN might enhance association by bridging the interacting cells. The presence of excess soluble FN during MPH-parasite interaction also inhibited the association, possibly by blocking FN receptors on the MPH and parasite surfaces. Pretreatment of the MPH with FN enhanced the capacity of these cells to associate with either untreated latex beads or killed T. cruzi. These findings indicated, on the one hand, that the FN-mediated enhancement was not unique to living T. cruzi and, on the other, that this enhancement was not likely due to an FN-induced alteration of the MPH membrane that would render it more susceptible to active penetration by the parasites. Taken together, these results suggest that FN, produced by MPH, may play a role in infection of this cell type by T. cruzi.     

Stimulatory effects of retinoic acid on macrophage interaction with blood forms of Trypanosoma cruzi: involvement of transglutaminase activity

The effects of retinoic acid (RA; vitamin A acid) on macrophage function were investigated by measuring the capacities of mouse peritoneal macrophages to associate with (i.e., bind and internalize) and kill the unicellular parasite Trypanosoma cruzi. The presence of 10(-8) to 10(-6) M RA in co-cultures of macrophages and blood forms of the parasite markedly increased their interaction as evidenced by significant increases in both the percentage of phagocytes associating with parasites and the average number of parasites per 100 cells. A similar effect was produced when either the macrophages or the trypanosomes were pretreated with RA, suggesting that both cell types could contribute to the noted effect. Although RA might have enhanced parasite-macrophage association by binding to both, its ability to stimulate phagocytosis was independently evidenced by a significant increase in the uptake of latex particles. RA-treated macrophages also took up larger numbers of dead T cruzi, denoting that parasite viability (i.e., infectivity) was not necessary for the production of the RA effect. The minimum pretreatment time for RA to significantly stimulate macrophage association with T. cruzi was 30 min, although a 45-min pretreatment was necessary for a maximal effect to be seen under our experimental conditions. The RA effect was reversible because, once optimally induced, it remained demonstrable for only 30 to 60 min after removal of the reagent; however, the effect persisted for at least 3 hr if RA was not removed. Transglutaminase activity appeared to be involved in the RA effect, because the latter was abrogated when the macrophages were treated with RA in the presence of cystamine, methylamine, or monodansylcadaverine, all of which inhibit transglutaminase activity by different mechanisms. RA also increased the capacity of macrophages to kill parasites internalized before the treatment. This cytotoxic capacity was inhibited by catalase, indicating that H2O2 played a role in the killing mechanism. RA treatment significantly increased the proportion of macrophages capable of reducing nitroblue tetrazolium. The present results indicated that RA was capable of activating macrophages, leading to greater uptake and killing of a protozoan parasite.     

Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane

The presence of phospholipase A2 (PLA2) significantly increased the association between Trypanosoma cruzi and macrophages. This effect reflected alterations to the parasite membrane since it was reproduced only when the parasite but not the macrophage was pretreated with PLA2. That PLA2 activity was responsible for the noted enhancement was indicated by the ability of the specific substrate phosphatidylcholine to block it. The presence of the PLA2 inhibitors quinacrine, 4-bromophenacyl bromide or phentermine markedly inhibited parasite-macrophage association. Quinacrine also inhibited association of the parasite with a non-phagocytic host cell. These results suggested a role for endogenous PLA2 in the initial stages of cell infection by T. cruzi.     

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.     

Differential effect of culture epimastigotes and blood-form trypomastigotes on normal mouse splenocyte responsiveness to mitogens

Blood form trypomastigotes of the Y strain of T. cruzi, produced a strong inhibition of the blastogenic response to T and B cell mitogens, of the C3H/He, C57BL/6 and BALB/cJ strains of mice, while culture epimastigotes of the Y strain kept in a medium that allows parasite growth at 26 degrees, 30 degrees, 34 degrees and 37 degrees C produced a strong stimulatory effect that was even higher than the effect of the mitogens alone. Both the inhibitory or the stimulatory effects were dose-dependent. The stimulatory effect of epimastigotes was also temperature-dependent producing increased stimulation indexes as the temperature of parasite cultures was raised. Metabolically active, living parasites seemed to be necessary for an improved lymphocyte stimulation suggesting a potential role of secreted metabolites as polyclonal activators of mouse lymphocytes.     

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.     

Does concanavalin A treatment of host cells enhance or inhibit their association with Trypanosoma cruzi trypomastigotes?

Concanavalin A (Con A) has frequently been used as a probe of cell surface molecules that mediate cell-cell interactions. There have been conflicting reports that Con A treatment of vertebrate host cells can subsequently increase or reduce the level of association (surface attachment and penetration) of Trypanosoma cruzi trypomastigotes with these cells. In this work, we have established that the type of effect produced by treatment of host cells with Con A depended on whether or not fetal bovine serum was present during the interaction of trypomastigotes and host cells; Con A treatment reduced host cell association with T. cruzi in the presence of the serum, but increased it when the serum was absent. In addition, ovalbumin, a glycoprotein with a high mannose content and the ability to specifically bind to Con A, was found capable of altering the effect of Con A treatment of host cells on parasite association levels in a manner similar to fetal bovine serum. These results suggested that glycoproteins present in the serum can modulate the effect of Con A, possibly by blocking free sites remaining on the Con A molecules which had bound to the surface of host cells. If free binding sites on the Con A molecule remained unblocked, they could conceivably form bridges between host cells and parasites resulting in an artifactual enhancement of their level of association in serum-free medium.     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

Trypanosoma cruzi: cytokine effects on macrophage trypanocidal activity

Mouse macrophages infected with Trypanosoma cruzi in vitro may be activated to reduce parasite infection by interferon gamma (IFN-gamma). The addition of up to 10,000 units of IFN-gamma however, does not result in a 100% reduction of intracellular parasites. We, therefore, investigated the possibility that macrophages require an additional signal or signals to completely clear T. cruzi infection. Because the combination of IFN-gamma with lipopolysaccharide greatly enhanced macrophages ability to decrease the number of intracellular parasites, the interaction of IFN-gamma with tumor necrosis factor (TNF) was examined. TNF alone and the combination of TNF with IFN-gamma did not have a significant effect on reducing parasite numbers below that obtained with IFN-gamma alone. This was also true for lymphotoxin, a lymphokine similar to TNF in structure and function. The effect of IFN-gamma in combination with a cytokine-rich supernatant containing IL-2, IL-3, IL-4, IL-5, and IFN-gamma on macrophage clearance of the parasite was also examined. The cytokine-rich supernatant alone had no effect on reducing parasite infection of the macrophages; indeed, in some experiments the addition of the supernatant resulted in an increase in the level of parasite infection. However, 1000 units of IFN-gamma combined with the complex cytokine mixture caused a decrease in parasite infection of nearly 100% compared to that of control cultures treated with media alone. To determine which cytokine or cytokines in the supernatant were responsible for this synergistic activity, anti-cytokine antibodies were added to the supernatant prior to its addition with IFN-gamma to the cultures. Anti-IL-4 was the only antibody found to inhibit the synergism of IFN-gamma with the cytokine-rich supernatant. IL-4, however, did not significantly enhance the ability of IFN-gamma to induce macrophage clearance of the parasite, and IL-4 alone caused a slight increase in parasite infection in vitro. These results further define the role that cytokines play in T. cruzi infection of macrophages in vitro and suggest that the interaction of cytokine networks within this system is complex.     

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.     

Trypanosoma cruzi: differences in cell surface interaction of circulating (trypomastigote) and culture (epimastigote) forms with macrophages

Characteristics of the association of circulating (trypomastigote) and cultured (epimastigote) forms of Trypanosoma cruzi with macrophages were studied. Treatment of mouse macrophages with the anti-microfilament drug cytochalasin D severely reduced the ability of these cells to bind either trypomastigotes or epimastigotes. Instead, treatment with the antimicrotubule drug colchicine or 2-deoxyglucose afforded differential effects because epimastigote but not trypomastigote association with the macrophages was significantly inhibited. Prior treatment of epimastigotes with either trypsin or neuraminidase decreased their uptake by macrophages whereas treatment of trypomastigotes with either enzyme increased it. Pretreatment of macrophages with neuraminidase did not affect epimastigote uptake but reduced that of trypomastigotes. Pretreatment of macrophages with trypsin reduced the uptake of both forms of the parasite. However, quantitative differences in the extent of such reduction were noted when varying concentrations of trypsin were used, epimastigote uptake being more drastically affected. These results suggest that the initial interaction of virulent circulating trypomastigote and the attenuated cultured epimastigote forms of T. cruzi to macrophages may involve attachment via different surface structures.     

Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of non-professional phagocytic epithelial cell lines by Trypanosoma cruzi

At the site of infection, pro-inflammatory cytokines locally produced by macrophages infected with Trypanosoma cruzi can activate surrounding non-professional phagocytes such as fibroblasts, epithelial and endothelial cells, which can be further invaded by the parasite. The effect of secreted soluble factors on the invasion of these cells remains, however, to be established. We show here that two epithelial cell lines become significantly susceptible to the infection by the Y strain of T. cruzi after tumour necrosis factor (TNF) treatment. The increase in the invasion was correlated with the increasing concentration of recombinant TNF added to cultures of HEK293T or LLC-MK2 cells. Supernatants taken from PMA-differentiated human monocytes infected with T. cruzi also increased the permissiveness of epithelial cells to subsequent infection with the parasite, which was inhibited by a TNF monoclonal antibody. Furthermore, the permissiveness induced by TNF was inhibited by TPCK, and led to significant decrease in the number of intracellular parasites, providing evidence that activation of NF-κB induced by TNF favours the invasion of the epithelial cell lines by T. cruzi through yet an unidentified mechanism. Our data indicate that soluble factors released from macrophages early in the infection favours T. cruzi invasion of non-professional phagocytic cells.     

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally infected mice, using limiting dilution analysis.

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57Bl/10 mice. The results showed that, in Swiss and Bl/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a greater number of parasites were observed in heart tissue from Swiss mice than from Bl/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and Bl/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.     

The role of Ca2+ in the process of cell invasion by intracellular parasites

In order to replicate, many parasites must invade host cells. Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) of different parasites and tissue culture cells during their interaction have been studied. An increase in cytosolic Ca(2+) in Trypanosoma cruzi trypomastigotes occurs after association of the parasites with host cells. Ca(2+) mobilization in the host cells also takes place upon contact with T. cruzi trypomastigotes, Leishmania donovani amastigotes or Plasmodium falciparum merozoites. When Ca(2+) transients are prevented by intracellular Ca(2+) chelators, a decrease in parasite association to host cells is observed. This reveals the importance of [Ca(2+)](i) in the process of parasite-host cell interaction, as discussed here by Roberto Docampo and Silvia Moreno.     

Modulatory effect of guanosine-3':5' cyclic monophosphate on macrophage susceptibility to Trypanosoma cruzi infection

The effects of agents that elevate intracellular levels of cGMP on macrophage internalization of the unicellular parasite Trypanosoma cruzi and latex particles were examined in an attempt to define characteristics of the infective process. Presence of imidazole, a drug that prevents degradation of the cGMP by inhibiting cGMP phosphodiesterase activity, during macrophage-T. cruzi interaction resulted in a marked increase in the number of parasites associated with the cells and the percentage of infected cells. Similar results were obtained when sodium nitroprusside (SNP), which increases cGMP levels by an as yet undefined mechanism, dibutyryl-cGMP, or both imidazole and dibutyryl-cGMP were added to the system. In contrast, the presence of imidazole, SNP, or dibutyryl-cGMP had no significant consequence on latex particle uptake by the macrophages. Whereas pretreatment of macrophages with imidazole plus dibutyryl-cGMP readily increased T. cruzi infection, pretreatment of the parasite with these drugs had no significant effect on the interaction. Furthermore, results of radioimmunoassays showed that imidazole and SNP indeed elevated cGMP levels in the macrophages but not in the parasites. Taken together, these results indicate that cGMP plays a facilitating role in macrophage infection by T. cruzi that contrasts with the lack of effect on the uptake of inert latex particles and the previously reported inhibitory effect of cAMP in the same system. Thus, cyclic nucleotides appear to play a role in modulating internalization of the parasite but not in the uptake of an inert particle by macrophages.     

Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele

We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/-) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.     

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma cruzi: parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.     

Trypanosoma cruzi: partial prevention of the natural infection of guinea pigs with a killed parasite vaccine

Guinea pigs are natural reservoirs of Chagas' disease. Domestic breeding and local trade of these animals are common practices among andean communities in South America. Infection by Trypanosoma cruzi occurs when the animals live in triatomine-infested houses or yards. The preventive effect of a vaccine consisting of cultured T. cruzi killed by freezing and thawing plus saponin was tested both in mice and in the guinea pig ecosystem. Resistance against T. cruzi challenge in mice was improved by increasing the trypomastigote/epimastigote ratio in live attenuated vaccines but not in killed parasite vaccines. Although the killing of attenuated parasites sharply reduced their immunogenicity for mice, a protective effect against natural T. cruzi infection was detected in guinea pigs. A total of 88 guinea pigs were vaccinated in four intradermal sites on three occasions. Eighty controls received similar inoculations of culture medium plus saponin. All animals were kept in a triatomine-infested yard. Parasitemia was studied with the capillary microhematocrit method. After an exposure time averaging 4 months, natural T. cruzi infection occurred in 55% (44/80) of the controls and in 33% (29/88) of the vaccinated group (P less than 0.01). The number of highly parasitemic guinea pigs was also significantly decreased (6/80 vs 0/88, P less than 0.01). Thus, immunizing protocols which are only partially protective against artificial callenge with T. cruzi may nevertheless constrain the exchange of parasites between natural hosts and vectors.