Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Serological Diagnosis of Trypanosoma rangeli Infected Patients. A Comparison of Different Methods and its Implications for the Diagnosis of Chagas' Disease

Venous blood from 65 Panamanian schoolchildren living in an area endemic for both Trypanosoma cruzi and T. rangeli were screened for the presence of these parasites. Trypanosoma rangeli were isolated and cultured from four individuals. Serological tests of all 65 sera were performed, including immunohaemagglutination (IHA), indirect immunofluorescence assay (IF) and ELISA using both T. rangeli and T. cruzi as antigens, as well as T. cruzi synthetic peptides in an ELISA assay. Results indicated a higher immunoreactivity to T. rangeli preparations than to T. cruzi within the studied population, which could be divided into four 'serological responder' groups. Interestingly, the panel of SAPA and other T. cruzi synthetic peptides were not useful in the discrimination of patients. Furthermore, patients from whom parasites had been isolated could not be distinguished from those of two other groups. Significant immunoreactivity to T. cruzi preparations was displayed in all responder sera, despite total lack of evidence of infection with this parasite. The immunobiological significance of T. rangeli infection is unclear, but these data indicate that it is a compounding problem in the accurate diagnosis of pathological T. cruzi infection by serological analysis. The relationship of these cohabiting species, in respect to infection outcome and immunological activation, is discussed.     

Radiolabeled total parasite DNA probe specifically detects Trypanosoma cruzi in mammalian blood.

A DNA-DNA hybridization procedure is described in which radiolabeled total parasite DNA is used to detect Trypanosoma cruzi spotted directly onto nylon membrane. Under conditions that favor hybridization of repetitive DNA sequences, the radiolabeled total DNA was able to detect as few as five parasites spotted onto nylon membrane. Trypanosomes were detectable in blood from mice with T. cruzi parasitemias spotted directly onto membranes. All 10 strains of T. cruzi examined, which came from different areas of the United States and South America, were readily detected with a total DNA probe from any 1 strain of the parasite. No signals were detected with up to 20,000 Trypanosoma rangeli, T. brucei, Crithidia fasciculata, or Leishmania parasites or with normal mouse blood or mammalian DNA. The hybridization method is sensitive, specific, rapid, and inexpensive and is potentially applicable to the detection of other parasites.     

Specific binding of human plasma high density lipoprotein (cruzin) to Trypanosoma cruzi

Binding of high density lipoprotein (HDL) to Trypanosoma cruzi was examined because of its ability to specifically inhibit the parasite's neuraminidase. 125I-Labeled HDL bound to live and glutaraldehyde-fixed parasites equally well either at 37 degrees C or at 4 degrees C. Binding was saturable and inhibited by unlabeled HDL but not by unrelated plasma proteins. Specificity of the T. cruzi-HDL interaction was confirmed using fluorescein labeled HDL which bound to T. cruzi but not to T. rangeli, a species whose neuraminidase is not inhibited by HDL. Binding of HDL to T. cruzi paralleled the neuraminidase activity exhibited by the parasite's different stages and strains. In agreement with this finding, Steck and Wallach analysis of the binding data showed that the number of HDL binding sites was greater in infective trypomastigotes and on strains with high neuraminidase activity. However, the association constant of the binding did not change within the various developmental forms and strains of T. cruzi, suggesting that HDL bound to the same receptor, presumably having neuraminidase activity.     

Interaction of Trypanosoma rangeli Tejera, 1920 with different cell lines in vitro

Intracellular multiplication of Trypanosoma rangeli was evaluated in vitro using experimental infection of Vero cells, murine macrophages, and promonocytes with T. rangeli Choachi, Macias, and SC-58 clone B1 strains. Our results revealed a low infectivity of all T. rangeli strains to these cell lines. Macrophages showed the highest infection rate; however, intracellular forms were no longer observed 48 h post infection Despite the observation of intracellular parasites up to 144 h post infection, the infection rates of Vero cells and J774G.8 promonocytes with these parasite strains were always below 5%. Pre-incubation of parasites with normal mouse serum increased the initial infectivity but not the time course of the infection. Under our experimental conditions, we did not observe any evidence of intracellular multiplication of T. rangeli within these cell lines.     

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes.

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.     

Cloning,heterologous expression,and substrate specificities of protein farnesyltransferases from Trypanosoma cruzi and Leishmania major

Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the alpha- and beta-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.     

Immunoparasitological Studies of Trypanosoma Cruzi Low Virulence Clones from Panama: Humoral Immune Responses and Antigenic Cross-Reactions with Trypanosoma Rangeli in Experimentally Infected Mice

The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1: 10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30–50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.     

trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs

The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.     

Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei

Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.     

Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers

Six discrete phylogenetic lineages were recently identified in Trypanosoma cruzi, on the basis of multilocus enzyme electrophoresis and random amplified polymorphic DNA (RAPD) characterisation. The objective of the present study was to develop specific PCR-based markers for the identification of each of the six lineages. Eighty-seven T. cruzi stocks representative of all the lineages were characterised by RAPD with three primers, resulting in the identification of three fragments that were specifically amplified in the given sets of lineages. After cloning and sequencing these fragments, three pairs of sequence-characterised amplified region (SCAR) primers were designed. After PCR amplification using the SCAR primers, the initial polymorphism was retained either as the presence or absence of amplification, or as size variation between the PCR products. Although most PCR products, taken individually, were distributed across several lineages, the combination of the three SCAR markers resulted in characteristic patterns that were distinct in the six lineages. Furthermore, T. cruzi lineages were distinguished from Trypanosoma rangeli, T. cruzi marinkellei and T. cruzi-like organisms. The excellent correspondence of these new PCR markers with the phylogenetic lineages, allied with their sensitivity, makes them reliable tools for lineage identification and strain characterisation in T. cruzi. The approach described here could be generalised to any species of microorganism harbouring clear-cut phylogenetic subdivisions.     

Immunization against Trypanosoma cruzi: adjuvant effect of glucan

Trypanosoma cruzi, the causative agent of Chagas' disease, infects humans and animals in tropical, subtropical and some temperature regions of the western hemisphere. At present, there is no effective vaccine for T. cruzi infection. Glucan, a beta-1,3 polyglucose biological response modifier, possesses significant adjuvant activity. The present study investigated the adjuvant activity of particulate glucan when combined with a vaccine of glutaraldehyde-killed T. cruzi culture forms. ICR/HSD mice (20 g) were injected s.c. with glutaraldehyde-killed T. cruzi on days 21, 14 and 7 prior to challenge with 50 T. cruzi blood forms. Particulate glucan (1 mg/mouse) was administered s.c. either alone or in conjunction with T. cruzi vaccine. Isovolumetric dextrose served as control. Dextrose, glucan or T. cruzi vaccine as single treatment regimens showed 100% mortality with 20.5, 21.4 and 21.6 day median survival times, respectively. In contrast, glucan administered with T. cruzi vaccine showed an 85% (P less than 0.01) survival at 275 days post-challenge. In addition, the number of T. cruzi observed in the blood of glucan--T. cruzi immunized mice was lower than the appropriate controls. However, immunized mice which survived at 275 days were positive for the presence of T. cruzi by xenodiagnosis. Histopathologic evaluation of glucan--T. cruzi mice revealed no parasites or cardiac pathology, but a mild splenic hyperplasia and inflammation of skeletal muscle were noted. In subsequent studies, mice were immunized with the same regimen of glucan--T. cruzi and challenged with 500 or 5000 T. cruzi. Glucan significantly (P less than 0.05) increased survival as denoted by 60% and 50% survival in the glucan-T. cruzi group vs 0% in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.     

Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera.

A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.     

NK cells contribute to the control of Trypanosoma cruzi infection by killing free parasites by perforin-independent mechanisms

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase leads to an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues, which causes the pathology of Chagas' disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we analyzed whether NK cells participated in the control of experimental T. cruzi infection. NK cells were depleted from C57BL/6 mice by antiasialo antibodies. This treatment caused an increased parasitemia during the acute phase, but tissue parasite burdens were not significantly altered according to quantitative real-time PCR. Our results demonstrated that NK cells were activated during the initial phase of a T. cruzi infection and exhibited a contact-dependent antiparasitic activity against extracellular parasites that was independent from perforin. Thus, NK cells limit the propagation of the parasite by acting on circulating T. cruzi trypomastigotes.     

Parasite-derived trans-sialidase binds to heart tissue in Trypanosoma cruzi-infected animals

Trypanosoma cruzi is an obligate intracellular protozoan parasite that actively penetrates into non-phagocytic mammalian cells. To accomplish this, the parasite relies on the binding of cell surface ligands. It is reported herein that the T. cruzi trans-sialidase (TS), which is exposed on the parasite surface, binds to mouse heart cells, and should therefore be further studied as a possible cell penetration-related ligand. In addition, as has been proposed elsewhere, the binding of T. cruzi to tissues may turn them into targets for parasite-specific immune reactions. Washed heart sections from T. cruzi-infected mice were subjected to immunoenzymatic staining with antisera against whole T. cruzi and with polyclonal or monoclonal antibodies against TS. The anti-TS antibodies stained both parasites and uninfected heart cells in the vicinity of T. cruzi nest remains/trypomastigotes. On the other hand, an anti-T. cruzi serum, which did not recognize TS, only stained the parasites. In addition, normal heart sections from uninfected nude mice were shown to react with both enzymatically active and inactive recombinant TS molecules, probably through their amino-terminal region, since a recombinant TS lacking this region failed to bind.     

Trypanosoma cruzi carrying a monoallelic deletion of the calreticulin (TcCRT) gene are susceptible to complement mediated killing and defective in their metacyclogenesis

Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/- epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/- parasites was observed. Moreover, TcCRT+/- parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system.     

Trypanosoma cruzi cell invasion and traffic: influence of Coxiella burnetii and pH in a comparative study between distinct infective forms

Previous studies have shown that Coxiella burnetii, an intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics, is an effective modulator of the intracellular traffic of trypomastigote forms of Trypanosoma cruzi. In addition, vacuolar and cellular pH are related to fusion events that result in doubly infected phagosomes. T. cruzi, the etiological agent of Chagas' disease, occurs as different strains grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle, and T. cruzi II, linked to the human disease. In this work we compared extracellular amastigotes (EA), metacyclic trypomastigotes (MT) and tissue culture derived trypomastigotes (TCT) belonging to T. cruzi I or T. cruzi II for their ability to invade and escape from their parasitophorous vacuole (PV), in Vero cells or Vero cells harboring the bacterium, C. burnetti. Distinct invasion patterns were observed between different infective stages and between infective forms of different strains. Studies on the transference kinetics revealed that pH modulates the intracellular traffic of each infective stage, but this influence is not exclusive for each phylogenetic group. Endosomal to lysosomal sequential labeling with EEA-1 and LAMP-1 of the PV formed during the entry of each infective form revealed that the phagosome maturation processes are distinct but not strain-dependent. Due to their low hemolysin and trans-sialidase activities, MTs are retained for longer periods in LAMP-1 positive vacuoles. Our results thus suggest that despite the contrasting invasion capabilities, parasites of distinct phylogenetic group behave in similar fashion once inside the host cell.     

Purification of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies.

Recent studies on the humoral immune response to Trypanosoma cruzi have shown that antibodies which are able to bind living parasites and lyse them in conjunction with complement are associated with host protection. Antibodies which support complement-mediated lysis (CML) of trypomastigotes are elicited as a result of an active infection and not after immunization with killed parasites. In spite of the requirement for immune antibodies, lysis proceeds mainly via the alternative complement pathway. We have purified a 160-kilodalton (kDa) glycoprotein from T. cruzi metacyclic trypomastigotes which appears to be a specific target for lytic antibodies. Rabbit antiserum to the purified 160-kDa protein was prepared, and we have determined that these antibodies will support CML of tissue-culture-derived trypomastigotes. The percentage of killing (65 to 70%) was consistent among three different T. cruzi strains tested. In order to examine the specificity of antibody-dependent CML, antibodies to T. cruzi neuraminidase, an unrelated trypomastigote membrane glycoprotein, were tested in the CML, assays and were not found lytic. Viable trypomastigotes bound anti-160-kDa antibodies uniformly as demonstrated by immunofluorescence, whereas antineuraminidase antibodies were extensively capped. The 160-kDa glycoprotein is specifically produced in infectious trypomastigotes (tissue culture derived and metacyclic) and was not detected in epimastigotes or amastigotes. The identification of the 160-kDa glycoprotein as a specific target for lytic antibodies, as well as its expression only in the infectious stage of the parasite, suggests an important role for this protein in eliciting host immunity.