TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi.

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.

Enzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas' disease.

In the present report we describe the use of Trypomastigote Excreted-Secreted Antigens (TESA) as antigen in ELISA for Chagas' disease serodiagnosis. The study was carried out on 284 patients, 164 of whom were nonchagasic subjects including individuals with leishmaniasis or other pathologies, and 120 chagasic patients, being 53 in the acute (with positive IgA and IgM antibodies to T. cruzi) and 67 in the chronic phase. TESA-ELISA showed 100% positivity in the survey of IgG antibodies in chagasic patients (acute and chronic) and 100% positivity for IgM antibodies in acute phase sera. TESA preparation does not require biochemical purification procedures and does not present the cross-reactivity of leishmaniasis sera observed when ELISA with epimastigote alkaline extract (EAE) is performed. ELISA competition assays showed that anti-T. cruzi antibodies of sera from chagasic patients that react with TESA are different from those that react with EAE. Besides, partial characterization of TESA showed that several epitopes present in this fraction are absent in EAE.

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates.

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.

Reactivity of chagasic antigal antibodies with noninfected cells treated with Trypanosoma cruzi secreted/excreted antigens.

Here, we show that antigal antibodies from Chagas' disease patients react with noninfected host cells previously treated with antigens secreted by the trypomastigote forms of Trypanosoma cruzi. With the exception of human and Old World monkey cells, which are GAL-negative, cells of all mammals express the GAL epitope (Gal alpha (1-3)Gal beta (1-4)GlcNAc-R) on their surface. Thus only the former ones develop antigal antibodies. Antigal antibodies increase during infection with T. cruzi, which expresses GAL epitopes on the surface of the infective forms. Here, we show that incubation of noninfected, GAL-negative cells with antigens shed by T. cruzi renders these cells reactive to antigal antibodies purified from chagasic sera. Neither chagasic sera depleted of antigal antibodies nor antigal antibodies purified from normal sera display reactivity with treated cells. Cell reactivity of chagasic antigal was abolished in the presence of melibiose (Gal alpha (1-6)Glc) or gal-gal (methyl 3-O-alpha-D-galactopyranosyl alpha-D-galactopyranoside). Since shedding of T. cruzi antigens can occur in vivo, these antigens may induce reactivity of chagasic antigal with noninfected human cells. The reactivity of noninfected, GAL-negative cells observed only with chagasic antigal antibodies can amplify the range of reactivity of these antibodies and consequently adds to their importance in the pathogenesis of human Chagas' disease.

Reactivity of Trypanosoma cruzi strains with peanut agglutinin (PNA) correlates with number of in vitro infected host cells.

Reactivities of 4 lectins with intact trypomastigote forms derived from 8 different Trypanosoma cruzi strains were compared with their capacity to infect in vitro cultured LLC-MK(2)cells. A sensitive and reproducible titration method for lectin binding sites (ELLA: Enzyme Linked Lectin Assay) was employed, in which reactivities were scored through optical densities in an ELISA reader. Tissue culture trypomastigotes from the strains Y, CL, SC4, SC24, SC25, SC28, SC32 and SC33 were investigated for expression of different cell surface carbohydrate residues using Concanavalin A (ConA), Peanut agglutinin (PNA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA) conjugated to peroxidase. The reactivity of the strains to PNA lectin was SC28 > SC32 > SC33 > SC25> SC24 > Y> CL> SC4. The optical density values obtained were highly correlated (r2=0.986, p< 10(-4)) with the number of parasitized LLC-MK(2) cells 24 hours after infection by trypomastigotes from each corresponding strain. We concluded that galactose and N-acetyl-D-galactosamine residues that are present on the surface of trypomastigotes are important in host-cell recognition.

Changes in isotype composition and antigen recognition of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease.

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.

In situ immunoassay for the assessment of Trypanosoma cruzi interiorization and growth in cultured cells.

Interiorization and multiplication of Trypanosoma cruzi within its host cells are usually assessed by counting parasites in fixed and stained cover slip preparations, a subjective and time-consuming method. Here we describe an immunoenzymatic assay (ELISA) for assessing the number of internalized parasites in infected LLC-MK2 seed on chamber slides (NUNC). ELISA was performed employing a rabbit polyclonal serum against trypomastigote components (MOP) and anti-rabbit IgG conjugated to peroxidase. The bottom of the chamber slide was then detached and processed for quantification of internalized parasites by the conventional method. Data analysis showed a linear relationship between optical densities and number of internalized parasites (r2 = 93.99, p < 0.001). The assay was also efficient to assess inhibition of parasite interiorization induced by the monosaccharide NAc-D-glucosamine.

Trypanosoma cruzi: identification of proteinases in shed components of trypomastigote forms.

Trypanosoma cruzi trypomastigotes were shown to predominantly release high molecular weight components (above 50 kDa) when allowed to shed for 1 hour in protein-free media. Under these conditions, parasites were not damaged or lysed, as was indicated by: (a) their normal mobility; (b) their retaining of some of the labelled proteins; (c) the unchanged pattern of biotinylated surface proteins after shedding. Shed components were shown to display proteinase activities, detected at 97 and 50/60 kDa in gelatin gels. These proteolytic activities were completely inhibited by E-64, indicating that they were due to cysteine proteinases.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to Dot-ELISA.

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Trypanosoma cruzi: studies on the reactivity of antibodies bound to the surface of blood forms at the early phase of infection.

The specificity and reactivity of antibodies bound to the surface of Trypanosoma cruzi blood forms at the very early acute phase of murine infection was investigated. Surface-bound antibodies of the IgG and IgM isotypes were recovered from blood forms upon incubation at 37 degrees C. The eluted antibodies immunoprecipitated several trypomastigote surface polypeptides from 80 to 100 kDa. In contrast, for epimastigotes a very faint reactivity was detected only for antigens of 50 and 95 kDa. The shed antibodies promoted in vitro complement-mediated lysis of live blood forms and reacted with fixed trypomastigotes by immunofluorescence. Thus, blood forms are already coated with active trypomastigote-specific antibodies with a potential role in the host defense, although the low levels of serum antibodies have prevented the demonstration of humoral protection at the early stages of infection.

Trypanosoma cruzi: different surface antigens of trypomastigotes are targets of lytic antibodies.

Polyclonal antisera were obtained in rabbits following immunization with disrupted epimastigote or trypomastigote forms; 8-methoxypsoralen-inactivated trypomastigotes; and surface trypomastigote antigens shed into the medium. High antibody levels were induced by all preparations as observed by indirect immunofluorescence and ELISA. However, antibodies promoting complement-mediated lysis of bloodstream forms were only detected in animals immunized with inactivated living trypomastigotes and shed surface antigens. Immunoprecipitation of radioiodinated parasites showed that sera with lytic antibodies bound strongly to a wide range of membrane polypeptides from 72 to 160 kDa. Immunoadsorption of antibodies from a serum with high lytic activity on specific classes of trypomastigote polypeptides indicated that independent antigens are targets of lytic antibodies and that common epitopes may exist in different trypomastigote components.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting).

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment.

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice.

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.

In vitro synthesis of Trypanosoma cruzi antigenic polypeptides.

A ribosomal fraction of Trypanosoma cruzi was isolated with detergent lysis and differential ultracentrifugation. This ribosomal fraction directed in vitro protein synthesis, in a heterologous incorporation system (rat liver pH 5 fraction), leading to values up to 10 times higher than endogenous control. The ideal concentrations of Mg2+, K+, and ribosomal proteins and the time of incubation of the incorporation mixtures were 6 mM, 21 mM, 60 microliters, and 45 min, respectively. The product thus obtained was analyzed by fluorography after polyacrylamide-sodium dodecyl sulfate gel electrophoresis and showed the presence of many protein bands, but few bands were present in the immunoprecipitate obtained with serum from Chagas' disease patients. This product was able to react with anti-T. cruzi antibodies when submitted to an immunoenzymatic assay.