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Unionicola formosa is a symbiotic water mite that passes most of its life cycle in the mantle cavity of freshwater mussels. Although mites of this genus are often referred to as parasitic, little is known about their nutritional biology. A few species reportedly pierce the gill of a host mussel and ingest tissue or hemolymph. The present study was undertaken to identify possible sources of nutrition for U. formosa. To determine if mites ingested particulate matter in the mucous strand produced by a mussel during feeding, mussels with resident mites were exposed to a suspension of fluorescent microspheres. There was no evidence that U. formosa ingested the beads. Histochemical staining did, however, indicate a mucous material present in the midgut of the mites. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays revealed a high molecular weight component, consistent with a mucopolysaccharide, present both in the mussel gill and the mites. Results from western blots and an immunoaffinity binding assay with antibodies against mussel gill tissue and hemolymph also indicated that mites ingested host tissue. Whereas U. formosa probably does not ingest particulate material acquired by its host's suspension feeding, it is apparent that this mite utilizes host mucus, gill tissue, or hemolymph for at least part of its nutrition.

Macrophages from mice that are infected with various intracellular pathogens including Leishmania major, Trypanosoma cruzi, and Salmonella typhimurium are stimulated to produce large quantities of nitric oxide (NO). Both viable and heat-treated L. major amastigotes have been shown to be effective co-signals for NO production in vitro. NO produced by macrophages has anti-microbial and immunosuppressive functions in an immune response. We have shown previously that NO plays a complicated role in T. cruzi infections since macrophages are important both in mediating an immune response against the parasite as well as in mediating immunosuppression. In this study we examined how T. cruzi affects NO production by macrophages from C3HeB/FeJ and C57BL/6 mice in vitro. We found that live trypomastigotes neither stimulate nor decrease NO production by interferon (IFN)-gamma-activated macrophages. However, heat-treated or glutaraldehyde-fixed trypomastigotes of T. cruzi significantly decrease NO production by IFN-gamma-activated macrophages and as a result decrease macrophage-mediated trypanocidal and immunosuppressive activity. We have determined that this decrease in NO production by T. cruzi is not due to stimulation of transforming growth factor-beta production and involves tumor necrosis factor-alpha only in C3HeB/FeJ macrophages. This study demonstrates the complexity of the T. cruzi-macrophage interaction as well as confirms previously demonstrated differences between macrophages from 2 strains of mice.

Binding of the complement components C3 and C5 to epimastigote and trypomastigote stages of the Brazil strain of Trypanosoma cruzi was examined using radioligand binding and flow cytometric assays. Fibroblast-derived trypomastigotes bound approximately 40% fewer molecules of [125I]C3 per parasite than did epimastigotes. The predominant molecular species of C3 deposited on fibroblast-derived trypomastigotes was the inactive form iC3b. Addition of parasite-specific antisera failed to enhance the number of molecules of [125I]C3 per parasite or the proportion of active to inactive C3b. Flow cytometric studies revealed that only 50% of trypomastigotes (fibroblast-derived or blood-form) bound C3. In contrast to results of the [125I]C3 binding studies, flow cytometric analysis showed that the percentage of trypomastigotes binding C3 actually increased upon incubation with parasite-specific antisera. C5 was found also to bind to only a percentage of trypomastigotes.

A parasite-specific, enzyme-linked immunosorbent assay and immunoblot analysis were used to examine the development of humoral immunity in Trypanosoma cruzi-infected C3H mice that survive acute infection when held at elevated environmental temperature. Both parasite-specific antibody levels and numbers of antigens identified increased during infection in mice held at 36 C, with the greatest reactivity measured in sera from mice that had resolved parasitemias. Heat shock of culture forms of T. cruzi resulted in production of different antigens, but there was no strong difference in the antigens recognized by sera from mice held at room temperature and those recognized by sera from mice held at 36 C. Immunoblot analysis using blood-form trypomastigote antigens identified a band of approximately 61 kDa produced by trypomastigotes in mice held at 36 C that was strongly detected by sera from mice held at 36 C. Little if any reactivity to this antigen was observed using sera from mice held at room temperature.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.

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 cerebrospinal fluid (CSF) obtained from patients suspected of having neurocysticerosis (79 samples), as well as from control patients (without neurological symptoms), was separated using a high performance liquid chromatography gel filtration column. During the chromatographic separation, the eluted fractions were collected separately according to distinctive peaks. The elution characteristics of CSF components were identified by aligning more than 100 chromatograms and 6 distinctive peaks, eluting in consistent positions. Samples of each peak were tested in an enzyme-linked immunosorbent assay (ELISA) for the presence of larval antigens. Forty-four of the suspected 79 cases were found to have larval antigens in their CSF and these antigens were detected in peak no. 2, the mean of which is approximately 110,000 molecular weight. Also, in some cases, larval antigens were found in peak no. 1; however, we were able to detect them in only 23 CSF samples out of 44 CSF samples in which larval antigens were present in peak no. 2. Nine of these 23 CSF samples (derived from 79 patients in which neurocysticercosis was suspected) were later confirmed by histopathology. Values of ELISA readings of 5 CSF samples obtained from control patients (0.054 +/- 0.064) were considered negative. Thus, in 44 of 79 CSF samples from patients suspected of having neurocysticercosis, the ELISA values were highly positive (0.551 +/- 0.293). The remaining 35 CSF samples gave ELISA readings of 0.092 +/- 0.062, which were not significantly different from values obtained with CSF of control patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum from mice infected with Trypanosoma cruzi-suppressed (=SSS) lipopolysaccharide (LPS)-, phytohemagglutinin (PHA)-, and concanavalin A (Con A)-induced lymphoblast transformation when added to cultures of spleen cells or lymph node cells. This serum maximally suppressed blastogenic responses in spleen cell and lymph node cell cultures that contained supportive fetal bovine serum concentrations of 2% and 4%, respectively. Preincubation of lymphoid cells with SSS for 18 to 48 hr prior to initiation of the blastogenesis assay led to suppression of LPS-, PHA-, and Con A-induced proliferation at the optimal concentration of supportive fetal bovine serum (5%), whereas adsorption of lymphoid cells with SSS at 4 C for 30 min before stimulation with mitogen led to suppression of LPS-induced proliferation in spleen cells only. There was a close temporal correspondence between the induction and manifestation of suppression to the T-cell mitogens (PHA and Con A), but the manifestation of suppression preceded the induction of suppression to the B-cell mitogen (LPS) by approximately 12 hr. The SSS-induced suppression of proliferative responses, except in spleen cell cultures stimulated with LPS, was shown to be dependent on the presence of macrophages during the preincubation and stimulation phases of the assay system. The combined results of experiments in which macrophages were preincubated with SSS, or in which macrophages from the spleen were cultured with lymphocytes from the lymph nodes and vice versa (before and after preincubation with SSS), clearly demonstrated the presence of SSS-activated suppressor cells in the spleen, but not in the lymph nodes. Furthermore, the activation of these suppressor macrophages was reliant upon interactions with splenic lymphocytes.

A method is described for estimating levels of cytophilic antibody against Trypanosoma cruzi in the sera of infected mice. 51Cr-labeled trypanosomes were incubated at 4 C in the presence of adherent, peritoneal cells which had been preincubated with immune or normal serum. It was found that immune, but not normal, serum mediated the enhanced binding of parasites in a manner dependent upon the amount of immune serum present during the preincubation period with peritoneal macrophages. Experiments were done comparing the development of anti-T. cruzi cytophilic antibody during the course of infection in two strains of mice which differ in susceptibility T. cruzi. It was found that neither C57BL/6 (resistant) nor C3H (susceptible) mice accumulated high levels cytophilic antibody, and no significant differences were noted in the level of cytophilic antibody in the sera of these two strains of infected mice. Attempts to stimulate higher levels of anti-T. cruzi cytophilic antibody by immunization with several preparations of culture-forms of the parasite were only minimally effective.

The onset and development of anti-Trypanosoma cruzi cytolytic antibody during the course of infection were examined in two strains of mice that differ in their susceptibility to T. cruzi. The relative amounts of cytolytic antibody in the sera of mice were determined by a terminal radiolabeling procedure that measures the amount of 3H-thymidine incorporatedinto culture-form parasites that survive treatment with antibody and complement. The results showed that low levels of anti-T. cruzi cytolytic antibody developed during the course of infection in mice, and that there was no apparent difference in cytolytic antibody responses during the acute phase of infection in relatively resistant C57BL/6 mice and highly susceptible C3H(He)mice.

Various assay conditions were employed in experiments examining the susceptibility of trypomastigote forms of the Brazil strain of Trypanosoma cruzi to antibody-dependent complement-mediated lysis. To identify optimal assay conditions, both guinea pig serum and normal human serum were used as complement sources, and fibroblast-derived or blood-form trypomastigotes were either coincubated with immune sera and complement together, or the parasites were first precoated with antibodies and then were incubated in complement. Under conditions promoting maximal lysis by antibodies and complement, 60-90% of the trypomastigote forms were not lysed. These results indicate that trypomastigotes of certain isolates of T. cruzi, such as the Brazil strain, may possess an escape mechanism by which they evade complement-mediated lysis.

Experiments were conducted to identify antigens of Trypanosoma cruzi (Brazil strain) to which antibodies are directed during the course of experimental Chagas' disease in C3H(He) (susceptible) and C57BL/6J (resistant) female mice. An extract of culture forms of the parasite was subjected to SDS-polyacrylamide gel electrophoresis, transferred to a solid phase matrix of nitrocellulose and used as antigens to detect antibodies in the sera of infected mice. Reactive antibodies were detected using an avidin-biotin peroxidase test. Two antigens were consistently detected with sera of normal, uninfected C57BL/6 and C3H(He) mice (51,000 and 44,000; and 53,000 and 46,000 daltons, respectively). A total of 32 antigens with m.w. of 230,000 to 25,000 daltons reacted with antibodies in sera of C3H mice infected for 25 days. Both the number of antigens detected and intensity of reactions increased with time of infection in C3H mice. An early (day 5), rapid, although weak response was observed to antigens of 85,000, 56,000, 53,000, 46,000 and 41,000 daltons. Throughout infection intense responses to antigens of 75,000, 67,000, 45,000, 41,000 and 36,000 daltons were detected. A similar number of components (a total of 34) with m.w. of 210,000 to 20,000 daltons were detected as being antigenic during the course of T. cruzi infection of C57BL/6 mice. A high number of antigens (25) was observed early in infection of C57BL/6 mice by day 10, including components with m.w. of 90,000, 85,000 and 70,000 daltons. Only minor changes were detected, however, after day 20 until day 120, when increases in the number of antigens and the intensity of certain reactions (e.g., antigens of 75,000, 46,000 and 26,000 daltons) were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro antisheep erythrocyte (SRBC) and antitrinitrophenyl (TNP) antibody responses of spleen cells obtained from C57BL/6 mice infected with Trypanosoma cruzi were reduced as early as 6 days postinfection and not detectable after 18 days of infection. Lymph node cells had normal antibody responses to SRBC and TNP in vitro until the 11th day of infection, after which responses were diminished. By day 31 of infection, lymph node cells were unresponsive to both SRBC and TNP in vitro. Not only were the antibody responses of spleen and lympho node cells to T-cell-dependent and -independent antigens progressively reduced as the period of infection increased, but in addition, the effect of lymphoid cell density and antigen dose on antibody production underwent several sequential changes. As the infection advanced, low densities of cultured lymphoid cells and low doses of antigen were ineffective in eliciting a detectable immune response, whereas high densities of lymphoid cells and high doses of antigen resulted in responses approximately equivalent to that observed with normal cells under the same conditions. Results of cell mixing studies have shown that a plastic-adherent, macrophage-like cell plays a major role in the suppressed humoral responses observed in this host-parasite system.

In this report we show that there are 37 polypeptides of larval Taenia solium which react with antibodies from humans with neurocysticercosis. Six of these 37 polypeptides are recognized by antibodies present in the sera of both patients and control individuals. Thus, a minimum of 31 antigens are specific for cysticerci. We describe herein the antigens more frequently recognized by the patients, and the immunoglobulin classes favored in antibody production against each of the cysticercal antigens. It was found that there are 10 major polypeptides with molecular weights of 200,000, 64,000, 62,000-61,000, 53,000, 45,000, 41,000, 36,000-35,000, 30,000 and 16,000 daltons. About 65% of the larval components found to be antigenic are glycoproteins with oligosaccharide chains containing N-acetyl-D-glycosamine and alpha-D-galactose. These results suggest that the sugar moieties of glycoproteins may play a role in the antigenicity of larval T. solium. Based on these observations polypeptides with molecular weights of 64,000, 53,000, and 32,000-30,000 daltons are probably the best choice as sources of antigen to develop an optimal immunological test for the serological diagnosis of neurocysticercosis.

Serum from mice infected with Trypanosoma cruzi (SSS) is known to interact with normal spleen cells to induce an immunosuppressed condition and activate splenic suppressor cells. The induction of immunosuppression by SSS was shown to be independent of, and precede the activation of, suppressor cells. Suppressor-cell activation, however, was demonstrable only after the induction of immunosuppression. Furthermore, mice that were given two aliquots of SSS at different intervals of time, exhibited suppression of humoral responses of similar duration and magnitude, regardless of the SSS transfer regimen, whereas both the length and degree of suppressor-cell activity was critically dependent on the interval of time between SSS transfers. SSS interacted with spleen cells via a trypsin-sensitive membrane site which was regenerable within a 4- to 5-hr period, yet the suppressive effects of SSS on spleen cells following interaction was resistant to treatment with trypsin. The interaction between SSS and spleen cells during brief adsorption protocols leads to immunosuppression only because extensive washing of SSS-treated spleen cells did not reverse the immunosuppression process, but did prevent the development of detectable suppressor cells. The phenomenon of suppressor-cell activation was further distinguished from immunosuppression in that supernates from culture of spleen cells derived from SSS-treated mice or T. cruzi-infected contained a factor that activated suppressor cells, but did not directly induce a state of suppression in the responding cell population.

Trypanosoma cruzi releases a factor (SCAF) when grown in vitro which decomplements normal mouse, human, and guinea pig sera. The production and potency of SCAF was dependent on the density of cultured parasites, parasite viability and proliferative capacity, and duration of culture. The in vitro interaction between SCAF and serum complement (C') occurred rapidly and was complete within 30 min of mixing. The administration of SCAF to normal mice resulted in up to 50% reduction in hemolytic C' activity, whereas SCAF had no effect on the C' levels in mice infected wit T. cruzi for more than 10 days. The active moiety of SCAF was shown to be a nonproteinaceous substance(s) with a molecular weight of approximately 23,000 daltons.

Previous work from this laboratory documented the presence of a suppressor substance in the serum of mice infected with Trypanosoma cruzi. Currently, however, we have been unable to demonstrate the presence of this suppressor factor, although infected mice were still profoundly suppressed in their ability to respond to SRBC, and suppressor cells were present in the spleens of these animals. Strains of T. cruzi other than the one presently in use in our laboratory gave similar results. Several possible explanations for our inability to demonstrate the presence of the suppressor factor in sera are discussed.

Six clones and 4 subclones were isolated from the Brazil strain of Trypanosoma cruzi and were passaged in C3H(He) mice. Parasitemia levels and survival times of mice infected with 8 of the isolates were equivalent to the Brazil strain in virulence. Two clones, designated WFTc-5.1 and WFTc-6.1 (WFTc = Wake Forest Trypanosoma cruzi) were of lower virulence in C3H mice than the other isolates and the Brazil strain. C57BL/6 mice infected with WFTc-5.1 had significantly lower parasitemias and higher survival rates than C57BL/6 mice infected with the Brazil strain or a clone designated WFTc-3.2. Levels of anti-T. cruzi IgM and IgG antibodies were the same in mice infected with higher virulence or lower virulence isolates. Based on these results the Brazil strain of T. cruzi is composed of distinct subpopulations which are heterogeneous with respect to virulence.

Spleen cells from normal mice and mice infected with Trypanosoma cruzi undergo mitogen-induced lymphoblast transformation in different ways. Some of these differences may be interpreted as suppression of the blastogenic response, whereas others cannot. Close examination of the culture conditions has enabled us to discriminate between in vivo-induced suppression and in vitro-determined (artifactual) suppression. Regardless of the type of suppression observed, it was shown to be dependent on duration of culture, length of labeling period, dose of mitogen, density of spleen cells, and method of expressing lymphoblast transformation data. The titration of mitomycin C-treated spleen cells from infected mice into cultures of normal spleen cells revealed two, separate, regulatory activities of spleen cells from infected mice as follow: (1) an enhancing effect which appeared late in infection and was obtained with low input of cells, and (2) a suppressive effect which appeared early in infection and was obtained with high input of cells. The enhancing effect was mediated by cells with properties characteristic of T-lymphocytes, whereas macrophages were found to be responsible for the suppressive effects.