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The effect of sub-lethal doses of coronaviruses on the course of disease in CBA mice experimentally infected with a mildly pathogenic strain of Trypanosoma cruzi was investigated. Mice were inoculated with either T. cruzi, 0.1 median lethal dose (LD50) of coronavirus (mouse hepatitis virus [MHV-3] or virus X), or both pathogens. Levels of parasitemia, mortality, and the extent of pathologic alterations in lymphoid organs were determined. Mice inoculated with T. cruzi had mild alterations in their lymphoid organs and survived infection. In contrast, mice inoculated with both pathogens died, and had significantly higher levels of parasitemia and profound alterations in lymphoid organs. These results indicate that the pathologic profile of T. cruzi infection can be profoundly altered by subclinical infection with coronaviruses.

Trypanosoma cruzi-infected mice show disturbance in the peripheral immune system such as polyclonal lymphocyte activation, autoantibody production, and immunosuppression of T lymphocytes. Previous observations in our laboratory showed that some stocks of T. cruzi can be contaminated with mouse hepatitis virus type 3 (MHV-3). Literature has shown that MHV-3 infection induces immunologic disorders characterized by thymic involution with marked cell depletion. However, the effects of interactions between MHV-3 and the parasite on the immune system are not well understood. In the present study specific-pathogen-free CBA mice were inoculated with MHV-3, alone or associated with different stocks of T. cruzi. Concurrent murine virus infection resulted in increased pathogenicity of T. cruzi infection shown by profound thymic atrophy; loss of cortical thymocytes; depletion of Thy1.2+, CD4+, and CD8+ cells; enhancement of in situ labeling of nuclear DNA fragmentation; and eventually, death of the animals. Such lines of evidence show that the mechanism underlying this thymic atrophy is associated with apoptosis. These results also suggest that MHV-3 can account for the increased immunosuppression observed during experimental infection with the parasite.

The possibility that some virus contaminants could be altering host response to Trypanosoma cruzi experimental infection was investigated. Data obtained showed that CBA/J mice infected with stocks of parasite maintained in mice (YIUEC) presented higher level of parasitemia and shorter survival times than those infected with a stock (YITC) which was also maintained in mice but had been previously passaged in cell culture. Mouse antibody production tests, performed with the filtered plasma of mice infected with YIUEC, indicated the presence of mouse hepatitis virus (MHV) while no virus was detected when testing the plasma of YITC infected mice. Filtered plasma of YIEUC infected mice was shown to contain a factor able to enhance the level of parasitemia and to reduce the mean survival time of mice challenged with 10(5) YITC. This factor, that could be serially passaged to naïve mice was shown to be a coronavirus by neutralization tests.

To verify whether anti-Trypanosoma-cruzi-proteinase antibodies are able to play a role in the development of Chagas' disease, CBA/J and C57BL/6 mice were immunized with purified proteinase fractions: antibody production was studied and passive immunization experiments were carried out. No significant differences were observed in the titre, isotype composition and specificity of the antibodies produced by the mouse strains. Immune sera produced in one strain was able to protect mice of both strains, provided that the challenge did not exceed the number of parasites corresponding to 30-fold the LD50. The data presented suggest that anti-proteinase antibodies may play a role in immunoprotection.

Previous observations in this laboratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 10(5) blood trypomastigotes (BT) corresponding to 2000 LD50%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permitting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 +/- 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Mice were given increasing doses of sheep serum albumin emulsified with Freund's complete adjuvant in hind footpads. The kinetics of development of cells which synthesize antibody or immunoglobulins without antibody function was studied in the draining lymph nodes. Results show the relationship between the two cell populations: as the number of antibody synthesizing cells increased, the number of immunoglobulin synthesizing cell increased, and conversely. Between 0.050 and 2.5 mg of antigen, the number of cells of each population increased with the dose injected whereas with 5 and 10 mg, this number is smaller than that obtained with smaller doses and decreased with an increasing dose.

The primary immune response to SRBC in BALB/c mice was depressed when they were injected with a fraction (FAd) obtained from Trypanosoma cruzi epimastigotes grown in LIT medium. Plaque-forming cell (PFC) number was 50% less than controls when FAd was injected i.v. 15 min before antigen in doses ranging from 70 microgram up to 400 microgram of protein. Similar depression was observed when 100 microgram FAd was injected up to 6 h before antigen. There was no shift in the peak response to SRBC, neither was depression detected, when a total of 100 microgram FAd protein was given in 20 microgram amounts twice a day before immunization. Mice injected with FAd fraction only showed no increase in background PFC. Both secondary IgM and secondary IgG PFC were depressed when FAd was given before the boosting injection. However, only IgG PFC were depressed when FAd was injected before the priming dose. The delayed-type hypersensitivity reaction to DNFB was depressed when animals were injected either during the 3 days after sensitization or with a single dose of 100 microgram of protein of FAd on day of challenge. Bone marrow colony-forming units in spleens of mice injected with FAd were depressed and nodules in the treated animals were smaller than in controls. We conclude that FAd affects humoral and cell-mediated immune responses by interfering with cell division at some stage of the cell cycle.

Mice injected with 10(5) culture trypomastigotes (CT) survived the 30-day observation period while those injected with the same number of bloodstream trypomastigotes (BT) died in 10 +/- 1 days. As early as 24 h after injection of CT the mice displayed an increased resistance to Trypanosoma cruzi as indicated by an increased longevity when challenged with lethal doses of BT. The phagocytic activity of mice injected 24-72 h earlier with CT is increased, while that of mice injected with BT is decreased. The data suggest that parasite factors acting on the early phase of the immune response play a role in the outcome of the infection.

Trypanosoma cruzi epimastigote lysates presented proteolytic activity both at pH 7.0 (Km = 2.5 mg casein/ml, Km = 12.2 mg hemoglobin/ml) and at pH 3.0 (Km = 2.5 mg hemoglobin/ml). A proteinase was isolated from these lysates by using three different steps: 1) Precipitation at -20 degrees C, pH 4.5, with 80% acetone; 2) Sephadex G200 chromatography and 3)Affinity chromatography on columns of Sepharose 4B coupled to p-aminophenylmercuri-acetate. The isolated proteinase, which is probably an SH-dependent enzyme, was able to hydrolyze hemoglobin at pH 3.0 and both casein and hemoglobin at pH 7., but was unable to hydrolyze the esterase synthetic substrate tested. A single enzyme of molecular weight 60,000 could be detected in purified preparations when analysed by disc gel electrophoresis, crossed immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis and Sephadex chromatography. Antibodies induced by the purified proteinase, shown to be specific for proteinase molecules by line immunoelectrophoresis experiments, reacted with epimastigota of the Y strain and with trypomastigota and amastigota of five different strains tested. Electron microscopy observations of peroxidase labeled preparations indicated that the proteinase could be found at the surface of different forms of the parasite.

An adhesin with fimbria-like properties was present in four strains of verocytotoxigenic Escherichia coli isolated from diarrhoeic calves in Brazil.

Three enterotoxigenic Escherichia coli (ETEC) strains (coded 62, 104, and 567/7) isolated from piglets with neonatal diarrhea produced only a thermostable enterotoxin. Although these strains showed mannose-resistant microhemagglutination (MRMH), the responsible factor was serologically different from the known hemagglutinating colonization factors from porcine strains (K88, K99, and F41). Bacterial cells from these strains adhered to HeLa cells and pig brush borders. Electron microscope studies revealed the presence of fimbria-like structures on bacterial cells grown at 37 C but not on cells grown at 18 C. The antiserum prepared from partially purified fimbrial antigen (provisionally called F42) inhibited chicken erythrocyte MRMH caused by these strains as well as adherence of strain 567/7 to HeLa cells and to pig brush borders. These data taken together suggest the existence of a new hemagglutinating adhesin that is different from those so far described for porcine ETEC.

A fraction (FA) has been isolated from the sediment obtained from Trypanosoma cruzi of epimastigote lysates centrifuged at 1500 x g, for 30 min. This fraction, obtained by extracting sediments, for 10 min, with ice-cold 0.1 N NaOH, exhibited a single component, a glycoprotein, when analysed by electrophoresis in 1% agarose gel and presented a single faint precipitation line in immunoelectrophoresis experiments. FA components were unable to penetrate polyacrylamide gel matrix, even when 1% SDS 4% polyacrylamide gels were used, unless previously hydrolyzed by parasite proteinase. Under this condition FA preparations presented at least four glycoproteins components as detected by electrophoresis in 1% SDS 15% polyacrylamide gels. FA obtained from Y strain was able to inhibit agglutination reactions between anti-epimastigote sera and epimastigote of either Y or Nic strains. Anti-FA antibodies, elicited in 4 out of 12 rabbits inoculated with this fraction, gave positive immunofluorescence and immunoperoxidase reactions with blood trypomastigota and tissue amastigota, obtained from mice infected with any of 6 different strains of T. cruzi. These reactions which were inhibited by FA preparations were completely abolished if antisera were absorbed with living epimastigota of Y or Nic strains.

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37 degrees C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandem-crossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100 degrees C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.