Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi.

A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.

Immunopathology of experimental Chagas' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process.

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice.

Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 microgram to 1000 micrograms. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.

Absorption of Mycobacterium bovis BCG administered by the oral route.

The action of gastric and duodenal juices on BCG as well as on its absorption and its distribution in the organs after intragastric administration in mice were studied. A significant decrease in BCG oxygen uptake and a moderate loss of viability were found after 2 h treatment with gastric juice. Using duodenal juice, a marked decrease of respiration and a notable fall in viability were observed. Labelling of BCG with carbon-14 was accomplished using [14C]glycerol as a precursor of mycobacterial lipids. Similar levels of radioactivity were recovered in organs of mice 24 h after intragastric administration of 14C-BCG, sonicated 14C-BCG and [14C]glycerol. The level of 14C-BCG remained stable from 6 to 24 days, while sonicated 14C-BCG and [14C]glycerol defined a biological decay process. Studies of biological decay from the small intestine and liver indicated that the absorptive process started rapidly and reached its highest level at 24 h, declining thereafter according to the complexity of the material given to mice. However, living bacilli were not cultured from organs of mice given single doses of unlabelled BCG. Therefore, judging from the above data it may be concluded that the majority of BCG bacilli absorbed intact were not alive.

[Humoral and cellular immunity in children vaccinated with oral B.C.G. (author's transl)]. / Imunidade humoral e cellular em crianças vacinadas com B.C.G. pela via oral.

The humoral and cellular immunity of 23 children with ages between 1 and 5 years, nonreactors to 10 mu of PPD, were investigated after oral vaccination with one dose of 100 mg of fresh oral BCG, Rio de Janeiro strain (Moreau strain). The tests performed shortly before and 70 days after vaccination showed that the schedule used was neither sufficient to stimulate the production of antibody anti-PPD, nor to change the levels of serum immunoglobulins and T, T-active and B blood lymphocytes. However, about 60% of the children became responsive to 10 mu of PPD after treatment and all gave positive reactions to PPD on "in vitro" assays of leukocyte migration inhibition. New schedules for oral vaccination with fresh BCG are in progress in our laboratory.