Epimastigotes multiplying extracellularly and metacyclic trypomastigotes, stages that correspond to the cycle of Trypanosoma cruzi in the intestinal lumen of its insect vector, were consistently found in the lumen of the anal glands of opossums Didelphis marsupialis inoculated subcutaneously with infective feces of triatomid bugs.

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SYNOPSIS. A cytostome is described in culture forms and in intracellular stages of Trypanosoma cruzi and in culture forms of T. conorhini. The organelle opens into the flagellar pocket or close to it and runs deeply into the cell.

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Groups of mice received double infections with the Y and F strains of Trypanosoma cruzi, the first inoculum of either strain being followed by a second inoculum of the other strain on day 5, 15, 30-40, or 60-65. Parasites were re-isolated from blood into culture, either directly or with an intermediate passage in gamma-irradiated mice, at intervals between 7 and 35 days after the second inoculation. Strain identification in the re-isolated material was by electrophoresis of kDNA fragments generated by the EcoRI restriction endonuclease and by electrophoresis for glucosephosphate isomerase isozymes. Both strains were identified in 22% of re-isolates originating from the experimental mice and only one of them was present in the remaining re-isolates, strain F being the most frequent. In some instances either Y or F was re-isolated from the same blood source, depending on whether culturing had been preceded or not by passage through a mouse. These results are certainly related to strain differences in the various aspects of host-parasite relationship and, possibly, growth rates in culture. The results demonstrate that: (1) more than one strain of T. cruzi can coexist in the same host; (2) the timing and method of parasite isolation from the vertebrate host act as selective factors, and further passages (in mice or cultures) may completely eliminate one (or more) strain from originally mixed trypanosome population, and (3) kDNA restriction "fingerprints" and isozyme profiles are simple, sensitive, and reliable techniques for strain identification both in single and mixed preparations.

The use of an indirect fluorescent antibody test (IFAT) performed in a "sandwich" technique has demonstrated: (i) the usefulness of the test for the diagnosis of Trypanosoma cruzi infection in the opossum Didelphis marsupialis; (ii) the existence of differences in the serological response of the opossum, that were related to the parasite strain and were clearly evident during the follow-up of experimental infections in laboratory born specimens; (iii) that, despite a good correlation between serological and parasitological examinations, IFAT was the most sensitive diagnostic test used, followed by xenodiagnosis; and, (iv) that in general, the opossum D. marsupialis seems to be a good responder to T. cruzi antigens.