Immunization of mice by intracutaneous inoculation with viable virulent Cryptococcus neoformans: immunological and histopathological parameters

Abstract

Immune responses, including protection and delayed hypersensitivity, were evaluated in experimental murine cryptococcosis. Mice were immunized by the intracutaneous inoculation of viable virulent Cryptococcus neoformans yeasts. Response to the cutaneous infection was evaluated histologically and by cultural assays of the internal organs, as well as by intravenous challenge with the same strain. Protection was assessed by survival, histopathology, and quantitative organ culture. The intracutaneous inoculation of cryptococci resulted in a local inflammatory response that effectively limited dissemination of the organisms systemically and induced the development of delayed hypersensitivity demonstrable with a membrane extract of C. neoformans and with soluble cytoplasmic substances. A protective response was induced by the cutaneous inoculation of cryptococci as well, in that immunized animals survived longer, with about 25% of the challenged group ridding themselves completely of the cryptococci. Protection could be demonstrated by cultural analyses, but all animals, whether control or immunized, allowed considerable multiplication of the inoculum during the first 4 weeks after intravenous challenge. It would appear, therefore, that the protective mechanism(s) required additional antigenic stimulation before it could eventually function to eliminate all cryptococci from tissues. Histologically, there were no differences in pathology of the internal organs between immunized and unimmunized animals. Although the model described herein for the induction of immune responses in murine cryptococcosis has at least one drawback, viz., the presence of cryptococci in the skin lesion of many animals throughout the duration of the experiment, it does have the advantage that the immune responses were stimulated by a virulent strain and only minimal dissemination occurred. Therefore, lymphocytes could be removed from animals that were not contaminated with cryptococci for in vitro and in vivo transfer.