Experimental systemic cryptococcosis in SCID mice

Journal of Medical & Veterinary Mycology 1996, 34, 331-335 Accepted 8 March 1996 Experimental systemic cryptococcosis in SCID mice K. V. CLEMONS,*~§ R. A Z Z I t & D. A. STEVENS*~§ *Department of Medicine, Division of Infectious Diseases and tDepartment of Pathology, Santa Clara Valley Medical Center, San Jose, CA 95128; ~.California Institute for Medical Research, San Jose, CA 95128; and §Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford CA 94305, USA Keywords cryptococcosis, Cryptococcus neoformans, meningitis, SCID mice Introduction Cryptococcus neoformans is a ubiquitous opportunistic fungal pathogen that can cause serious disease in both normal and immunocompromised hosts [1]. To examine the mechanisms of host resistance, various experimental models of cryptococcosis have been described utilizing both immunocompetent and immunodeficient animals [1-7]. In addition, models of cryptococcal infection have been used to determine the potential therapeutic value of new antifungal or immunomodulatory agents [2,8,9]. Because patients with acquired immunodeficiency syndrome or other causes of severe immunodeficiency are at risk from systemic infection with involvement of the central nervous system [10,11], models of disease in immunocompromised animals are important, especially for the study of therapeutics. Severe combined immune deficiency (SCID) in mice is a result of the scid mutation, which causes these animals to be severely deficient in functional T and B lymphocytes, although NK cells, monocytes and granulocytes are normal [reviewed in refs 12-14]. These animals provide a model system that can be used to study the role of different arms of acquired immunity to infective agents. Huffnagle et al. [15] have reported that SCID mice Correspondence: Dr K. V. Clemons, Division of Infectious Diseases, Santa Clara Valley Medical Centre, 751 South Bascom Ave., San Jose, CA 95128, USA. Tel.: (408) 998 4557; Fax: (408) 998 2723. (~;) 1996 ISHAM are more susceptible to pulmonary challenge with C. neoformans and that pulmonary clearance of the organism was increased by almost 100-fold by adoptive transfer of immune T cells. However, less extrapulmonary dissemination to the spleen and brain occurred in SCID mice than in athymic nude mice [15]. Because of this unexpected resistance to extrapulmonary dissemination displayed by SCID mice [15], and because severely immunodeficient patients frequently have central nervous system infection, the pulmonary model may not be useful for the study of the efficacy of therapeutic agents. To further examine the susceptibility of SCID mice to infection with C. neoJormans, we have established systemic infection to mimic disseminated human disease and assessed the comparative lethality of different numbers of inoculated yeast given to SCID and their immunocompetent counterparts. Materials and m e t h o d s Five- to six-week-old female C.B-17/IcrTac-scidDF (SCID) and C.B-17/IcrTac immunocompetent congenic control mice were obtained from Taconic (Germantown, NY). SCID mice were housed in microisolator cages. All caging, food and water were sterilized by autoclaving; food and water were provided ad libitum. Immunocompetent control mice were housed under conventional conditions and provided sterilized food and water ad The susceptibility of severe combined immune deficiency (SCID) mice to experimental systemic cryptococcosis was studied. SCID mice were at least 16-fold more susceptible to lethal infection than were immunocompetent control mice (P < 0"001). Histological assessment showed that control mice responded with a granulomatous inflammatory response, whereas SCID mice showed no evidence of a cellular host response. In a therapeutic study, 5 mg k g - I day I of fluconazole caused a significant reduction in the infectious burden in the spleen, liver, kidney and lungs of treated mice compared with no treatment. These results indicate that a model of systemic cryptococcosis with meningitis in SCID mice may have utility in the study of immunology and therapy of this disease in the severely immunocompromised host. U Clemonset al. I00 80 <D z; L I , , , , I , , , , 5 I 10 , ] , [ 15 20 25 Days Postinfection 30 libitum. On day 0, randomly chosen groups of 12 SCID mice and ten control mice were infected intravenously with C neoformans strain 9759 given in 0-25 ml of saline prepared as described previously [8,9]. This strain of C neoformans was originally provided to us by Christine Morrison (Centers for Disease Control and Prevention, Atlanta, GA) and is an encapsulated serotype A strain of moderate virulence. One group of both SCID and control mice received either 1 x 105, 2.5 x 10 4, 6 x 103 or 1'5 x 10 3 viable yeasts of C. neoformans. Two SCID mice were not infected and served as controls for the possible development of secondary infection during the course of the experiment. The comparative susceptibility of the SCID and control mice was determined by tallying the number of deaths to 42 days of infection. Statistical evaluation of the mortality curves was done using a Wilcoxon rank sums test [16]. At various times after infection, two SCID mice from the various groups were killed by CO2 asphyxiation just prior to death and the organs removed for histological assessment of disease involvement. Tissues were placed in buffered formalin, embedded in paraffin, sectioned and stained by periodic acid Schiff (PAS) and haematoxylin and eosin for histological examination. A second experiment was carried out to evaluate the potential utility of this model for experimental therapeutic work. Infected animals were treated with a dose of fluconazole similar to previously described models studied in immunocompetent mice [8]. Five- to six-week-old female SCID mice were infected i.v. with 1125 viable colony-forming units (CFU) of C. neoformans 9759. Therapies were initiated 2 days after infection with ten mice receiving no treatment and ten mice receiving 5 mg kg - I day 1 of fluconazole (Pfizer, Groton, CT) given by gavage. Fluconazole powder was dissolved in , 35 , , , I 40 , , , Fig. 1 Cumulative mortality of SCID and control C.B-17 mice infected with various numbers of viable C neoformansyeasts. sterile water and administered twice daily in 0' 1 ml doses of 2.5 mg kg ~ per dose for 10 consecutive days. Previous experiments have shown no differences in outcome between mice given gavage with water compared with those not treated. One day after the cessation of therapy all mice were killed and the burden of C. neoforrnans in various organs was determined by quantitative plating [8,9]. Comparative burdens of organisms in the organs were analysed using a Mann-Whitney U-test [16]. Results The results of the survival study are presented in Fig. 1. Both SCID and control mice were susceptible to lethal systemic infection with C. neoformans in a doseresponsive manner. Ni (...truncated)