Summary The protective role of humoral antibodies in the resolution of systemic candidiasis remains controversial. Investigation of the humoral immune responses in mouse strains of varying susceptibility to infection may demonstrate a link between mouse strain susceptibility, antibody production and specificity, and the ability to resolve an infection. The antibody response in five different strainsof mice during a primary imune response to systemic infection with Candida albicans was investigated. Immune sera were fractionated by protein A affinity chromatography to yield fractions containing IgGl, IgG2a and IgG2b immunoglobulins. BALB/cmiceof low susceptibility to the infection and DBA/2J mice of high susceptibility produced increased levels of the IgGl isotype and decreased levels of the IgG2a isotype. AKR, CBA/H and C57B1/6J mice of moderate susceptibility produced antibodies predominantly of the IgG2a isotype. The patterns of antigen recognition by antibodies in immune sera and in fractions obtained after protein A chromatography of immune sera were investigated by western blotting and immunostaining. Antibodies from AKR(H-2K) and CBA/H (H-2k) mice reacted strongly after immunoblotting with antigens of 87 and 96 kDa. In contrast, immune sera from both the highly susceptible DBA/2J (H-2d) mice and the resistant BALB/c (H-2d) mice reacted strongly with an antigen of 48 kDa. C57BI/6J (H-2b) mice produced variable antibody reactivity to antigens of 48, 65, 66 and 79 kDa depending on the IgG subclass tested. The IgG subclass responses and the patterns of antigen recognition in these mice suggest that humoral responses to C albicans may be restricted by H-2 haplotype. There was no clear correlation between humoral immunity and resistance or susceptibility to infection with C. albicans.

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Reported targets of the specific immune responses to Candida albicans in human candidiasis include a 47-kDa breakdown product of a 90-kDa heat shock protein (HSP 90) (R.

Key terms: Colony forming units, viability testing, systemic candi-is, cell respirationInvestigation of the immune responses in a murine model of systemic candidiasis is currently being performed in our laboratory. AKR, BALB/c, CBNH, C57Bl/6J, and DBA/2J mice vary in their ability to resolve a systemic infection with Candida albicans (1,5,7). The mice are routinely inoculated intravenously (iv) with a sublethal dose of C albicans blastospores. Following inoculation the number of viable cells in the inoculum is usually estimated by determining the number of colony forming units (cfu) per milliliter. To ensure that the mice receive uniform numbers of viable C. albicans blastospores, the viability of C albicans in overnight cultures needed to be determined accurately prior to iv inoculation.Enumeration of cfu on solid phase culture medium is a common method of ascertaining the viability of C albicans in suspensions (2,4,13,15,17). Optical density ( 15), fluorescence microscopy ( 14,16), and/or a hemocytometer have been used to determine the viability of C albicans blastospores. More efficient methods of estimating C albicans viability have been developed by using fluorescent dyes and flow cytometry (3,6, Tetrabromofluorescein (eosin Y) is a vital stain which fluoresces green with an absorption maximum between 5 15 and 5 18 nm (9). The present study was performed to assess whether eosin Y, a cheap and readily available dye, could be used in conjunction with flow cytometry to assess the viability of C. albicans. Eosin Y and flow cytometry were utilized in combination to develop a rapid and reproducible method of accurately evaluating the viability of C. afbicans blastospores. 10,12). MATERIALS AND METHODS Culture ConditionsA single clinical isolate of C. albicans (KEMH 5) was obtained from the Department of Pathology, King Edward Memorial Hospital, Subiaco, Perth, Western Australia. The organism was cultured in yeast extract peptone dextrose (YEPD) broth (1% yeast extract, 2% peptone, 2% glucose) overnight at 30°C in a shaking water bath. A 1 ml aliquot of the overnight culture was pelleted by centrifugation at 2,OOOg for 5 min and washed twice with 0.15 M sodium chloride (saline). The cell pellet was resuspended in 10 ml of saline. To ensure that the viable control suspensions contained 100% viable organisms, cultures in mid to late log phase were used. Staining ConditionsWashed C albicans blastospores were resuspended in 0.01% tetrabromofluorescein in 0.1 5 M sodium chloride (eosin Y solution) and incubated at 25°C for 10 min. VIABILITY TESTING O F C. ALBICANS USING EOSIN Y 3714°C. Fluorescence activity and cell staining characteristics of the stock eosin Y solution were found to decrease with time (unpublished observations). Flow CytometryPrior to analysis of C. albicans suspensions, the flow cytometer was standardized using Immunocheck beads (Cbulter Cytometry, Hialeah, FL). Stained suspensions of C. albicans were measured for fluorescence on a Coulter EPICS-PROFILE I1 using an air-cooled argon ion laser operati...