Experimental Oropharyngeal and Gastrointestinal Candida Infection in Mice

Chapter 79

Experimental Oropharyngeal and Gastrointestinal Candida Infection in Mice A. M. Flattery, G. K. Abruzzo, C. J. Gill, J. G. Smith and K. Bartizal

Background of model Maintenance of Candida albicans in the gastrointestinal tract of non-immune-compromised mice is short-lived, with the host immune system rapidly clearing the yeast. Evidence suggests that cell-mediated immunity, and more specifically, CD4 + T lymphocytes, play an important role in resistance to mucosal candidiasis (Rogers and Balish, 1980; Balish et al., 1990; Cantorna and Balish, 1991). In particular, the decrease in CD4 + T-lymphocyte counts associated with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) has been correlated with the rise in cases of alimentary tract candidiasis (Epstein et al., 1984; Klein et al., 1984; Glatt et al., 1988; Pankhurst and Peakman, 1989). Other mouse models of mucosal candidiasis have been described which utilize combinations of chemically induced immune suppression, elimination or alteration of host microflora by administration of antibiotics, high inocula, trauma, infant animals or animals with congenital, functional, physiological, immunological or metabolic defects to facilitate colonization of the gastrointestinal tract by C. albicans (Nolting, 1975; Jorizzo et al., 1980; Field et al., 1981; Guentzel and Herrera, 1982; Ekenna and Fader, 1989; Cole et al., 1990; Narayanan et al., 1991). This model of oropharyngeal and gastrointestinal candidiasis was designed to represent the immune status of the patient population in which mucosal candidiasis is prevalent. It employs a combination of selective CD4 + Tcell depletion to initiate a specific immune deficiency and antibiotic reduction of the normal gastrointestinal microflora to allow colonization of the alimentary tract by Candida. Efficacy of therapy with a variety of known and novel antifungals may then easily be assessed in this model.

Animal species Mice deficient in CD4 + T-lymphocyte function facilitate prolonged colonization of the alimentary tract by C. albicans. This immune deficiency may be created in a variety of Handboo k of Animal Models of Infection

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ways in both immune-competent and immune-deficient mice as well as in outbred and inbred strains. Mice may be selectively depleted of CD4 + T-lymphocytes by treatment with a rat immunoglobulin G2 monoclonal antibody (MAb) secreted by GK1.5 hybridoma cells (American Type Culture Collection, Rockville, MD, Culture #TIB 207), which is specific for mouse CD4 + T cells. Alternately, mice may be injected directly with the hybridoma cells subcutaneously, leading to in vivo secretion of MAb and depletion of the CD4 + population. We commonly use complement component 5-deficient female DBA/2 mice (Taconic, Germantown, NY) since these mice show acceptable duration of depletion of CD4 § T cells and are easily colonized with C. albicans both orally and in a disseminated model of candidiasis (Bartizal et al., 1992). Similar methods of CD4 + T-cell depletipn have also been used in C3Heb/FeJ mice with comparable results and in BALB/c mice with a shorter duration of CD4 + T-cell depletion (McFadden et al., 1994). Presumably other strains of mice may be used; however the efficacy of the immune suppression would need to be verified by fluorescence-activated cell sorter (FACS) analysis and perhaps the level of MAb administered adjusted accordingly. Recently, transgenic mice have been developed which lack certain genes necessary for CD4 +T-lymphocyte production and thus which are CD4 + T-lymphocyte deficient. These mice are available from multiple sources, including Jackson Laboratories (Bar Harbor, ME) and eliminate the need for antibody treatment altogether. Fecal C. albicans colonization levels after oral inoculation in these transgenic animals are similar to those in MAb-induced CD4 +T-lymphocytopenic mice.

Immune suppression In DBA/2 mice three intraperitoneal (i.p.) injections of 300pg purified MAb per mouse in sterile saline administered 3 days prior to, the day of and 1 week after challenge maintain depletion of CD4 + T cells through 14 days after challenge. A single subcutaneous (s.c.) injection of 9 x 106 GK1.5 hybridoma cells 4 days prior to challenge maintains CD4 +T-cell depletion through 21 days after challenge. Copyright 9 1999AcademicPress All rights of reproduction in any form reserved

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Cell culture GK1.5 hybridoma cells are cultured in high glucose Dulbecco's Modified Eagle's Medium (D-MEM, Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 1% L-glutamine (Sigma), 100 units/ml penicillin (Sigma) and 100 lag/ml streptomycin (Sigma) at 37~ under 5% CO 2. Cells for injection should be passaged and incubated for log phase growth. Cultures are then harvested, centrifuged for 8 minutes at 400g to pellet cells and washed twice with the above medium. Resuspended cells should be counted on a hemacytometer, with viability confirmed by trypan blue dye exclusion, and adjusted to the appropriate concentration for CD4 + T-cell depletion by s.c. injection or for generation of MAb by ascites production.

Ascites production Athymic nu/nu mice are used for ascites production. In our experience outbred athymic nu/nu Swiss Webster mice (Taconic) work well. Mice are primed by i.p. injection with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Sigma). Ten days after pristane priming, mice are injected i.p. with 5 • 106 GK1.5 hybridoma cells prepared as stated above. Ascites is then collected from the mice, centrifuged for 10 minutes at 400g to remove cellular debris, and then stored frozen at-20~ until use for antibody purification. MAb is purified from ascites by passage over a protein G column (MabTrap G, Pharmacia LKB, Piscataway, N]) and quantitated by Bio-Rad Protein Assay (Bio-Rad, Rockville Center, NY) using a bovine gamma globulin standard. Purified MAb is then stored a t - 2 0 ~ until used for immune suppression.

FACS analysis The depletion of CD4 + T lymphocytes is monitored using FACS analysis. Splenic T lymphocytes may be stained with fluoresceinated MAbs specific for mouse CD4 +, CD8 + or CD3 + T cells. Spleens are removed aseptically and using frosted glass microscope slides splenic tissue is teased apart to create a cell suspension. Cells are suspended in 3 ml phosphate-buffered saline (PBS, GIBCO, Grand Island, NY) and centrifuged for 5 minutes at 400g. The supernatant is decanted and the cell pellet resuspended in 1 ml ACK lysing buffer (GIBCO), vortexed for 1 minute to lyse red blood cells, then diluted in 3 ml PBS and centrifuged for 5 minutes at 400g. The supernatant is again decanted and cells are resuspended in 4 ml PBS. Then, 100 Ill of the spleen cell suspension is incubated with rat anti-mouse MAb at a concentration of 5 tag/ml for 30 minutes at room temperature. The cells are stained either with fluorescein isothiocyanate (FITC) conjugated L3T4 (CD4) (PharMingen, San Diego, CA) and R-phycoerythrin (PE) conjugated Ly-2 (CD8a) (PharMingen), or with FITC conjugated Thy-l.2

(PharMingen), which reacts with 100% of T cells in mice expressing the Thy-l.2 allele. Cells are washed with 3 ml PBS and centrifuged for 5 minutes at 400g. The supernatant is then decanted and the cell pellet resuspended in 200 lal propidium iodide (Sigma) at 1 tag/ml. Samples are run on the FACScan analyzer (Becton Dickinson, San lose, CA) to determine percentage of total lymphocytes which are CD4 +, CD8 +or CD3 +T cells.

Preparation of animals Housing Animals should be housed in sterile microisolator cages and given sterile feed and water ad libiturn. Mice are pretreated with gentamicin (Garamycin injectable, Schering, Kenilworth, NJ), a non-absorbable broad-spectrum antibacterial, at 0.1 mg/ml in the drinking water from 4 days prior to C. albicans challenge through 3 days postchallenge to reduce the normal gastrointestinal microflora, allowing less competition for colonization of the gastrointestinal tract by C. albicans.

Preparation of inocula and infection process Stock cultures of C. albicans MY1055 (Merck Culture Collection) are maintained by monthly transfers on Sabouraud dextrose agar (SDA, BBL, Cockeysville, MD). Growth from an 18-24-hour SDA culture of C. albicans is suspended in sterile saline to a concentration of 108 cells/ml determined by hemacytometer count and verified by plate counts. Mice are challenged by gavage with 0.2 ml of the yeast suspension (approximately 2 • 107 cells/mouse) and additionally by swabbing their oral cavities with the yeast suspension, while gently abrading the buccal mucosa by rotation of the swab. This model should be amenable to use of other strains of C. albicans as well as other species of Candida; however, colonization levels may vary between strains and species and challenge amounts may need to be adjusted accordingly.

Key parameters to monitor infection and response to treatment Infection in this model is confined to the oropharynx and gastrointestinal tract and translocation of the yeast out of the gastrointestinal tract is rare, and in fact difficult to induce. Despite high levels of colonization mice show no outward signs of illness. The extent of colonization of the alimentary tract after challenge may then easily be moni-

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tored throughout the study by culturing fecal samples and by swabbing the oral mucosa. This model also allows the investigator to follow individual mice throughout the course of treatment since mice need not be euthanized for sampling. Fresh fecal pellets are collected from each mouse, weighed, homogenized in sterile saline, serially diluted and plated on Sabouraud's dextrose agar containing 50pg/ml chloramphenicol (SDAC) for inhibition of bacterial growth. Oral swabs may also be taken from each mouse and plated on SDAC for a qualitative estimate of oral Candida load. At termination of the study segments of the alimentary tract may also be cultured as above to determine levels of colonization. Typically DBA/2 mice carry a Candida load of between 4 and 5 log colony-forming units (cfu) per gram feces through at least 14 days. Oral Candida cfu may vary markedly depending on the technique of the individual swabbing the oral cavity and the method of applying the swabbed material to the culture plate.

therapy the rapidity and extent of efficacy may easily be determined. Also, rebound in growth of any uncleared yeasts may be seen if fecal cultures are taken 24 hours after the termination of therapy at day 14 after challenge. We have used this model to assess the efficacy of novel antifungal agents as compared to the efficacy of agents currently marketed for oropharyngeal candidiasis. The activity of currently marketed agents in this model correlated well with their efficacy in humans. Fluconazole administered for 10 days in the drinking water at 100-400 pg/ml (approximately 25-100mg/kg per day) rapidly decreases the fecal and oral Candida load in this model. The polyene nystatin administered in the drinking water was somewhat less efficacious, with fecal and oral colonization levels decreasing more slowly and to a lesser extent than the azole (Flattery et al., 1996).

Antifungal therapy

There is no difference in gastrointestinal colonization by Candida in mice depleted of CD4 + T cells by either the hybridoma or antibody method; however, there are certain side-effects associated with each method. In long-term experiments, multiple injections of rat GK 1.5 MAb may cause some mortality in mice. A single injection of hybridoma cells requires no antibody purification and less animal handling but it also has some disadvantages. Approximately 50% of DBA/2 mice injected with hybridoma cells develop visible tumors beginning 3 weeks following hybridoma injection. Many of these tumors become so large that they are lethal, while approximately one in eight tumors regresses. All mice with visible tumors are depleted of CD4 +T cells and depletion may continue for up to 5 weeks after tumor regression. In mice with no visible tumors CD4 § T cells begin to reappear at 4 weeks after injection of hybridoma cells. For experimental procedures requiring less than 3 weeks to complete, these tumors pose no problem. However, in long-term experiments, significant mortality and the reappearance of CD4 +T cells after 4 weeks must be anticipated. These tumors are observed using DBA/2 mice; however, in other strains of mice such as BALB/c and C3Heb/FeJ mice, visible tumors have not formed and the length of CD4 + T-cell depletion varied (McFadden et al., 1994). For these reasons use of transgenically CD4 + T-lymphocyte deficient mice, although possibly more costly, might be preferable to either antibody or hybridoma treatment, especially for long-term experiments. Although this model mimicks the immune status of the patient population in which the infection commonly occurs, it may not be truly representative of the disease state seen in humans. Histology of the oropharynx and gastrointestinal tract of DBA/2 mice shows little adhesion of C. albicans to the mucosal surface and little, if any, inflammatory

Many different routes of administration of antifungal therapy may be used in this model, including parenteral injection, administration by garage (p.o.) and administration in the drinking water. Administration i.p., s.c. and p.o. allows exactly measured amounts of drug to be delivered, while administration in the drinking water gives an approximate dose level, given that mice drink approximately 5 ml of water per day (Ralston Purina Company, 1961). However, the drinking water route of administration allows for therapy similar to the "swish-and-swallow" routes now used clinically with both Diflucan (fluconazole for oral suspension) and Fungizone (amphotericin B oral suspension). The route chosen may be affected by the properties of the compound given. For example, administration in the drinking water requires that the compound be relatively soluble in a formulation suitable for oral administration to mice, since insoluble compounds tend to settle at the bottom of the water bottle. These compounds must also be relatively stable at room temperature. An experimental chemotherapeutic not having these characteristics may need to be administered by an alternate route. Typically, antifungal therapy in this model is begun 3 days after challenge so that the yeast has sufficient time to colonize the alimentary tract and also so that a pretreatment colonization level may be determined. Therapy is then begun by any of the above routes and continued for varying lengths of time based on the characteristics of the antifungal under study. A dosing regimen of 10 days (days 3-13 after challenge) allows maximal time for the antifungal to reduce the Candida load, while also maintaining the test to within the 14 days in which antibody-treated mice are CD4depleted and colonization levels in control mice are still high. Since mice are monitored throughout the course of

Advantages and disadvantages of the model

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response, indicating a colonization rather than true infection. In 85% of DBA/2 mice intragastrically inoculated with C. albicans and administered antibiotics over a long term, small self-limiting loci of mucosal involvement in the stomach were seen, but C. albicans was eventually cleared without dissemination (Bistoni et al., 1993). In fact it has been shown that in mice a combination of both cell-mediated immunity and phagocytic cell defects is necessary for extensive infection or invasion of the gastrointestinal tract (Cantorna and Balish, 1990). The CD4 +T-lymphocyte-deficient DBA/2 mice offer a good model of chronic Candida without dissemination, in which in vivo antifungal activities correlate well with in vitro susceptibility of the isolate to the antifungal compounds.

Contributions of the model to infectious disease therapy With the increase in numbers of immunocompromised patients, there has been a significant rise in the incidence of fungal infections, including alimentary tract candidiasis. Unlike antibacterials, few antifungal agents exist which are both safe and efficacious. Also, with the incidence of azoleresistant Candida strains on the rise, the need for novel antifungal agents is increasing. This model of oropharyngeal and gastrointestinal candidiasis may give a good indication of the efficacy of novel agents as compared to currently marketed antifungals. These agents may then also be useful in more severe models of disseminated fungal disease.

References Balish, E., Filutowicz, H., Oberly, T. D. (1990). Correlates of cellmediated immunity in Candida albicans-colonized gnotobiotic mice. Infect. Immun., 58, 107-113. Bartizal, K., Abruzzo, G., Trainor, C. et al. (1992). In vitro antifungal activities and in vivo efficacies of 1,3-~-D glucan synthesis inhibitors L-671,329, L-646991, tetrahydroechinocandin B, and L-687,781 a papulacandin. Antimicrob. Agents Chemother., 36, 1648-1657. Bistoni, F., Cenci, E., Mencacci, A. et al. (1993). Mucosal and systemic T helper cell function after intragastric colonization of adult mice with Candida albicans. ]. Infect. D#., 168, 1449-1457. Cantorna, M. T., Balish, E. (1990). Mucosal and systemic candidiasis in congenitally immunodeficient mice. Infect. Immun., 58,

1093-1100. Cantorna, M. T., Balish, E. (1991). Role of CD4 + lymphocytes in resistance to mucosal candidiasis. Infect. Immun., 59, 2447-2455. Cole, G. T., Lynn, K. T., Seshan, K. R. (1990). An animal model for oropharyngeal, esophageal, and gastric candidosis. Mycoses, 33,7-19. Ekenna, O., Fader, R. C. (1989). Effect of thermal injury and immunosuppression on the dissemination of Candida albicans from the mouse gastrointestinal tract.J. Burn Care Rehabil., 10, 138-145. Epstein, J. B., Truelove, E., Izutzu, K. T. (1984). Oral candidiasis: pathogenesis and host defense. Rev. Infect. D#., 6, 96-106. Field, L. H., Pope, L. M., Cole, G. T., Guentzel, M. N., Berry, L. J. (1981). Persistence and spread of Candida albicans after intragastric inoculation of infant mice. Infect. Immun., 31, 783-791. Flattery, A. M., Abruzzo, G. K., Gill, C. J., Smith, J. G., Bartizal, K. (1996). New model of oropharyngeal and gastrointestinal colonization by Candida albicans in CD4 § T-cell-deficient mice for evaluation of antifungal agents. Antimicrob. Agents Chemother., 40, 1604-1609. Glatt, A. E., Chirgwin, K., Landesman, S. H. (1988). Treatment of infections associated with human immunodeficiency virus. N. Engl. ]. Med., 3, 1439-1448. Guentzel, M. N., Herrera, C. (1982). Effects of compromising agents on candidosis in mice with persistent infections initiated in infancy. Infect. Immun., 35,222-228. Jorizzo, J. L., Sams, W. M., Jr, Jegasothy, B. V., Olansky, A. J. (1980). Cimetedine as an immunomodulator: chronic mucocutaneous candidiasis as a model. Ann. Intern. Med., 92, 192-195. Klein, R. S., Harris, C. A., Small, C. B., Moll, B., Lesser, M., Friedland, G. H. (1984). Oral candidiasis in high-risk patients as the initial manifestation of the acquired immunodeficiency syndrome. N. Engl. J. Med., 311,354-358. McFadden, D. C., Powles, M. A., Smith, J. G., Flattery, A. M., Bartizal, K., Schmatz, D. M. (1994). Use of anti-CD4 + hybridoma cells to induce Pneumocystis carinii in mice. Infect. Immun., 62, 4887-4892. Narayanan, R., Joyce, W. A., Greenfield, R. A. (1991). Gastrointestinal candidiasis in a murine model of severe combined immunodeficiency syndrome. Infect. Immun., 59, 2116-2119. Nolting, S. (1975). Effect of antibiotics and cytostatic drugs on experimental candidiasis in mice. Mykosen. , 18, 309-313. Pankhurst, C., Peakman, M. (1989). Reduced CD4 + T cells and severe oral candidiasis in absence of HIV infection. Lancet, i, 672. Ralston Purina Company (1961). Laboratory Animal Care: Management of Laboratory Animals, the Mouse, section 2, p. 5. Ralston Purina Co., St. Louis, MO. Rogers, T. J., Balish, E. (1980). Immunity to Candida albicans. Microbiol. Rev., 44, 660-682.