Mouse Intravenous Challenge Models and Applications
Murine intravenous (IV) challenge models have been widely used in medical mycology to study fungal virulence, host responses, and antifungal efficacy. This chapter describes the well-characterised Candida albicans IV challenge model, where fungal cells ar
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1. Introduction Mouse intravenous (IV) challenge models have been a feature of medical mycology for over 50 years, with the virulence of many pathogenic fungi assessed in this way (1–10). Although the model bypasses the requirement for the fungus to escape the gut and cross endothelial barriers to enter the bloodstream, it reliably generates acute, disseminated infections involving deep organs. However, some species, e.g. Candida glabrata, are incapable of generating lethal infections in immunocompetent mice, and immunosuppression is required before a lethal infection can develop, even when “hypervirulent” fungal strains are created (11). The best characterised mouse fungal infection model is the Candida albicans IV challenge model (1, 2, 12, 13). This is a reliable, reproducible model and mimics severe human disseminated disease (13). Because this model is so well characterised, it has been extensively used to study C. albicans virulence (14, 15), host responses (16), and antifungal therapy efficacy (17).
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_35, © Springer Science+Business Media, LLC 2012
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D.M. MacCallum
In this chapter, I describe the C. albicans murine IV challenge model, where a bolus of fungal cells is administered into the lateral tail vein of mice, and infection progression is monitored by weight change and fungal organ burdens. Issues surrounding the use of different C. albicans strains/isolates and mouse strains, as well as various downstream uses for mouse samples post-infection, are also discussed. Although this chapter concentrates on C. albicans, the principles are the same for IV infection of mice with other pathogenic fungi.
2. Materials 2.1. Candida albicans Growth and Preparation of Inocula
1. C. albicans clinical isolates or laboratory strains (see Note 1) 2. YPD: 1% (w/v) yeast extract, 2% (w/v) mycological peptone, and 2% (w/v) glucose. 3. Sabouraud agar plates: 1% (w/v) mycological peptone, 4% (w/v) glucose, and 2% (w/v) agar. 4. NGY medium: 0.1% (w/v) Neopeptone, 0.4% (w/v) glucose, and 0.1% (w/v) yeast extract (see Note 2). 5. Wire inoculation loop or disposable plastic inoculation loops. 6. Sterile physiological saline (see Note 3). 7. Neubauer haemocytometer.
2.2. Infection of Experimental Animals
1. Female BALB/c mice, 6–8 weeks old (specific pathogen-free) (Harlan) (see Notes 4–9). 2. Weighing balance. 3. Heat lamp or warm box (see Note 10). 4. Mouse restraining device (see Note 11). 5. 26-G or 27-G syringe needles (see Note 12). 6. 1-mL Leur-Lok syringes. 7. Tissues/cotton wool.
2.3. Assessing Experimental Infection
1. Blunt-ended microcentrifuge tubes (2 mL) containing 0.5 mL sterile physiological saline (optional: protease inhibitors added to saline) (weights recorded). 2. 70% Ethanol. 3. Sterile scissors and forceps. 4. CAT homogeniser (model: X1030D) and dispersal tool.
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Murine IV Challenge Model
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3. Methods 3.1. Experimental
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