Invasive Models of Histoplasmosis
Histoplasmosis results from infection with the fungal organism Histoplasma capsulatum. Most Histoplasma research today uses models based on primary pulmonary infection. Following inhalation, conidia are rapidly ingested and intracellularly convert to the
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1. Introduction Histoplasmosis results from infection with the fungal organism Histoplasma capsulatum. Exposure to the organism occurs via the respiratory route following inhalation of infectious particles (1). Micro- or macroconidia, sexual spores, or hyphal fragments act as the infectious propagules giving rise to infection. Following inhalation, conidia are rapidly ingested and convert to the yeast form intracellularly. After initial pulmonary infection, organisms are carried throughout the body giving rise to disseminated infection. Most individuals spontaneously clear the infection and recover but live organisms can remain within the body and reactivate at a later stage. Most Histoplasma research today uses models based on primary pulmonary infection, although older studies utilized intraperitoneal and intravenous routes of infection (2–4). A model of central
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_37, © Springer Science+Business Media, LLC 2012
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nervous system (CNS) infection following intracranial inoculation of organisms has also been described (5). This chapter focuses primarily on infections initiated via the respiratory tract following intranasal inoculation of organisms. Three murine models of infection have been developed which mimic human disease; primary infection following initial exposure to the organism, secondary exposure to Histoplasma organisms following spontaneous recovery from primary disease and a model of reactivated infection following spontaneous clearance of primary disease (3, 6, 7).
2. Materials 1. H. capsulatum strain(s) (see Note 1). 2. Histoplasma macrophage (HM) medium: 10.6 g Hams F12powered tissue culture medium, 5.96 g HEPES, 1 g glutamic acid, 10 mL 100× cysteine solution (0.84 g cysteine, 50 mL 1 N HCl, adjust to 100 mL with water and filter sterilize), and 25 μM ferrous sulfate; adjust to 1,000 mL, pH to 7.5 and filter sterilize. 3. HMM plates: HM medium as above but adjust volume to 500 mL to give 2× HMM media and warm to 50–60°C. Prepare 500 mL agarose by adding 10 g SeaKem ME® agarose to water and autoclave for 20 min. Allow agarose to cool to 50–60°C, mix with 2× HMM and pour plates. 4. Phosphate-buffered saline (PBS). 5. Hemocytometer. 6. Sterile centrifuge tubes. 7. Anesthesia equipment. 8. Mice (see Note 2). 9. Rat anti-mouse-CD4, rat anti-mouse-CD8, and (optional) rat anti-mouse-Th1.2 antibodies (National Cell Culture Center).
3. Methods 3.1. Preparation of H. capsulatum Inoculum (see Note 3)
All manipulation of H. capsulatum should be performed in a biosafety cabinet using approved protocols (8). 1. Scrape H. capsulatum yeast phase organisms from a plate or slant of actively growing colonies. Inoculate cells into 1–2 mL HMM media, vortex briefly to generate a single cell suspension, and remove an aliquot for enumeration using a hemocytometer.
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Animal Models of Histoplasmosis
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2. Inoculate 5–50 mL HM m
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