Animal Models for Candidiasis

Heather R. Conti1, Anna R. Huppler2, Natasha Whibley1, Sarah L. Gaffen1

1 Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 2 Division of Pediatric Infectious Diseases, Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.6
DOI:  10.1002/0471142735.im1906s105
Online Posting Date:  April, 2014
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Abstract

Multiple forms of candidiasis are clinically important in humans. Established murine models of disseminated, oropharyngeal, vaginal, and cutaneous candidiasis caused by Candida albicans are described in this unit. Detailed materials and methods for C. albicans growth and detection are also described. Curr. Protoc. Immunol. 105:19.6.1‐19.6.17. © 2014 by John Wiley & Sons, Inc.

Keywords: Candida albicans ; mouse model; fungal infection

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Disseminated Candidiasis in Adult Mice
  • Alternate Protocol 1: Gastric Disseminated Candidiasis in Adult Immunocompromised Mice
  • Basic Protocol 2: Oropharyngeal Candidiasis (OPC) in Mice
  • Basic Protocol 3: Vaginal Candidiasis in Mice
  • Basic Protocol 4: Dermal/Cutaneous Candidiasis in Adult Mice
  • Support Protocol 1: Candida Albicans Growth and Maintenance
  • Support Protocol 2: Preparation of Cortisone Acetate Suspension
  • Support Protocol 3: Periodic Acid–Schiff Staining of Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Disseminated Candidiasis in Adult Mice

  Materials
  • C. albicans blastospores, SC5314 or CAF2‐1 strain ( protocol 6)
  • Phosphate‐buffered saline ( ), sterile
  • 70% ethanol in a spray bottle
  • 6‐ to 8‐week‐old C57BL/6 mice
  • Tissue homogenizer (GentleMACS Dissociator, Miltenyi Biotec)
  • YPD agar (BD Difco) 100 mm × 15 mm plates (optionally containing 50 µg/ml ampicillin)
  • 1‐ml tuberculin syringes
  • 27‐G, 1/2‐in. needles
  • Dissecting pins
  • Sterile dissecting tools
  • Scale for weighing organs
  • GentleMACS C‐tubes (Miltenyi Biotec)
  • Additional reagents and equipment for preparing Candida suspension ( protocol 6), injection of mice (unit ), euthanasia of mice (unit ), and PAS staining ( protocol 8)

Alternate Protocol 1: Gastric Disseminated Candidiasis in Adult Immunocompromised Mice

  Materials
  • Adult male and female BALB/c (H‐2d) mice, 6 to 8 weeks old
  • 0.2 mg/ml vancomycin in drinking water
  • C. albicans blastospores, 3153 serotype A (3153A; ATCC)
  • Phosphate‐buffered saline (PBS; )
  • RB6‐8C5 MAb (anti‐mouse Ly6G from BioXCell)
  • Cyclophosphamide (optional)
  • 9‐cm Saboraud's dextrose plates (see recipe) containing 50 µg/ml chloramphenicol
  • 24‐G feeding needle (Popper & Sons) attached to a 1.0‐ml syringe or 1‐ml tuberculin syringe with a blunt‐end 21‐G needle tipped with polyethylene tubing (25‐mm i.d.; 61‐mm o.d.; Clay Adams)
  • Sterile dissecting tools including metal punch
  • Tissue homogenizer (Polytron‐Kinematica)
  • Additional reagents and equipment for restraint of mice (unit ), injection of mice (unit ), and euthanasia of mice (unit )

Basic Protocol 2: Oropharyngeal Candidiasis (OPC) in Mice

  Materials
  • Cortisone acetate suspension ( protocol 7)
  • 0.05% (v/v) Tween 20/PBS: add 10 µl Tween 20 to 20 ml sterile PBS (see for PBS)
  • C. albicans blastospore suspension, CAF2‐1 strain (2 × 107 cells/ml; protocol 6)
  • Phosphate‐buffered saline (PBS; ), sterile
  • 5‐ to 7‐week‐old C57BL/6 mice
  • Ketamine
  • Xylazine
  • 0.9% (w/v) NaCl solution, sterile
  • YPD plates of C. albicans ( protocol 6)
  • Sterile lubricant eye ointment
  • 10% neutral buffered formalin
  • RNALater (Life Technologies) or liquid N 2
  • Scale for weighing mice and tissues
  • 26‐G needle
  • 1‐ml tuberculin syringe
  • Cotton plugs
  • Cotton‐tip applicators, autoclaved (3 in., pack of 1000, Fisher)
  • Cage illuminated with heat lamp
  • Sterile dissecting tools including straight scissors and medium‐point forceps
  • GentleMACS C‐tubes (Miltenyi Biotec)
  • Straight razor blades
  • Additional reagents and equipment for preparing cortisone acetate suspension ( protocol 7), injection of mice (unit ), euthanasia of mice (unit ), and processing the kidneys for yeast infection ( protocol 1)

Basic Protocol 3: Vaginal Candidiasis in Mice

  Materials
  • Female CBA/J (J‐2α) mice, 8 to 10 weeks of age
  • Estradiol valerate (Sigma, cat. no. E1631) dissolved in sesame oil (Sigma, cat. no. S3547) immediately before use
  • C. albicans blastospores, 3153 serotype A (3153A; ATCC) ( protocol 6)
  • Phosphate‐buffered saline (PBS; ), sterile
  • 70% ethanol
  • Refrigerated centrifuge
  • Sterile filter (0.22‐µm)
  • Dissecting equipment, including curved forceps
  • Additional reagents and equipment for injection of mice (unit ), preparation of C. albicans suspension ( protocol 6), anesthesia of mice ( protocol 3), determination of fungal burden ( protocol 1, steps 13 to 14), paraffin sectioning (unit ), and PAS staining ( protocol 8)

Basic Protocol 4: Dermal/Cutaneous Candidiasis in Adult Mice

  Materials
  • 5‐7 week old C57BL/6 mice
  • C. albicans pseudohyphae, CAF2‐1 strain ( protocol 6)
  • YPD medium (e.g., BD Difco) containing 10% (v/v) fetal bovine serum (FBS)
  • 10% neutral buffered formalin
  • RNALater (Life Technologies) or liquid N 2
  • Phosphate‐buffered saline (PBS; ), sterile
  • Inverted microscope
  • Electric razor
  • Hemacytometer
  • 1‐ml tuberculin syringe and 25‐G needle
  • Dissecting pins
  • Sterile dissecting tools including straight scissors and forceps4‐mm punch biopsy tool
  • Pellet pestles (Sigma, cat. no. Z359947)
  • Additional reagents and equipment for preparation of Candida suspension ( protocol 6), injection of mice (unit ), and sacrifice of mice and plating of organ homogenates ( protocol 1),

Support Protocol 1: Candida Albicans Growth and Maintenance

  Materials
  • Candida albicans [laboratory strain CAF2‐1 (Conti et al., ) or SC5314 (ATCC no. MYA‐2876)] or other strain as called for in protocol
  • YPD broth (BD Difco)
  • YPD agar (BD Difco) in 100 × 15–mm plates
  • 15% (v/v) glycerol
  • Phosphate‐buffered saline (PBS; )
  • 50‐ml conical tubes, sterile (e.g., BD Falcon)
  • Centrifuge
  • Additional reagents and equipment for counting cells with a hemacytometer ( )

Support Protocol 2: Preparation of Cortisone Acetate Suspension

  Materials
  • Cortisone 21‐acetate (Sigma)
  • 0.05% Tween 20/PBS: add 10 µl Tween 20 to 20 ml sterile PBS (see for PBS)
  • 50‐ml conical tube (BD Falcon)
  • Probe‐type sonicator

Support Protocol 3: Periodic Acid–Schiff Staining of Tissue Sections

  Materials
  • Fixed, paraffin‐embedded, 4‐µm tissue sections (e.g., with modified Bouin's fixative; unit )
  • Xylol
  • 95% and 100% (v/v) ethanol
  • Periodic acid–Schiff (PAS) stain (see recipe) consisting of:
    • 1% periodic acid
    • Basic fuchsin solution
    • Zinc (or sodium) hydrosulfite solution
    • Light‐green stain
  • Mounting medium
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Figures

Videos

Literature Cited

Literature Cited
   Bär, E. , Gladiator, A. , Bastidas, S. , Roschitzki, B. , Acha‐Orbea, H. , Oxenius, A. , and LeibundGut‐Landmann, S. 2012. A novel Th cell epitope of Candida albicans mediates protection from fungal infection. J. Immunol. 188:5636‐5643.
   Bistoni, F. , Cenci, E. , Mencacci, A. , Schiaffella, E. , Mosci, P. , Puccetti, P. , and Romani, L. 1993. Mucosal and systemic T helper cell function after intragastric colonization of adult mice with Candida albicans . J. Infect. Dis. 168:1449‐1457.
   Carvalho, A. , Giovannini, G. , De Luca, A. , D'Angelo, C. , Casagrande, A. , Iannitti, R.G. , Ricci, G. , Cunha, C. , and Romani, L. 2012. Dectin‐1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis. Cell Mol. Immunol. 9:276‐286.
   Chaffin, W.L. , Lopez‐Ribot, J.L. , Casanova, M. , Gozalbo, D. , and Martinez, J.P. 1998. Cell wall and secreted proteins of Candida albicans: Identification, function, and expression. Microbiol. Mol. Biol. Rev. 62:130‐180.
   Clancy, C. , Cheng, S. , and Nguyen, M. 2009. Animal models of candidiasis. In Candida albicans: Methods and Protocols, Vol. 499 ( R. Cihlar and R. Calderone , eds.) pp. 65‐76. Humana Press, Totowa, New Jersey.
   Conti, H.R. and Gaffen, S.L. 2010. Host responses to Candida albicans: Th17 cells and mucosal candidiasis. Microbes Infect. 12:518‐527.
   Conti, H. , Shen, F. , Nayyar, N. , Stocum, E. , JN, S. , Lindemann, M. , Ho, A. , Hai, J. , Yu, J. , Jung, J. , Filler, S. , Masso‐Welch, P. , Edgerton, M. , and Gaffen, S. 2009. Th17 cells and IL‐17 receptor signaling are essential for mucosal host defense against oral candidiasis. J. Exp. Med. 206:299‐311.
   Fidel, P.L. Jr. and Sobel, J.D. 1996. Immunopathogenesis of recurrent vulvovaginal candidiasis. Clin. Microbiol. Rev. 9:335‐348.
   Glocker, E. and Grimbacher, B. 2010. Chronic mucocutaneous candidiasis and congenital susceptibility to Candida . Curr. Opin. Allergy Clin. Immunol. 10:542‐550.
   Hernández‐Santos, N. and Gaffen, S.L. 2012. Th17 cells in immunity to Candida albicans . Cell Host Microbe 11:425‐435.
   Hernández‐Santos, N. , Huppler, A.R. , Peterson, A.C. , Khader, S.A. , KC, M. , and Gaffen, S.L. 2013. Th17 cells confer long term adaptive immunity to oral mucosal Candida albicans infections. Mucosal Immunol 6:900‐910.
   Ho, A. , Shen, F. , Conti, H. , Patel, N. , Childs, E. , Peterson, A. , Hernandez‐Santos, N. , Kolls, J. , Kane, L. , Ouyang, W. , and Gaffen, S. 2010. IL‐17RC is required for immune signaling via an extended SEFIR domain in the cytoplasmic tail. J. Immunol. 185:1063‐1070.
   Huang, W. , Na, L. , Fidel, P.L. , and Schwarzenberger, P. 2004. Requirement of interleukin‐17A for systemic anti‐Candida albicans host defense in mice. J. Infect. Dis. 190:624‐631.
   Huppler, A.R. , Bishu, S. , and Gaffen, S.L. 2012. Mucocutaneous candidiasis: The IL‐17 pathway and implications for targeted immunotherapy. Arthritis Res. Ther. 14:217.
   Iliev, I.D. , Funari, V.A. , Taylor, K.D. , Nguyen, Q. , Reyes, C.N. , Strom, S.P. , Brown, J. , Becker, C.A. , Fleshner, P.R. , Dubinsky, M. , Rotter, J.I. , Wang, H.L. , McGovern, D.P. , Brown, G.D. , and Underhill, D.M. 2012. Interactions between commensal fungi and the C‐type lectin receptor Dectin‐1 influence colitis. Science 336:1314‐1317.
   Johnson, C.C. , Yu, A. , Lee, H. , Fidel, P.L. Jr. , and Noverr, M.C. 2012. Development of a contemporary animal model of Candida albicans–associated denture stomatitis using a novel intraoral denture system. Infect. Immun. 80:1736‐1743.
   Kagami, S. , Rizzo, H.L. , Kurtz, S.E. , Miller, L.S. , and Blauvelt, A. 2010. IL‐23 and IL‐17A, but not IL‐12 and IL‐22, are required for optimal skin host defense against Candida albicans . J. Immunol. 185:5453‐5462.
   Kamai, Y. , Kubota, M. , Kamai, Y. , Hosokawa, T. , Fukuoka, T. , and Filler, S. 2001. New model of oropharyngeal candidiasis in mice. Antimicrob. Agents Chemother. 45:3195‐3197.
   Lionakis, M.S. , Lim, J.K. , Lee, C.C. , and Murphy, P.M. 2011. Organ‐specific innate immune responses in a mouse model of invasive candidiasis. J. Innate Immun. 3:180‐199.
   MacCallum, D.M. and Odds, F.C. 2005. Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice. Mycoses 48:151‐161.
   MacCallum, D.M. , Castillo, L. , Nather, K. , Munro, C.A. , Brown, A.J. , Gow, N.A. and Odds, F.C. 2009. Property differences among the four major Candida albicans strain clades. Eukaryot. Cell 8:373‐387.
   Noble, S.M. , French, S. , Kohn, L.A. , Chen, V. , and Johnson, A.D. 2010. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat. Genet. 42:590‐598.
   Odds, F.C. 1998. Pathogenesis of candidosis. In Candida and Candidosis ( F.C. Odds , ed.) pp. 252‐278. Bailliere Tindall, Oxford.
   Patel, D.D. , Lee, D.M. , Kolbinger, F. , and Antoni, C. 2013. Effect of IL‐17A blockade with secukinumab in autoimmune diseases. Ann. Rheum. Dis. 72:116‐123.
   Puel, A. , Picard, C. , Cypowyj, S. , Lilic, D. , Abel, L. , and Casanova, J.L. 2010. Inborn errors of mucocutaneous immunity to Candida albicans in humans: A role for IL‐17 cytokines? Curr. Opin. Immunol. 22:467‐474.
   Puel, A. , Cypowji, S. , Bustamante, J. , Wright, J. , Liu, L. , Lim, H. , Migaud, M. , Israel, L. , Chrabieh, M. , Audry, M. , Gumbleton, M. , Toulon, A. , Bodemer, C. , El‐Baghdadi, J. , Whitters, M. , Paradis, T. , Brooks, J. , Collins, M. , Wolfman, N. , Al‐Muhsen, S. , Galicchio, M. , Abel, L. , Picard, C. , and Casanova, J.‐L. 2011. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin‐17 immunity. Science 332:65‐68.
   Samonis, G. , Anaissie, E.J. , Rosenbaum, B. , and Bodey, G.P. 1990. A model of sustained gastrointestinal colonization by Candida albicans in healthy adult mice. Infect. Immun. 58:1514‐1517.
   Solis, N.V. and Filler, S.G. 2012. Mouse model of oropharyngeal candidiasis. Nat. Protoc. 7:637‐642.
   Spellberg, B. , Ibrahim, A.S. , Edwards, J.E. Jr. , and Filler, S.G. 2005. Mice with disseminated candidiasis die of progressive sepsis. J. Infect. Dis. 192:336‐343.
   Suegara, N. , Siegel, J.E. , and Savage, D.C. 1979. Ecological determinants in microbial colonization of the murine gastrointestinal tract: Adherence of Torulopsis pintolopesii to epithelial surfaces. Infect. Immun. 25:139‐145.
   Sun, J.N. , Solis, N.V. , Phan, Q.T. , Bajwa, J.S. , Kashleva, H. , Thompson, A. , Liu, Y. , Dongari‐Bagtzoglou, A. , Edgerton, M. , and Filler, S.G. 2010. Host cell invasion and virulence mediated by Candida albicans Ssa1. PLoS Pathog. 6:e1001181.
   Szabo, E.K. and MacCallum, D.M. 2011. The contribution of mouse models to our understanding of systemic candidiasis. FEMS Microbiol. Lett. 320:1‐8.
   Taylor, B.N. , Fichtenbaum, C. , Saavedra, M. , Slavinsky, I.J. , Swoboda, R. , Wozniak, K. , Arribas, A. , Powderly, W. , and Fidel, P.L. Jr. 2000. In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus. J. Infect. Dis. 182:955‐959.
   Yano, J. and Fidel, P.L. Jr. 2011. Protocols for vaginal inoculation and sample collection in the experimental mouse model of Candida vaginitis . J. Vis. Exp. pii: 3382. doi: 10.3791/3382.
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