Adult Zebrafish Model of Streptococcal Infection

Hilary A. Phelps1, Donna L. Runft2, Melody N. Neely2

1 Assay Designs, Inc., Ann Arbor, Michigan, 2 Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9D.1
DOI:  10.1002/9780471729259.mc09d01s13
Online Posting Date:  May, 2009
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Abstract

Streptococcal pathogens cause a wide array of clinical syndromes in humans, including invasive systemic infections resulting in high mortality rates. Many of these pathogens are human specific, and therefore difficult to analyze in vivo using typical animal models, as these models rarely replicate what is observed in human infections. This unit describes the use of the zebrafish (Danio rerio) as an animal model for streptococcal infection to analyze multiple disease states. This model closely mimics the necrotizing fasciitis/myositis pathology observed in humans from a Streptococcus pyogenes infection. The use of a zoonotic pathogen, Streptococcus iniae, which replicates systemic infections caused by many streptococcal pathogens, including dissemination to the brain, is also described. Protocols describing both intraperitoneal and intramuscular infections, as well as methods for histological and quantitative measurements of infection, are also described. Curr. Protoc. Microbiol. 13:9D.1.1–9D.1.27. © 2009 by John Wiley & Sons, Inc.

Keywords: Streptococcus; zebrafish; necrotizing fasciitis; S. pyogenes; S. iniae; infection; histology; systemic; animal model

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Infection of Adult Zebrafish (Danio rerio)
  • Basic Protocol 2: Dissection for Collection of Infected Tissues
  • Basic Protocol 3: Enumeration of Bacteria in Tissues on Solid CNA Medium
  • Basic Protocol 4: Cytospin Preparations of Streptococcus‐Infected Tissues
  • Basic Protocol 5: Histology of Infected Tissues
  • Basic Protocol 6: Virulence Analysis by Competitive Index Assay
  • Support Protocol 1: Preparation of Streptococcal Cultures for Zebrafish Infection
  • Basic Protocol 7: Quantitative Determination of Virulence by Lethal Dose 50
  • Basic Protocol 8: Analysis of Bacterial Dissemination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Infection of Adult Zebrafish (Danio rerio)

  Materials
  • 168 µg/ml and 336 µg/ml Tris‐buffered tricaine, pH 7.0 (3‐aminobenzoic acid ethylester, Sigma)
  • Sterilized deionized water supplemented with aquarium salts (60 mg/liter; see recipe)
  • Streptococcal bacterial culture (see protocol 7)
  • TP broth (see recipe)
  • Zebrafish (see )
  • TP plates with and without antibiotics (see protocol 7)
  • Plastic dissecting board
  • Petri dishes
  • Hemostat
  • Gauze tape
  • 400‐ml beakers
  • Perforated lids
  • 1.5‐ml microcentrifuge tubes
  • Fish nets
  • Small transportable holding tank with perforated lid (typical plastic mouse container)
  • 28°C glass front incubator
  • 3/10‐ml U‐100 ultrafine insulin syringe with a 0.5‐in. long, 29‐G needle
  • Plastic spoon

Basic Protocol 2: Dissection for Collection of Infected Tissues

  Materials
  • 95% ethanol in 250‐ml beaker (∼100 ml)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Infected zebrafish (see protocol 1)
  • 336 µg/ml Tris‐buffered tricaine, pH 7.0 (see recipe)
  • Sterile dissection instruments including:
    • Dissecting board (Styrofoam or cork cutting board)
    • Dissecting pins
    • Small, pointed‐tip surgical scissors
    • Tissue forceps
    • Scalpel
  • 1.5‐ml microcentrifuge tubes, sterile

Basic Protocol 3: Enumeration of Bacteria in Tissues on Solid CNA Medium

  Materials
  • Columbia colistin‐nalidixic acid (CNA) agar plates (see recipe for CNA)
  • Infected, dissected tissue (see protocol 2)
  • Sterile THY B medium (see recipe)
  • Tissue homogenizer (Kontes hand‐held cordless motor)
  • Disposable pellet pestles, sterile
  • 37°C, 5% CO 2 incubator or 37°C incubator and GasPak jars

Basic Protocol 4: Cytospin Preparations of Streptococcus‐Infected Tissues

  Materials
  • Isolated zebrafish organs (see protocol 2)
  • Sterile PBS ( appendix 2A)
  • Protocol Hema‐3 stain system (Fisher)
  • Mounting medium
  • Vortexer
  • 1‐ml syringes
  • 18‐ and 26‐G needles
  • 1.5‐ml microcentrifuge tubes
  • Cytofunnels (Fisher)
  • Cytofuge (Shandon Elliot Cytospin 1 centrifuge)
  • Glass microscope slides
  • Coverslips
  • Light microscope

Basic Protocol 5: Histology of Infected Tissues

  Materials
  • Infected zebrafish (see protocol 1)
  • 336 µg/ml Tris‐buffered tricaine, pH 7.0 (see recipe)
  • Deitrich's fixative (see recipe)
  • 30%, 50%, 70%, 80%, 95%, and 100% ethanol solutions (freshly made)
  • Clearify (American Master Tech)
  • Tissue infiltration medium (Surgipath), 60°C
  • Formula R paraffin (Surgipath)
  • Acid‐alcohol solution (see recipe)
  • Dish detergent (e.g., Dawn)
  • Liquid fabric softener (e.g., Downy)
  • Gelatin
  • PBS ( appendix 2A)
  • Hematoxylin, freshly filtered
  • Eosin
  • Toluene
  • Mounting medium
  • Plastic spoon
  • Dissecting board
  • Scalpel, sterile
  • 15‐ml polypropylene conical tube
  • Rocking platform shaker
  • Histology tissue cassette (Fisher cat. no. 15‐182‐500E)
  • Plastic screw‐cap container
  • 50° to 55°C and 60° to 64°C water baths
  • Metal base mold for tissue cassette
  • 60°C heating block
  • Glass microscope slides
  • Microtome (Richardson model no. HM 315)
  • Glass staining dishes
  • Staining rack to hold slides
  • Coverslips

Basic Protocol 6: Virulence Analysis by Competitive Index Assay

  Materials
  • Agar plates with and without antibiotic
  • 1‐ml microcentrifuge tubes containing 200 µl PBS labeled with strain, organ, and time point
  • Streptococcal bacterial cultures (see protocol 7)
  • Sterile medium
  • Zebrafish
  • 1‐ml microcentrifuge tubes
  • Additional reagents and equipment for injections (see protocol 1) and dissections (see protocol 2)

Support Protocol 1: Preparation of Streptococcal Cultures for Zebrafish Infection

  Materials
  • S. pyogenes and S. iniae frozen stocks in glycerol
  • Todd‐Hewitt yeast peptone broth (TP; see recipe)
  • Antibiotics
  • Todd‐Hewett agar plates (THY A; see recipe for THY B)
  • 15‐ml sterile, screw‐capped conical tubes
  • 37°C incubator
  • Spectrophotometer
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge

Basic Protocol 7: Quantitative Determination of Virulence by Lethal Dose 50

  Materials
  • Bacterial cultures (see protocol 7)
  • Sterile medium
  • Zebrafish (6 to 9 months)
  • Agar plates
  • 336 µg/ml Tricaine (see recipe)
  • 28°C incubator
  • 37°C, 5% CO 2 incubator or GasPak jars at 37°C
  • Additional reagents and equipment for injections and euthanization (see protocol 1)

Basic Protocol 8: Analysis of Bacterial Dissemination

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Colistin‐nalidixic acid (CNA) agar Petri plates (see recipe)
  • Infected zebrafish (see protocol 1)
  • 336 µg/ml Tris‐buffered Tricaine, pH 7.0 (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Additional reagents and equipment for injection (see protocol 1) and dissection, tissue homogenization, and enumeration (see protocol 2)
CAUTION: While all personal protective equipment should be worn for all protocols in this unit, particular caution should be used during injections and dissections of the zebrafish.
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Figures

Videos

Literature Cited

   Bisno, A.L. and Stevens, D.L. 1996. Streptococcal infections of skin and soft tissues. New Engl. J. Med. 334:240‐245.
   Buchanan, J.T., Stannard, J.A., Lauth, X., Ostland, V.E., Powell, H.C., Westerman, M.E., and Nizet, V. 2005. Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development. Infect. Immun. 73:6935‐6944.
   Cockerill, F.R. 3rd, Thompson, R.L., Musser, J.M., Schlievert, P.M., Talbot, J., Holley, K.E., Harmsen, W.S., Ilstrup, D.M., Kohner, P.C., Kim, M.H., Frankfort, B., Manahan, J.M., Steckelberg, J.M., Roberson, F., and Wilson, W.R. 1998. Molecular, serological, and clinical features of 16 consecutive cases of invasive streptococcal disease. Southeastern Minnesota Streptococcal Working Group. Clin. Infect. Dis. 26:1448‐1458.
   Cosma, C.L., Swaim, L.E., Volkman, H., Ramakrishnan, L., and Davis, J.M. 2006. Zebrafish and frog models of Mycobacterium marinum infection. Curr. Protoc. Microbiol. 3:10B.2.1‐10B.2.33.
   Cunningham, M.W. 2000. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13:470‐511.
   Hidalgo‐Grass, C., Dan‐Goor, M., Maly, A., Eran, Y., Kwinn, L.A., Nizet, V., Ravins, M., Jaffe, J., Peyser, A., Moses, A.E., and Hanski, E. 2004. Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft‐tissue infections. Lancet 363:696‐703.
   Locke, J.B., Colvin, K.M., Datta, A.K., Patel, S.K., Naidu, N.N., Neely, M.N., Nizet, V., and Buchanan, J.T. 2007a. Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish. J. Bacteriol. 189:1279‐1287.
   Locke, J.B., Colvin, K.M., Varki, N., Vicknair, M.R., Nizet, V., and Buchanan, J.T. 2007b. Streptococcus iniae beta‐hemolysin streptolysin S is a virulence factor in fish infection. Dis. Aquat. Organ. 76:17‐26.
   Locke, J.B., Aziz, R.K., Vicknair, M.R., Nizet, V., and Buchanan, J.T. 2008. Streptococcus iniae M‐like protein contributes to virulence in fish and is a target for live attenuated vaccine development. PLoS ONE 3:e2824.
   Lowe, B.A., Miller, J.D., and Neely, M.N. 2007. Analysis of the polysaccharide capsule of the systemic pathogen Streptococcus iniae and its implications in virulence. Infect. Immun. 75:1255‐1264.
   Miller, J.D. and Neely, M.N. 2004. Zebrafish as a model host for streptococcal pathogenesis. Acta Trop. 91:53‐68.
   Miller, J.D. and Neely, M.N. 2005. Large‐scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae. Infect. Immun. 73:921‐934.
   Neely, M., Pfeifer, J., and Caparon, M.G. 2002. Streptococcus‐zebrafish model of bacterial pathogenesis. Infect. Immun. 70:3904‐3914.
   Phelps, H.A. and Neely, M.N. 2005. Evolution of the zebrafish model: From development to immunity and infectious disease. Zebrafish 2:87‐103.
   Phelps, H.A. and Neely, M.N. 2007. SalY of the Streptococcus pyogenes lantibiotic locus is required for full virulence and intracellular survival in macrophages. Infect. Immun. 75:4541‐4551.
   Reed, L.J. and Muench, H. 1938. A simple method of estimation of fifty percect endpoints. Am. J. Hyg. 27:493‐497.
   Thulin, P., Johansson, L., Low, D.E., Gan, B.S., Kotb, M., McGeer, A., and Norrby‐Teglund, A. 2006. Viable group A streptococci in macrophages during acute soft tissue infection. PLoS Med. 3:e53.
   Weinstein, M.R., Low, D.E., and McGeer, A. 1996a. Invasive infection due to Streptococcus iniae: A new or previously unrecognized disease. CCDR 22:129‐132.
   Weinstein, M.R., Low, D.E., McGeer, A., Willey, B., Rose, D., Coulter, M., Wyper, P., Borczyk, A., and Lovgren, M. 1996b. Invasive infection with Streptococcus iniae‐Ontario, 1995‐1996, MMWR Weekly. 45:650‐653.
   Weinstein, M.R., Litt, M., Kertesz, D.A., Wyper, P., Rose, D., Coulter, M., McGeer, A., Facklam, R., Ostach, C., Willey, B., Borczyk, A., and Low, D.E. 1997. Invasive infections due to a fish pathogen, Streptococcus iniae. New Engl. J. Med. 337:589‐594.
Key References
   Neely et al., 2002. See above.
  This manuscript describes the development of the zebrafish infectious disease model with infection procedures, typical infection parameters, and course of infection.
   Phelps and Neely, 2005. See above.
  This is a comprehensive review of bacterial species used to infect zebrafish and the zebrafish immune response to infection.
   Westerfield, M. 2000. The Zebrafish Book. Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th ed. University of Oregon Press, Eugene, Oregon.
  This describes basic protocols for zebrafish breeding, maintenance, and analysis. Available online at http://www.zfin.org.
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