Zebrafish and Frog Models of Mycobacterium marinum Infection

Christine L. Cosma1, Laura E. Swaim1, Hannah Volkman1, Lalita Ramakrishnan1, J. Muse Davis2

1 University of Washington School of Medicine, Seattle, Washington, 2 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10B.2
DOI:  10.1002/0471729256.mc10b02s3
Online Posting Date:  December, 2006
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Abstract

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium‐host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

Keywords: Mycobacterium; animal models; zebrafish; fluorescence microscopy; infection

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Infection of Adult Frogs (Rana Pipiens) and enumeration of M. marinum
  • Infection of Adult Zebrafish (Danio rerio) and Enumeration of M. Marinium
  • Basic Protocol 2: Infection of Adult Zebrafish with M. marinum
  • Basic Protocol 3: Enumeration of M. marinum from Adult Zebrafish
  • Basic Protocol 4: Histology of Adult Zebrafish Infected with M. marinum
  • Basic Protocol 5: Analysis of Tissues Infected with M. marinum by Fluorescence Microscopy
  • Support Protocol 1: Preparation of M. marinum Stocks for Infection of Adult Animals
  • Basic Protocol 6: Infection of Zebrafish Embryos with M. marinum by Microinjection
  • Basic Protocol 7: Enumeration of M. marinum from Infected Embryos
  • Support Protocol 2: Preparation of M. marinum Stocks for Infection of Larvae
  • Support Protocol 3: Plating Injection Volumes to Estimate M. marinum Inoculum
  • Support Protocol 4: Manual Dechorionation of Zebrafish Embryos
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Infection of Adult Frogs (Rana Pipiens) and enumeration of M. marinum

  Materials
  • Frogs (see )
  • 0.2% (w/v) tricaine (ethyl 3‐aminobenzoate, methanesulfonate salt), pH 7.0
  • 70% ethanol
  • 7H9 liquid culture medium (see recipe)
  • PBST: PBS ( appendix 2A) supplemented with 0.05% (v/v) Tween 80, sterile
  • Bacteriological plates: usually 7H10 agar plates (see recipe) supplemented with 10 mg/liter amphotericin B and antibiotics (see recipe for supplements to 7H10 agar plates) if appropriate
  • Plastic bags, ∼1‐gallon
  • 1‐ml syringes with 27‐G needles (tuberculin syringes)
  • Frog tanks (see )
  • 2‐liter beaker
  • Dissecting board and pins
  • Dissecting forceps, both with and without teeth
  • Surgical scissors for dissection
  • 50‐ml conical tube, preweighed
  • Omni homogenizer (model TH‐115; Omni International) with sterile Omni tips (disposable generator probes)
  • 33°C incubator
  • Additional reagents and equipment for culturing M. marinum for inoculation (see protocol 6)

Basic Protocol 2: Infection of Adult Zebrafish with M. marinum

  Materials
  • M. marinium (ATTC #BAA‐535 or other strain of interest)
  • PBS ( appendix 2A), sterile
  • Bacteriological plates: usually 7H10 agar plates (see recipe) supplemented with 10 mg/liter amphotericin B and antibiotics (see recipe for supplements to 7H10 agar plates) if appropriate
  • 0.02% (w/v) tricaine in fish water (see recipe for fish water)
  • Fish (see )
  • 50‐ml vaccine bottles
  • 1‐ml syringes with 21‐G (or larger) needles
  • 1/ 2 in., 30‐G needles
  • 33°C incubator
  • 250‐ml beakers
  • Plastic spoon
  • Petri dish
  • Additional reagents and equipment for culture of M. marinum for inoculation (see protocol 6)
NOTE: After inoculation, fish must be housed in a flow‐through system (see ). For most consistent results, use age‐matched fish of the same family, and acclimate fish prior to injection to ensure maximal health. See for details.

Basic Protocol 3: Enumeration of M. marinum from Adult Zebrafish

  Materials
  • Infected fish ( protocol 2)
  • PBS ( appendix 2A), sterile
  • Fish water (see recipe) supplemented with 1.5 mg/ml kanamycin sulfate
  • Fish water (see recipe) supplemented with 1.5 mg/ml kanamycin sulfate and 0.05% (w/v) tricaine
  • 70% ethanol
  • 7H10 agar plates (see recipe), 10 mg/liter amphotericin B, 25 mg/liter polymyxin B sulfate, 20 mg/liter trimethoprim, and 50 mg/liter carbenicillin disodium salt (see recipe for supplements to 7H10 agar plates)
  • 17 × 100–mm sterile polystyrene tubes
  • Petri dish
  • Omni homogenizer (model TH‐115; Omni International) with sterile Omni tips (disposable generator probes)
  • 1‐ml serological pipet with filter plug
  • Sterile glass test tubes
  • 33°C incubator

Basic Protocol 4: Histology of Adult Zebrafish Infected with M. marinum

  Materials
  • Infected fish ( protocol 2)
  • Dietrich's fixative (see recipe)
  • 70% ethanol
  • Embedding cassettes (e.g., Tru‐Flow, Fisher Scientific)

Basic Protocol 5: Analysis of Tissues Infected with M. marinum by Fluorescence Microscopy

  Materials
  • Infected frog tissues (see protocol 1, step ), or infected whole fish (see protocol 2)
  • 4% (w/v) paraformaldehyde in 1× PBS (see appendix 2A for PBS)
  • 5%, 15%, and 30% (w/v) sucrose in 1× PBS (see appendix 2A for PBS)
  • OCT compound (Tissue‐Tek)
  • Antifade reagent with DAPI (e.g., Slow‐Fade, Molecular Probes; or similar antiquenching reagent)
  • Nail polish
  • 17 × 60–mm (8‐ml) glass vials with screw caps (VWR)
  • Razor blade
  • Aluminum foil boats for freezing of tissue sections (Figure )
  • Microtome/cryostat
  • Fluorescence microscope

Support Protocol 1: Preparation of M. marinum Stocks for Infection of Adult Animals

  Materials
  • 7H9 liquid culture medium (see recipe)
  • M. marinum (ATCC # BAA‐535 or other strain of interest), frozen stock (unit 10.1)
  • Antibiotics as required
  • LB agar plate
  • 25‐cm2 tissue culture flasks
  • 33°C incubator

Basic Protocol 6: Infection of Zebrafish Embryos with M. marinum by Microinjection

  Materials
  • M. marinum (ATCC # BAA‐535 or other strain of interest, usually marked with a fluorescent marker)
  • 1.5× EMPT (see recipe)
  • Dechorionated zebrafish embryos at 30 hr post‐fertilization or later (see protocol 11 for dechorionation details)
  • Sterile zebrafish embryo medium (see recipe)
  • Micropipettor with microloader tips for loading needles (Eppendorf)
  • Injection needles: needles made from aluminosilicate capillaries (SM100F‐10, Harvard Apparatus) pulled on a Sutter P‐2000 micropipet puller, with the following settings: Heat = 350, FIL = 4, VEL = 50, DEL = 225, PUL = 150
  • Microscope with Microinjector (e.g., Eppendorf 5246) and Micromanipulator (e.g., Narishige GJ‐1)
  • Deep‐well microscope slides (VWR)
  • Extra fine jeweler's forceps (Miltex no. 17‐305 or Dumont no. 5)
  • Slide warmer, optional (e.g., Barstead no.26005 or 26020)
  • Microprobe (2 to 3 cm of 32‐G platinum wire mounted on the end of a Pasteur pipet)
  • Multi‐well culture plates, or small petri dishes
  • Large‐bore glass pipets (Chase Scientific Glass, Inc. #63AS3WT, distributed by Fisher #XC‐9993639, note that these are not typical Pasteur pipets)
NOTE: Standard borosilicate capillaries may also be used, but the authors find clumping of mycobacteria to be less problematic when using aluminosilicate capillaries. If only borosilicate is available, note that the settings on a Sutter P‐2000 micropipet puller are different for producing the same shape needle. Use: Heat=300, FIL=4, VEL=40, DEL=150, PUL=125.

Basic Protocol 7: Enumeration of M. marinum from Infected Embryos

  Materials
  • Sterile zebrafish embryo medium (see recipe) supplemented with 20 µg/ml kanamycin
  • Infected embryos (see protocol 7)
  • Fish water (see recipe) supplemented with 200 µg/ml tricaine
  • Trypsin‐EDTA (Invitrogen)
  • PBS ( appendix 2A)
  • 1% (v/v) Triton X‐100
  • 7H10 agar plates supplemented with OADC (see recipe) and antimicrobial agents (see recipe for supplements to 7H10 agar; see protocol 2)
  • 30° and 33°C incubators
  • Water bath ultrasonicator (Bransonic, model no. 1510R‐NT)

Support Protocol 2: Preparation of M. marinum Stocks for Infection of Larvae

  Materials
  • M. marinum (ATCC #BAA‐535 or other strain of interest) frozen stock (unit 10.1)
  • Zebrafish embryo medium (see recipe), sterile, or PBS ( appendix 2A)
  • 20% (w/v) phenol red dye stock solution in H 2O
  • 1‐ml tuberculin syringes with 27‐G needles
  • Additional reagents and equipment for preparing M. marimum (see protocol 6, steps to ) and loading the microinjector needle (see protocol 7, steps and )

Support Protocol 3: Plating Injection Volumes to Estimate M. marinum Inoculum

  Materials
  • PBS ( appendix 2A)
  • Middlebrook 7H10 agar plates (see recipe)
  • Injection needles loaded with bacterial suspension (see protocol 7 steps to )
  • Micromanipulator (e.g., Narishige GJ‐1) and microinjector (e.g., Eppendorf 5246)
  • 33°C incubator

Support Protocol 4: Manual Dechorionation of Zebrafish Embryos

  Materials
  • Zebrafish embryos (see )
  • Zebrafish embryo medium (see recipe), sterile
  • 100 × 15–mm petri dishes (or similar size)
  • Two pair extra‐fine jeweler's forceps (Miltex #17‐305 or Dumont #5)
  • Dissecting microscope
NOTE: Keep forceps for dechorionation separate from those used for injections.
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Figures

Videos

Literature Cited

   Aubry, A., Jarlier, V., Escolano, S., Truffot‐Pernot, C., and Cambau, E. 2000. Antibiotic susceptibility pattern of Mycobacterium marinum. Antimicrob. Agents Chemother. 44:3133‐3136.
   Cody, S.H. and Williams, D.A. 1997. A novel organ bath design enables the use of saline‐immersible lenses on inverted microscopes. J. Microscopy 185:94‐97.
   Cormack, B.P., Valdiva, R.H., and Falkow, S. 1996. FACS‐optimized mutants of the green fluorescent protein (GFP). Gene 173:33‐38.
   Cosma, C.L., Sherman, D.R., and Ramakrishnan, L. 2003. The secret lives of the pathogenic mycobacteria. Ann. Rev. Microbiol. 57:641‐76.
   Cosma, C.L., Humbert, O., and Ramakrishnan, L. 2004. Superinfecting mycobacteria home to established tuberculous granulomas. Nat. Immunol. 5:828‐835.
   Cosma, C.L., Klein, K., Kim, R., Beery, D., and Ramakrishnan, L. 2006. Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival. Infect. Immun. 74:3125‐3133.
   Danilova, N., Hohman, V.S., Sacher, F., Ota, T., Willett, C.E., and Steiner, L.A. 2004. T cells and the thymus in developing zebrafish. Dev. Comp. Immunol. 28:755‐767.
   Davidson, A.J. and Zon, L.I. 2004. The ‘definitive’ (and ‘primitive’) guide to zebrafish hematopoiesis. Oncogene 23:7233‐7246.
   Gauthier, D.T., Rhodes, M.W., Vogelbein, W.K., Kator, H., and Ottinger, C.A. 2003. Experimental mycobacteriosis in striped bass Morone saxatilis. Dis. Aquat. Organ 54:105‐117.
   Herbomel, P., Thisse, B., and Thisse, C. 1999. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126:3735‐3745.
   Kamei, M. and Weinstein, B.M. 2005. Long‐term time‐lapse fluorescence imaging of developing zebrafish. Zebrafish 2:113‐123.
   Kamei, M., Isogai, S., and Weinstein, B.M. 2004. Imaging blood vessels in the zebrafish. Methods Cell Biol. 76:51‐74.
   Lawson, N.D. and Weinstein, B.M. 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248:307‐318.
   Malone, R.F. and Rusch, K.A. 1997. Using the Bead Filter in Your Koi Pond: A Comprehensive Guide to Water Quality management. Louisiana Sea Grant College Program. Baton Rouge, La.
   Moe, M.A. 1992. The Marine Aquarium Reference, Systems and Invertebrates. Green Turtle Publications, Plantation, Fla
   Pozos, T.C. and Ramakrishnan, L. 2004. New models for the study of Mycobacterium‐host interactions. Curr. Opin. Immunol. 16:499‐505.
   Ramakrishnan, L. 2004. Using Mycobacterium marinum and its hosts to study tuberculosis. Curr. Sci. 86:82‐92.
   Streisinger, G., Singer, F., Walker, C., Knauber, D., and Dower, N. 1986. Segregation analyses and gene‐centromere distances in zebrafish. Genetics 112:311‐319.
   Swaim, L.E., Connolly, L.E., Volkman, H.E., Humbert, O., Born, D.E., and Ramakrishnan, L. 2006. Mycobacterium marinum infection of adult zebrafish produces caseating granulomatous tuberculosis that is moderated by adaptive immunity. Infect. Immun. In press.
   Talaat, A.M., Reimschuessel, R., Wasserman, S.S., and Trucksis, M. 1998. Goldfish, Carassium auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infect. Immun. 66:2938‐2942.
   Traver, D., Herbomel, P., Patton, E.E., Murphey, R.D., Yoder, J.A., Litman, G.W., Catic, A., Amemiya, C.T., Zon, L.I., and Trede, N.S. 2003. The zebrafish as a model organism to study development of the immune system. Adv. Immunol. 81:253‐330.
   Van der Sar, A.M., Abdallah, A.M., Sparrius, M., Reinders, E., Vandenbroucke‐Grauls, C.M.J.E., and Bitter, W. 2004. Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infect. Immun. 72:6306‐6312.
   Volkman, H.E., Clay, H., Beery, D., Chang, J.C., Sherman, D.R., and Ramakrishnan, L. 2004. Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant. PLoS Biol. 2:1946‐1956.
   Watkins, S. 1989. Cryosectioning. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.). John Wiley & Sons, Hoboken, N.J.
   Westerfield, M. 2000. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th ed. University of Oregon Press, Eugene.
   Wolinsky, E. 1979. State of the art: Nontuberculous mycobacteria and associated disease. Am. Rev. Respir. Dis. 119:107‐159.
Key References
   Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P., and Ramakrishnan, L. 2002. Real‐time visualization of Mycobacterium‐macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 17:693‐702.
  This paper presents a characterization of the zebrafish embryo infection model, including typical course of infection, cell behaviors, and bacterial responses.
   Ramakrishnan, L., Valdiva, R.H., McKerrow, J.H., and Falkow, S. 1997. Mycobacterium marinum causes both long term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infect. Immun. 65:767‐773.
  This paper describes the experimental inoculation of R. pipiens and subsequent progression of the infection.
   Swaim et al., 2006. See above.
  This paper describes the experimental inoculation of adult zebrafish and subsequent disease progression.
   Westerfield, 2000. See above.
  Available online at http://www.zfin.org. This work covers most basic aspects of zebrafish methodology and maintenance.
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