Helicobacter Animal Models

John G. Nedrud1, Thomas G. Blanchard1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.8
DOI:  10.1002/0471142735.im1908s36
Online Posting Date:  May, 2001
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Abstract

In this unit, protocols for growing Helicobacter organisms on plates or in liquid cultures are presented, followed by protocols for infecting mice with Helicobacter felis and H. pylori and for infecting ferrets with H. mustelae. Also, a procedure is described for adapting an H. pylori isolate to growth in mice. Support protocols describe methods for quantifying numbers of Helicobacter organisms, and how to create a growth curve for Helicobacter cultures. One important technique in investigating Helicobacter infection is assaying the disease processes that occur in the stomach, and a protocol is provided for preparing tissue sections for this purpose. It is also important to confirm that organisms recovered from tissue samples are, in fact, Helicobacter species, and a support protocol describes morphological and biochemical tests for this purpose. Helicobacter bacteria produce large amounts of the enzyme urease, and a support protocol describes how to perform a rapid urease test on animal‐tissue biopsies. Assays of Helicobacter‐specific immune responses require appropriate antigens, and preparation of both Helicobacter lysates and outer‐membrane proteins are detailed for use in these assays.

     
 
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Table of Contents

  • Basic Protocol 1: Culture of Helicobacter Organisms on Plates
  • Basic Protocol 2: Culture of Helicobacter Organisms in Liquid Cultures
  • Basic Protocol 3: Orally Infecting Mice with Helicobacter
  • Basic Protocol 4: Adapting an H. Pylori Isolate to Growth in Mice
  • Basic Protocol 5: Infecting Ferrets with H. Mustelae
  • Support Protocol 1: Quantifying Helicobacter Organisms in Stock Cultures
  • Support Protocol 2: Creating a Growth Curve
  • Support Protocol 3: Culturing Helicobacter Bacteria from Infected Animals
  • Support Protocol 4: Preparing Solid‐Phase Growth Media (Plates) for Helicobacter Cultures
  • Support Protocol 5: Preparing Tissue Strips of Mouse Stomach for Histological Examination
  • Support Protocol 6: Confirmation that Recovered Bacteria are Helicobacter Species
  • Support Protocol 7: Rapid Urease Test
  • Support Protocol 8: Preparation of Helicobacter Lysates (Sonicates)
  • Support Protocol 9: Preparation of Outer‐Membrane Proteins from Helicobacter Bacteria
  • Support Protocol 10: Collection of Gastric Secretions for Antibody Determination by Rinsing
  • Support Protocol 11: Collection of Gastric Secretions for Antibody Determination: Polywicks
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture of Helicobacter Organisms on Plates

  Materials
  • Freshly prepared blood‐agar plates (see protocol 9)
  • Helicobacter source material from a frozen stock, liquid culture (see protocol 2), or homogenized animal biopsy (see protocol 8)
  • Inoculating loop
  • Anaerobe jars with sealable lids (e.g., BBL GasPak model 100 from Becton Dickinson)
  • BBL CampyPak Plus Microaerophilic System Envelopes with Palladium Catalyst (Becton Dickinson) or equivalent
NOTE: All solutions and equipment coming into contact with living bacteria must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 2: Culture of Helicobacter Organisms in Liquid Cultures

  Materials
  • Brain Heart Infusion Broth or other suitable liquid medium (e.g., Difco Brucella Broth or Columbia Broth)
  • Fetal bovine serum ( appendix 2A)
  • Antibiotics/antimycotics (see protocol 9 and Table 19.8.1)
  • Plate of H. felis or H. pylori, with 3 days growth (see protocol 1)
  • 500‐ml Erlenmeyer or round‐bottom glass flasks or 500‐ml tissue culture bottles that will fit inside anaerobe jar
  • Bent glass rod or Pasteur pipet (“hockey stick”) for harvesting cultures
  • Sterile 5‐ml tubes
  • Anaerobe jars with sealable lids (e.g., BBL GasPak model 100 from Becton Dickinson)
  • BBL CampyPak Plus Microaerophilic System Envelopes with Palladium Catalyst (Becton Dickinson) or equivalent
  • Rotary shaker with 2‐liter brackets to accommodate the anaerobe jar
NOTE: All solutions and equipment coming into contact with living bacteria must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 3: Orally Infecting Mice with Helicobacter

  Materials
  • Mice of desired strain (see protocol 4 and )
  • Inoculum of H. felis or H. pylori diluted to give ≥107 cfu/0.5 ml, prepared immediately before use
  • Additional reagents and equipment for working with microisolator cages (unit 15.11), oral inoculation (as for Sendai virus oral gavage, protocol 3 in unit 15.11), cage changing and animal handling (unit 1.2), and assay of infection status (see Support Protocols protocol 61, protocol 105, and protocol 127).

Basic Protocol 4: Adapting an H. Pylori Isolate to Growth in Mice

  Materials
  • Culture of H. mustelae prepared as above ( protocol 1 or protocol 22).
  • Young adult ferrets (see Critical Parameters)
  • 5‐ml syringe
  • 16‐G ball‐tipped feeding needles: 3‐inch curved needle with 3‐mm ball tip (e.g., Perfectum, Popper and Sons, or equivalent)
  • Padded gloves

Basic Protocol 5: Infecting Ferrets with H. Mustelae

  Materials
  • Gastric biopsies obtained at necropsy or by endoscopy of experimental animals (e.g., see Basic Protocols protocol 33 and protocol 55)
  • Brucella broth (e.g., Difco) supplemented with 10% fetal bovine serum ( FBS; appendix 2A)
  • Blood agar plates supplemented with Helicobacter‐selective antibiotics (see protocol 9 and Table 19.8.1)
  • Disposable pellet pestles and 1.5‐ml microcentrifuge tubes (Kontes)
  • Additional reagents and equipment for culturing Helicobacter on plates (see protocol 1)
NOTE: All solutions and equipment coming into contact with living bacteria must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 1: Quantifying Helicobacter Organisms in Stock Cultures

  Materials
  • Blood agar base (e.g., Columbia Agar Base, Difco)
  • Sterile horse blood (e.g., Cleveland Scientific)
  • recipeSelective antibiotics (see recipe and Table 19.8.1)
  • 56°C water bath
  • 50‐ml conical centrifuge tubes, sterile
  • 100‐mm bacterial petri dishes
  • Plastic sleeve, for storing plates
NOTE: All solutions and equipment coming into contact with living bacteria must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 2: Creating a Growth Curve

  Materials
  • Infected mouse (see protocol 3)
  • 10% buffered formalin fixative
  • Dissecting pins and board
  • Sterile surgical tools
  • Rectangular or square pieces of Styrofoam that fit into 5‐ to 10‐cm deep plastic containers
  • Covered plastic containers, 5 to 10 cm deep
  • Hard polymer rectangular embedding cassettes (Fisher) or equivalent
  • 32 × 25 × 3–mm biopsy foam pads (Curtin Matheson or equivalent)
  • Additional reagents and equipment for sacrifice of mice (unit 1.8), embedding and sectioning of tissue (unit 21.4), and hematoxylin and eosin staining (unit 12.8)

Support Protocol 3: Culturing Helicobacter Bacteria from Infected Animals

  Materials
  • recipeUrease test broth (see recipe)
  • Experimental or infected animal (see Basic Protocols protocol 33 or protocol 55)
  • Sterile transparent or translucent glass or plastic tubes with tight‐fitting caps
  • Sterile, sharp dissecting scissors and forceps
NOTE: All solutions and equipment coming into contact with living bacteria must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 4: Preparing Solid‐Phase Growth Media (Plates) for Helicobacter Cultures

  Materials
  • Liquid culture of Helicobacter (see protocol 2) or suspension of bacteria scraped from plate culture (see protocol 1)
  • Dulbecco's phosphate‐buffered saline (DPBS; e.g., Life Technologies)
  • BCA protein assay kit (Pierce) or equivalent
  • 0.44‐µm syringe‐tip filters (Swinney‐type, either reusable or disposable)
  • Preparative, high‐speed, refrigerated centrifuge with a GSA‐type rotor or equivalent (holds 200‐ to 250‐ml centrifuge bottles) and SS34‐type rotor (holds 35‐ml Oak Ridge tubes)
  • Probe sonicator and ear protectors

Support Protocol 5: Preparing Tissue Strips of Mouse Stomach for Histological Examination

  Materials
  • 0.05 M Tris⋅Cl, pH 7.8 ( appendix 2A) with 1 mM EDTA
  • recipe1 mM RNase/DNase stock solution (see recipe)
  • 1% and 2% N‐lauryl sarcosine
  • 0.05 M sodium phosphate buffer, pH 7.2 ( appendix 2A)
  • Ultracentrifuge with Beckman 50 Ti rotor or equivalent
  • Additional reagents and equipment for preparing Helicobacter lysates (see protocol 13)

Support Protocol 6: Confirmation that Recovered Bacteria are Helicobacter Species

  Materials
  • Infected or experimental mouse (e.g., see protocol 3)
  • recipeCold protease inhibitor solution (see recipe)
  • Dissecting scissors and forceps
  • 60‐mm tissue‐culture dishes
  • Additional reagents and equipment for euthanasia of mice (unit 1.8)

Support Protocol 7: Rapid Urease Test

  Materials
  • Infected or experimental mouse (e.g., see protocol 3)
  • CO 2 source
  • recipeCold protease inhibitor solution (see recipe)
  • Dissecting scissors and forceps
  • Absorbent polywicks (Polyfiltronics, Whatman)
  • 0.5‐microcentrifuge tube with 18‐G needle hole bored through the bottom
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Figures

Videos

Literature Cited

Literature Cited
   Czinn, S.J. and Nedrud, J.G. 1991. Oral immunization against Helicobacter pylori. Infect. Immun. 59:2359‐2363.
   Eaton, K.A. 1999. Animal models of Helicobacter gastritis. In Gastroduodenal disease and Helicobacter pylori, Vol. 241 of Current Topics in Microbiology and Immunology (T.U. Westblom, S.J. Czinn and J.G. Nedrud, eds.) pp. 123‐154. Springer‐Verlag, Berlin.
   Enno, A., O'Rourke, J.L., Howlett, C.R., Jack, A., Dixon, M.F., Lee, A. 1997. MALToma‐like lesions in the murine gastric mucosal after long‐term infection with Helicobacter felis. Am. J. Pathol. 147:217‐222.
   Fox, J.G. and Lee, A. 1997. The role of Helicobacter species in newly recognized gastrointestinal tract diseases of animals. Lab. Anim. Sci. 47:222‐255.
   Fox, J.G., Correa, P., Taylor, N.S., Lee, A., Otto, G., Murphy, J.C., and Rose, R. 1990 Helicobacter mustelae–associated gastritis in ferrets: An animal model of Helicobacter pylori gastritis in humans. Gastroenterology. 99:352‐361.
   Genta, R.M., Robason, G.O., and Graham, D.Y. 1994. Simultaneous visualization of Helicobacter pylori and gastric morphology: A new stain. Hum. Pathol. 25:221‐226.
   Haneberg, B., Kendall, D., Amerongen, H.M., Apter, F.M., Kraehenbuhl, J.P., and Neutra, M.R. 1994. Induction of specific immunoglobulin A in the small intestine, colon‐rectum, and vagina measured by a new method for collection of secretions from mucosal surfaces. Infect. Immun. 62:15‐23.
   Kleanthous, H., Tibbets, T., Bakios, T.J., Georgakopoulos, K., Myers, G., Ermak, T.H., and Monath, T. 1995. In vivo. selection of a highly adapted H. pylori isolate and the development of an H. pylori mouse model for studying vaccine efficacy and attenuating lesions. Gut 137:A94.
   Kleanthous, H., Myers, G.A., Georgakopoulos, K.M., Tibbits, T.J., Ingrassia, J.W., Gray, H.L., Ding, R., Zhang, Z.Z., Lei, W., Nichols, R., Lee, C.K., Ermak, T.H., and Monath, T.P. 1998. Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection. Infect. Immun. 66:2879‐2886.
   Lee, A., O'Rourke, J., De Ungria, M.C., Robertson, B., Daskalopoulous, G., and Dixon, M.F. 1997. A standardised mouse model of Helicobacter pylori infection: Introducing the Sydney strain. Gastroenterology. 112:1386‐1397.
   Matsumoto, S., Washizuka, Y., Matsumoto, Y., Tawara, S., Ikeda, F., Yokota, Y., and Karita, M. 1997. Induction of ulceration and severe gastritis in Mongolian gerbil by Helicobacter pylori infection. J. Med. Microbiol. 46:391‐397.
   McColm, A.A. 1997. Nonprimate animal models of H. pylori infection. In Helicobacter pylori Protocols (C.L. Clayton and H.L.T. Mobley, eds.) pp. 235‐251. Humana Press, Totowa, New Jersey.
   Mohammadi, M., Redline, R., Nedrud, J., and Czinn, S. 1996. Role of the host in pathogenesis of Helicobacter‐associated gastritis: H. felis infection of inbred and congenic mouse strains. Infect. Immun. 64:238‐245.
   Mohammadi, M., Nedrud, J., Redline, R., Lycke, N., and Czinn, S. 1997. Murine CD4 T cell responses to Helicobacter infection: TH1 cells enhance gastritis and TH2 cells reduce bacterial load. Gastroenterology 113:1848‐1857.
   Sakagami, T., Dixon, M., O'Rourke, J., Howlett, R., Alderuccio, F., Vella, J., Shimoyama, T., and Lee, A. 1996. Atrophic gastric changes in both H. felis and H. pylori infected mice are host dependent and seperate from antral gastritis. Gut 39:639‐648.
   Schnaitman, C.A. 1970. Examination of the protein composition of the cell envelope of Escherichia coli by polyacrylamide gel electrophoresis. J. Bacteriol. 104:882‐889.
   Watanabe, T., Tada, M., Nagai, H., Sasaki, S., and Nakao, M. 1998. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils. Gastroenterology 115:642‐648.
Key References
   Lee, A. and Megraud, F. (eds.) 1996. Helicobacter pylori: Techniques for Clinical Diagnosis and Basic Research. W.B. Saunders, Philadelphia.
  This monograph contains a collection of clinical, diagnostic and basic research techniques for Helicobacter species.
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