Cultivation of Borrelia burgdorferi in Dialysis Membrane Chambers in Rat Peritonea

Melissa J. Caimano1

1 University of Connecticut Health Center, Farmington, Connecticut
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12C.3
DOI:  10.1002/9780471729259.mc12c03s00
Online Posting Date:  July, 2005
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Abstract

In order to be sustained within its enzootic cycle, B. burgdorferi must adapt to two strikingly different environments, the arthropod vector and the mammalian host. The ability to rapidly adapt to environmental changes is therefore presumed to be central to spirochete survival and pathogenic programs. Indeed, it has now been well established that tick feeding initiates extensive changes in both gene expression and protein composition, collectively referred to as “host adaptation,” a process that is thought to continue throughout infection. The paucibacillary nature of borrelial infections, however, has hampered our ability to study this bacterium in vivo. To circumvent this limitation, an animal model was developed for obtaining sufficient numbers of organisms to directly examine differential gene expression and antigenic composition of B. burgdorferi within the context of the mammalian host. The DMC model allows for a direct comparison of host‐adapted B. burgdorferi and their in vitro‐cultivated counterparts.

Keywords: B. burgdorferi; Lyme disease; spirochete; host‐adaptation; in vivo gene expression; pathogenesis; animal models

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Host‐Adapted B. burgdorferi using Dialysis Membrane Chamber Peritoneal Implants
  • Support Protocol 1: Preparation of Sterile Dialysis Membrane
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Host‐Adapted B. burgdorferi using Dialysis Membrane Chamber Peritoneal Implants

  Materials
  • Female Sprague‐Dawley rats weighing ∼150 to 174 g upon arrival
  • 70% ethanol in spray bottle
  • BSK‐H (or BSK‐II) medium (Sigma), prewarmed to room temperature
  • Borrelia burgdorferi growing in culture (unit 12.1)
  • Ketamine/xylazine anesthetic cocktail (see recipe)
  • Ophthalmic ointment, sterile (e.g., Puralube from Pharmaderm Animal Health)
  • 0.3 mg/ml buprenorphine·HCl (e.g., Buprenex Injectable)
  • Betadine surgical scrub solution
  • 10‐ml individually wrapped, sterile disposable pipets
  • Surgical drape, cut into ∼18‐in. (∼46‐cm) squares, sterile
  • Battery‐operated pipet filler (e.g., Pipet‐Aid, Drummond Scientific)
  • Petroff‐Hausser counting chamber (Hausser Scientific)
  • 50‐ml conical polypropylene centrifuge tubes
  • Surgical gloves, sterile, individually wrapped
  • Sterile blunt‐end forceps
  • Sterile dialysis tubing (see protocol 2)
  • Sterile petri dishes
  • 0.5‐ml syringes with 28‐G, 1/2‐in. (∼1.3‐cm) needles
  • Sterile 4 × 4–in. (∼10.2 × 10.2–cm) gauze
  • Surgical instruments, sterilized and kept within sterile packaging inside surgical field:
    • Scalpel blade and holder, no. 10
    • 4‐in. (∼10.2‐cm) iris scissors (optional)
    • Tissue forceps (1 × 2–tooth dissecting or Adson‐Brown, available from Henry Schein Medical)
    • 5 1/2‐in. (∼14‐cm) needle holder with built‐in scissors (e.g., Olsen Hegar, available from Henry Schein Medical)
    • Ethicon 4‐0, SH‐1, 27‐in. (∼68.6‐cm) coated Vicryl, violet‐braided suture (available from Henry Schein Medical)
    • Auto‐clip 9‐mm stainless steel wound closure clips and applicator (Fisher Scientific)
  • 10‐ml syringe with 18‐G, 1‐in. (∼2.54‐cm) needle, sterile
  • Additional reagents and equipment for injection (Donovan and Brown, ) and euthanasia (Donovan and Brown, ) of rodents

Support Protocol 1: Preparation of Sterile Dialysis Membrane

  Materials
  • 1 mM EDTA, pH 8.0
  • Three 2‐liter sterile Pyrex beakers
  • Regenerated dialysis membrane tubing (Spectra/Por 6, 6000 to 8000 MWCO, 32‐mm width; Spectrum Labs)
  • Surgical gloves, sterile, individually wrapped
  • 5 1/2 in. (∼14 cm) straight surgical scissors, sterile
  • 11 13/16 in. (∼30 cm) extra‐long blunt‐end forceps, sterile
  • 250‐ml sterile Pyrex beakers or 150‐mm sterile petri dishes with lids
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Figures

Videos

Literature Cited

   Akins, D.R., Bourell, K.W., Caimano, M.J., Norgard, M.V., and Radolf, J.D. 1998. A new animal model for studying Lyme disease spirochetes in a mammalian host‐adapted state. J. Clin. Invest. 101:2240‐2250.
   Anguita, J., Hedrick, M.N., and Fikrig, E. 2003. Adaptation of Borrelia burgdorferi in the tick and the mammalian host. FEMS Microbiol. Rev. 27:493‐504.
   Brooks, C.S., Hefty, P.S., Jolliff, S.E., and Akins, D.R. 2003. Global analysis of Borrelia burgdorferi genes regulated by mammalian host‐specific signals. Infect. Immun. 71:3371‐3383.
   Caimano, M.J., Eggers, C.H., Hazlett, K.R.O., and Radolf, J.D. 2004. RpoSBb is not central to the general stress response of Borrelia burgdorferi but does control expression of one or more central virulence determinants. Infect. Immun. 72:6433‐6445.
   de Silva, A.M., Fikrig, E., Hodzic, E., Kantor, F.S., Telford, S.R. III, and Barthold, S.W. 1998. Immune evasion by tickborne and host‐adapted Borrelia burgdorferi. J. Infect. Dis. 177:395‐400.
   Donovan, J. and Brown, P. 1995a. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1‐1.6.10. John Wiley & Sons, Hoboken, N.J.
   Donovan, J. and Brown, P. 1995b. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.8.1‐1.8.4. John Wiley & Sons, Hoboken, N.J.
   Elias, A.F., Stewart, P.E., Grimm, D., Caimano, M.J., Eggers, C.H., Tilly, K., Bono, J.L., Akins, D.R., Radolf, J.D., Schwan, T.G., and Rosa, P. 2002. Clonal polymorphism of Borrelia burgdorferi strain B31 MI: Implications for mutagenesis in an infectious strain background. Infect. Immun. 70:2139‐2150.
   Fox, J.G., Anderson, L.C., Loew, F.M., and Quimby, F.W. (eds.) 2002. Laboratory Animal Medicine. Academic Press, New York.
   Hefty, P.S., Jolliff, S.E., Caimano, M.J., Wikel, S.K., Radolf, J.D., and Akins, D.R. 2001. Regulation of OspE‐related, OspF‐related, and Elp lipoproteins of Borrelia burgdorferi strain 297 by mammalian host‐specific signals. Infect. Immun. 69:3618‐3627.
   Hodzic, E., Feng, S., Freet, K.J., and Barthold, S.W. 2003. Borrelia burgdorferi population dynamics and prototype gene expression during infection of immunocompetent and immunodeficient mice. Infect. Immun. 71:5042‐5055.
   Hurtenbach, U., Museteanu, C., Gasser, J., Schaible, U.E., and Simon, M.M. 1995. Studies on early events of Borrelia burgdorferi–induced cytokine production in immunodeficient SCID mice by using a tissue chamber model for acute inflammation. Int. J. Exp. Pathol. 76:111‐123.
   Jonsson, M., Elmros, T., and Bergstrom, S. 1995. Subcutaneous implanted chambers in different mouse strains as an animal model to study genetic stability during infection with lyme disease Borrelia. Microb. Pathog. 18:109‐114.
   Mashburn, L.M., Jett, A.M., Akins, D.R., and Whiteley, M. 2004. Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo co‐culture. J. Bacteriol. 187:554‐566.
   Miller, J.C., von Lackum, K., Babb, K., McAlister, J.D., and Stevenson, B. 2003. Temporal analysis of Borrelia burgdorferi Erp protein expression throughout the mammal‐tick infectious cycle. Infect. Immun. 71:6943‐6952.
   Pal, U. and Fikrig, E. 2003. Adaptation of Borrelia burgdorferi in the vector and vertebrate host. Microbes Infect. 5:659‐666.
   Parveen, N., Caimano, M., Radolf, J.D., and Leong, J.M. 2003. Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding. Mol. Microbiol. 47:1433‐1444.
   Purser, J.E., Lawrenz, M.B., Caimano, M.J., Howell, J.K., Radolf, J.D., and Norris, S.J. 2003. A plasmid‐encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host. Mol. Microbiol. 48:753‐764.
   Revel, A.T., Talaat, A.M., and Norgard, M.V. 2002. DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete. Proc. Natl. Acad. Sci. U.S.A. 99:1562‐1567.
   Schwan, T.G. and Piesman, J. 2000. Temporal changes in outer surface proteins A and C of the Lyme disease‐associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J. Clin. Microbiol. 38:382‐388.
   Stevenson, B., Schwan, T.G., and Rosa, P.A. 1995. Temperature‐related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 63:4535‐4539.
   Wang, X.G., Lin, B., Kidder, J.M., Telford, S., and Hu, L.T. 2002. Effects of environmental changes on expression of the oligopeptide permease (opp) genes of Borrelia burgdorferi. J. Bacteriol. 184:6198‐6206.
   Yang, X., Goldberg, M.S., Popova, T.G., Schoeler, G.B., Wikel, S.K., Hagman, K.E., and Norgard, M.V. 2000. Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi. Mol. Microbiol. 37:1470‐1479.
   Yang, X.F., Hubner, A., Popova, T.G., Hagman, K.E., and Norgard, M.V. 2003. Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi. Infect. Immun. 71:5012‐5020.
Key References
   Akins et al., 1998. See above.
  Original report describing use of dialysis membrane chamber technique with B. burgdorferi.
   Fox et al., 2002. See above.
  Contains practical advice administering anesthetics and analgesics in common laboratory animal species.
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