Isolation and Laboratory Maintenance of Treponema pallidum

Sheila A. Lukehart1, Christina M. Marra1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12A.1
DOI:  10.1002/9780471729259.mc12a01s7
Online Posting Date:  November, 2007
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Abstract

The spirochetal bacteria that cause syphilis, yaws, and bejel cannot be cultivated in vitro. This unit describes methods for the isolation of subspecies of Treponema pallidum and other pathogenic treponemes from clinical specimens, the propagation of these isolates in rabbits, isolation of clonal populations of T. pallidum, and techniques for maintenance of frozen stocks of these treponemes. Curr. Protoc. Microbiol. 7:12A.1.1‐12A.1.18. © 2007 by John Wiley & Sons, Inc.

Keywords: syphilis; treponeme; rabbit; chancre; cerebrospinal fluid

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Routine Propagation of Noncultivable Treponemes in Rabbits
  • Support Protocol 1: Dark‐Field Microscopy for Quantification of T. pallidum Suspensions
  • Basic Protocol 2: Isolation of Treponema pallidum from Human Specimens
  • Basic Protocol 3: Recovery of Treponema pallidum from Lymph Nodes of Rabbits
  • Basic Protocol 4: Long‐Term Maintenance of T. pallidum Strains as Frozen Stocks
  • Basic Protocol 5: Cloning of T. pallidum in the Rabbit
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Routine Propagation of Noncultivable Treponemes in Rabbits

  Materials
  • T. pallidum sample, frozen (e.g., obtained from a colleague): up to 2 to 3 ml per injection
  • Rabbits: seronegative in VDRL/RPR or FTA‐ABS tests, healthy, adult males with well developed testes and no history of antibiotic treatment: e.g., New Zealand White (NZW), ∼3 months old (2.5 to 3.0 kg)
  • 7.5% Betadine solution
  • 70% (v/v) ethanol
  • Saline (0.14 M NaCl; see recipe)
  • Normal rabbit serum (NRS; see recipe)
  • Disinfectant (e.g., phenol based)
  • Rabbit restraining board (see and Fig. )
  • 3‐cc sterile, disposable Luer‐lok syringe
  • 23‐G × 1‐in. sterile, disposable Luer‐hub needle
  • Cotton balls or gauze
  • Sharps disposal unit
  • 18°C to 20°C BSL‐2 rabbit facility
  • 5‐in. surgical scissors and 6‐in. Kelly hemostat forceps, sterile: packaged as a set prior to autoclaving; two sets required for each procedure
  • 100 × 15–mm petri dishes, sterile
  • Closed transport container (e.g., plastic box with lid)
  • Scalpel, sterile
  • 6‐ to 7‐in. forceps, sterile
  • 5‐ and 10‐ml sterile disposable pipets and pipetting aid
  • Platform rotator with disposable adsorbent material on platform
  • Additional reagents and equipment for sedating and euthanizing rabbits (e.g., see http://depts.washington.edu/auts/rabbit_handout_2007.pdf) and quantifying treponemes by dark‐field microscopy ( protocol 2)

Support Protocol 1: Dark‐Field Microscopy for Quantification of T. pallidum Suspensions

  Materials
  • Treponemes harvested from rabbit testes ( protocol 1)
  • Dark‐field microscope and condenser oil
  • Slide micrometer
  • Microscope slide and 22 × 22–mm coverslip

Basic Protocol 2: Isolation of Treponema pallidum from Human Specimens

  Materials
  • Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal rabbit serum (NRS; see recipe)
  • 1‐ml tuberculin syringe
  • 100 × 15–mm petri dishes, sterile
  • Scalpel, sterile
  • Platform rotator
  • 3‐cc sterile, disposable Luer‐lok syringe
  • 23‐G × 1‐in. needle
  • Additional reagents and equipment for performing venipuncture, lumbar puncture, and punch biopsy (qualified personnel), anesthetizing rabbits (e.g., see http://depts.washington.edu/auts/rabbit_handout_2007.pdf), inoculating rabbits and harvesting treponemes ( protocol 1), and performing VDRL/RPR and FTA‐ABS screening (Lukehart, ; Larson, )

Basic Protocol 3: Recovery of Treponema pallidum from Lymph Nodes of Rabbits

  Materials
  • Infected rabbit ( protocol 1)
  • 70% (v/v) alcohol
  • Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal rabbit serum (NRS; see recipe)
  • Rabbit restraining board (see and Fig. )
  • 4‐ to 5‐in. curved scissors, sterile
  • 100 × 15–mm petri dishes, sterile
  • Forceps, sterile
  • Stainless steel mesh screen (∼2 × 2 in; e.g., from hardware store), sterile
  • 3‐cc sterile, disposable Luer‐lok syringe
  • 23‐G × 1‐in. needle
  • Additional reagents and equipment for euthanizing rabbits (e.g., see http://depts.washington.edu/auts/rabbit_handout_2007.pdf) and passaging treponemes in rabbits ( protocol 1)

Basic Protocol 4: Long‐Term Maintenance of T. pallidum Strains as Frozen Stocks

  Materials
  • Saline/50% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 10 ml normal rabbit serum (NRS; see recipe)
  • Sterile glycerol or DMSO
  • Dry ice
  • 95% (v/v) ethanol
  • Sterile 2‐ml cryovials, labeled with strain name, concentration, and date.
  • Liquid nitrogen freezer
  • Additional reagents and equipment for harvesting T. pallidum suspension ( protocol 1)

Basic Protocol 5: Cloning of T. pallidum in the Rabbit

  Materials
  • Rabbits: seronegative in VDRL/RPR or FTA‐ABS tests, healthy, adult males with well developed testes and no history of antibiotic treatment: e.g., New Zealand White (NZW), ∼3 months old, 2.5 to 3.0 kg
  • 70% (v/v) ethanol or Betadine
  • Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal rabbit serum (NRS; see recipe)
  • 15‐ml sterile, conical, screw cap tubes
  • Low speed centrifuge with BSL‐2 containment
  • Rabbit restraining box (restraining board can be used, but commercially available restraining box preferred)
  • Closed transport container (e.g., plastic box with lid)
  • 3‐cc sterile, disposable Luer‐lok syringe
  • 23‐G × 1‐in. needle
  • Animal clippers for keeping rabbit back free of fur (size 40 blade, optimal)
  • 4‐mm disposable biopsy punches (e.g., Miltex)
  • 100 × 15–mm petri dishes, sterile
  • 4‐ to 5‐in. small curved scissors, sterile
  • Additional reagents and equipment for preparing T. pallidum suspensions ( protocol 1), determining suspension concentration by dark‐field microscopy ( protocol 2), sedating or anesthetizing and euthanizing rabbits (e.g., see http://depts.washington.edu/auts/rabbit_handout_2007.pdf), propagating organisms in rabbits ( protocol 1), maintaining treponemes as frozen stocks ( protocol 5), and sequencing DNA (e.g., see Centurion‐Lara et al., )
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Figures

Videos

Literature Cited

Literature Cited
   Antal, G.M., Lukehart, S.A., and Meheus, A.Z. 2002. The endemic treponematoses. Microbes Infect. 4:83‐94.
   Centurion‐Lara, A., Godornes, C., Castro, C., Van Voorhis, W.C., and Lukehart, S.A. 2000. The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles. Infect. Immun. 68:824‐831.
   Centurion‐Lara, A., LaFond, R.E., Hevner, K., Godornes, C., Molini, B.J., Van Voorhis, W.C., and Lukehart, S.A. 2004. Gene conversion: A mechanism for generation of heterogeneity in the tprK gene of Treponema pallidum during infection. Mol. Microbiol. 52:1579‐1596.
   Fieldsteel, A.H., Cox, D.L., and Moeckli, R.A. 1981. Cultivation of virulent Treponema pallidum in tissue culture. Infect. Immun. 32:908‐915.
   Fribourg‐Blanc, A., Niel, G., and Mollaret, H.H. 1963. Note sur quelques aspects immunologiques du cynocephale africain. Bull. Soc. Pathol. Exot. 56:474‐485.
   Gerbase, A.C., Rowley, J.T., Heymann, D.H., Berkely, S.F., and Piot, P. 1998. Global prevalence and incidence estimates of selected curable STDs. Sex Transm. Infect. 74:S12‐S16.
   Giacani, L., Sun, E.S., Hevner, K., Molini, B.J., Van Voorhis, W.C., Lukehart, S.A., and Centurion‐Lara, A. 2004. Tpr homologs in Treponema paraluiscuniculi cuniculi A strain. Infect. Immun. 72:6561‐6576.
   Graves, S. and Downes, J. 1981. Experimental infection of man with rabbit‐virulent Treponema paraluis‐cuniculi. Br. J. Vener. Dis. 57:7‐10.
   LaFond, R.E., Centurion‐Lara, A., Godornes, C., Rompalo, A.M., Van Voorhis, W.C., and Lukehart, S.A. 2003. Sequence diversity of Treponema pallidum subsp. pallidum tprK in human syphilis lesions and rabbit‐propagated isolates. J. Bacteriol. 185:6262‐6268.
   Larsen, S.A. 1999. Manual of tests for syphilis. American Public Health Association, Washington, D.C.
   Lukehart, S.A. 1982. Activation of macrophages by products of lymphocytes from normal and syphilitic rabbits. Infect. Immun. 37:64‐69.
   Lukehart, S.A., Baker‐Zander, S.A., Lloyd, R.M., and Sell, S. 1980. Characterization of lymphocyte responsiveness in early experimental syphilis. II. Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions. J. Immunol. 124:461‐467.
   Magnuson, H.J., Eagle, H., and Fleischman, R. 1948. The minimal infectious inoculum of Spirochaeta pallida (Nichols strain) and a consideration of its rate of multiplication in vivo. Am. J. Syphilis 32:1‐18.
   Miller, J.N. 1971. Spirochetes in Body Fluids and Tissues: Manual of Investigative Methods. Charles C. Thomas, Springfield, Ill.
   Nichols, H.J. and Hough, W.H. 1913. Demonstration of Spirochaeta pallida in the cerebrospinal fluid. JAMA 60:108‐110.
   Norris, S.J. 1982. In vitro cultivation of Treponema pallidum: Independent confirmation. Infect. Immun. 36:437‐439.
   Radolf, J.D. and Lukehart, S.A. (eds.) 2006. Pathogenic Treponemes: Molecular and Cellular Biology. Caister Academic Press, London.
   Turner, T.B. and Hollander, D.H. 1957. Biology of the Treponematoses. World Health Organization, Geneva.
   Turner, T.B., Hardy, P.H., and Newman, B. 1969. Infectivity tests in syphilis. Br. J. Vener. Dis. 45:183‐196.
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