Laboratory Cultivation and Maintenance of Borrelia miyamotoi

Brandee L. Stone1, Catherine A. Brissette1

1 Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12F.1
DOI:  10.1002/cpmc.12
Online Posting Date:  August, 2016
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Borrelia miyamotoi is a relapsing fever tick‐borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour‐Stoenner‐Kelly‐II (BSK‐II) medium. The ability to culture in vitro opens many avenues for investigating the genetics and physiology of bacterial species. This unit describes methods for the maintenance and cultivation of B. miyamotoi in liquid medium. © 2016 by John Wiley & Sons, Inc.

Keywords: Borrelia miyamotoi; in vitro cultivation; Ixodes; pathogen; spirochete

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cultivation of Borrelia miyamotoi IN MKP‐F
  • Basic Protocol 2: Preparation of frozen B. miyamotoi Stocks
  • Support Protocol 1: Preparation of Complete Modified Kelly‐Pettenkofer Medium (MKP‐F)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Cultivation of Borrelia miyamotoi IN MKP‐F

  • MKP‐F, prewarmed to 34°C (see protocol 3Support Protocol)
  • Late‐exponential phase B. miyamotoi culture or frozen stock (see protocol 2)
  • 16‐ml polystyrene round bottom tube with screw cap (e.g., Falcon, cat. no. 352025)
  • 34°C incubator with or without 5% CO 2
  • Dark‐field microscope
  • Petroff‐Hausser counting chamber (optional; see appendix 4A)

Basic Protocol 2: Preparation of frozen B. miyamotoi Stocks

  • Mid‐ to late‐exponential phase B. miyamotoi culture in MKP‐F (see protocol 1)
  • 50% (v/v) glycerol, filter‐sterilized or autoclaved
  • 1.8‐ml polyethylene cryogenic tubes (e.g., Thermo Scientific, cat. no.375418)
  • Vortex (optional)

Support Protocol 1: Preparation of Complete Modified Kelly‐Pettenkofer Medium (MKP‐F)

  • CMRL‐1066 without L‐glutamine powder (US Biological Life Sciences, cat. no. C5900‐01)
  • Neopeptone
  • Sodium bicarbonate
  • Glucose
  • Sodium pyruvate
  • Sodium citrate
  • N‐acetyl‐glucosamine
  • Heat‐inactivated rabbit serum
  • 5 N NaOH
  • Heat‐inactivated fetal bovine serum (FBS)
  • 65.57 g/liter bovine serum albumin (BSA), pH 7.6, sterile (see recipe)
  • 7% (w/v) gelatin, pH 7.7, sterile (see recipe)
  • 1‐liter beaker
  • 0.22‐μm filter units
  • Vacuum filtration system
  • Tissue culture hood
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Literature Cited

Literature Cited
  Barbour, A.G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521‐525.
  Barbour, A.G., Bunikis, J., Travinsky, B., Hoen, A.G., Diuk‐Wasser, M.A., Fish, D., and Tsao, J.I. 2009. Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. Am. J. Trop. Med. Hyg. 81:1120‐1131. doi: 10.4269/ajtmh.2009.09‐0208.
  Callister, S.M., Case, K.L., Agger, W.A., Schell, R.F., Johnson, R.C., and Ellingson, J.L. 1990. Effects of bovine serum albumin on the ability of Barbour‐Stoenner‐Kelly medium to detect Borrelia burgdorferi. J. Clin. Microbiol. 28:363‐365.
  Cochez, C., Heyman, P., Heylen, D., Fonville, M., Hengeveld, P., Takken, W., Simons, L., and Sprong, H. 2015. The presence of Borrelia miyamotoi, a relapsing fever spirochaete, in questing Ixodes ricinus in Belgium and in the Netherlands. Zoonoses and Public Health 62:331‐333. doi: 10.1111/zph.12154.
  Eshoo, M.W., Carolan, H.E., Massire, C., Chou, D.M., Crowder, C.D., Rounds, M.A., Phillipson, C.A., Schutzer, S.E., and Ecker, D.J. 2015. Survey of Ixodes pacificus ticks in California reveals a diversity of microorganisms and a novel and widespread Anaplasmataceae species. PLoS One 10:e0135828. doi: 10.1371/journal.pone.0135828.
  Fukunaga, M. and Koreki, Y. 1995. The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species. FEMS Microbiol. Lett. 134:255‐258. doi: 10.1111/j.1574‐6968.1995.tb07947.x.
  Fukunaga, M., Takahashi, Y., Tsuruta, Y., Matsushita, O., Ralph, D., McClelland, M., and Nakao, M. 1995. Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. Int. J. Syst. Bacteriol. 45:804‐810. doi: 10.1099/00207713‐45‐4‐804.
  Hamase, A., Takahashi, Y., Nohgi, K., and Fukunaga, M. 1996. Homology of variable major protein genes between Borrelia hermsii and Borrelia miyamotoi. FEMS Microbiol. Lett. 140:131‐137. doi: 10.1111/j.1574‐6968.1996.tb08326.x.
  Hue, F., Langeroudi, A.G., and Barbour, A.G. 2013. Chromosome sequence of Borrelia miyamotoi, an uncultivable tick‐borne agent of human infection. Genome Announc. 1:e00713‐13. doi: 10.1128/genomeA.00713‐13.
  Khasnatinov, M.A., Danchinova, G.A., Takano, A., Kawabata, H., Ohashi, N., and Masuzawa, T. 2016. Prevalence of Borrelia miyamotoi in Ixodes persulcatus in Irkutsk City and its neighboring territories, Russia. Ticks Tick Borne Dis. 7:394–397. doi:10.1016/j.ttbdis.2015.12.016.
  Margos, G., Stockmeier, S., Hizo‐Teufel, C., Hepner, S., Fish, D., Dautel, H., Sing, A., Dzaferovic, E., Rieger, M., Jungnick, S., Binder, K., Straubinger R.K., and Fingerle, V. 2015. Long‐term in vitro cultivation of Borrelia miyamotoi. Ticks Tick Borne Dis. 6:181‐184. doi: 10.1016/j.ttbdis.2014.12.001.
  Mays, S.E., Hendricks, B.M., Paulsen, D.J., Houston, A.E., and Fryxell, R.T.T. 2014. Prevalence of five tick‐borne bacterial genera in adult Ixodes scapularis removed from white‐tailed deer in western Tennessee. Parasit. Vectors 7:473. doi: 10.1186/s13071‐014‐0473‐y.
  Pollack, R.J., Telford, S.R., and Spielman, A. 1993. Standardization of medium for culturing Lyme disease spirochetes. J. Clin. Microbiol. 31:1251‐1255.
  Preac‐Mursic, V., Wilske, B., and Schierz, G. 1986. European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 263:112‐118. doi:10.1016/S0176‐6724(86)80110‐9.
  Richter, D., Schlee, D.B., and Matuschka, F.‐R. 2003. Relapsing fever‐like spirochetes infecting European vector tick of Lyme disease agent. Emerging Infect. Dis. 9:697. doi: 10.3201/eid0906.020459.
  Salkeld, D.J., Cinkovich, S., and Nieto, N.C. 2014. Tick‐borne pathogens in northwestern California, USA. Emerging Infect. Dis. 20:493‐494. doi: 10.3201/eid2003.130668.
  Scoles, G.A., Papero, M., Beati, L., and Fish, D. 2001. A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vector Borne Zoonotic Dis. 1:21‐34. doi: 10.1089/153036601750137624.
  Wagemakers, A., Oei, A., Fikrig, M.M., Miellet, W.R., and Hovius, J.W. 2014. The relapsing fever spirochete Borrelia miyamotoi is cultivable in a modified Kelly‐Pettenkofer medium, and is resistant to human complement. Parasit. Vectors 7:418. doi: 10.1186/1756‐3305‐7‐418.
  Wang, G., Iyer, R., Bittker, S., Cooper, D., Small, J., Wormser, G.P., and Schwartz, I. 2004. Variations in Barbour‐Stoenner‐Kelly culture medium modulate infectivity and pathogenicity of Borrelia burgdorferi clinical isolates. Infect. Immun. 72:6702‐6706. doi: 10.1128/IAI.72.11.6702‐6706.2004.
  Zückert, W.R. 2007. Laboratory maintenance of Borrelia burgdorferi. Curr. Protoc. Microbio. 4:12C.1.1‐12C.1.10. doi: 10.1002/9780471729259.mc12c01s4.
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