Genotyping Strains of Lyme Disease Agents Directly From Ticks, Blood, or Tissue

Part of the Methods in Molecular Biology book series (MIMB, volume 1690)


The tick-borne spirochetes that cause Lyme disease in North America and Eurasia display strong linkage disequilibrium between certain chromosomal and plasmid loci within each three major geographic areas of their distribution. For strain typing for epidemiologic and ecologic purposes, the commonly used genotypes based on a single locus are the spacer between the 16S–23S ribosomal RNA and the ospC gene of a plasmid. A simple genotyping scheme based on the two loci allows for discrimination between strains representing all the areas of distribution. The methods presented here are meant for genotyping directly from ticks and from blood and tissue samples from vertebrates.

Key words

Borrelia Borreliella B. burgdorferi Ixodes 


  1. 1.
    Adeolu M, Gupta RS (2014) A phylogenomic and molecular marker based proposal for the division of the genus Borrelia into two genera: the emended genus Borrelia containing only the members of the relapsing fever Borrelia, and the genus Borreliella gen. nov. containing the members of the Lyme disease Borrelia (Borrelia burgdorferi sensu lato complex). Antonie Van Leeuwenhoek 105(6):1049–1072. doi: 10.1007/s10482-014-0164-x CrossRefPubMedGoogle Scholar
  2. 2.
    Oren A, Garrity GM (2015) List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 65(7):2017–2025CrossRefGoogle Scholar
  3. 3.
    Wang IN, Dykhuizen DE, Qiu W, Dunn JJ, Bosler EM, Luft BJ (1999) Genetic diversity of ospC in a local population of Borrelia burgdorferi sensu stricto. Genetics 151(1):15–30PubMedPubMedCentralGoogle Scholar
  4. 4.
    Bunikis J, Garpmo U, Tsao J, Berglund J, Fish D, Barbour AG (2004) Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe. Microbiology 150:1741–1755. doi: 10.1099/mic.0.26944-0 CrossRefPubMedGoogle Scholar
  5. 5.
    Margos G, Gatewood AG, Aanensen DM, Hanincova K, Terekhova D, Vollmer SA, Cornet M, Piesman J, Donaghy M, Bormane A, Hurn MA, Feil EJ, Fish D, Casjens S, Wormser GP, Schwartz I, Kurtenbach K (2008) MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc Natl Acad Sci U S A 105(25):8730–8735. doi: 10.1073/pnas.0800323105. PMC: Pmc2435589CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hoen AG, Margos G, Bent SJ, Diuk-Wasser MA, Barbour A, Kurtenbach K, Fish D (2009) Phylogeography of Borrelia burgdorferi in the eastern United States reflects multiple independent Lyme disease emergence events. Proc Natl Acad Sci U S A 106(35):15013–15018. doi: 10.1073/pnas.0903810106. PMC: 2727481CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Brisson D, Vandermause MF, Meece JK, Reed KD, Dykhuizen DE (2010) Evolution of northeastern and midwestern Borrelia burgdorferi, United States. Emerg Infect Dis 16(6):911–917. doi: 10.3201/eid1606.090329. PMC: 3086229CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Travinsky B, Bunikis J, Barbour AG (2010) Geographic differences in genetic locus linkages for Borrelia burgdorferi. Emerg Infect Dis 16(7):1147–1150. PMC: 2841027CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Marti Ras N, Postic D, Foretz M, Baranton G (1997) Borrelia burgdorferi sensu stricto, a bacterial species “made in the U.S.A.”? Int J Syst Bacteriol 47(4):1112–1117CrossRefPubMedGoogle Scholar
  10. 10.
    Dykhuizen DE, Polin DS, Dunn JJ, Wilske B, Preac-Mursic V, Dattwyler RJ, Luft BJ (1993) Borrelia burgdorferi is clonal: implications for taxonomy and vaccine development. Proc Natl Acad Sci U S A 90(21):10163–10167CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Marconi RT, Samuels DS, Landry RK, Garon CF (1994) Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange. J Bacteriol 176(15):4572–4582CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Livey I, Gibbs CP, Schuster R, Dorner F (1995) Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia. Mol Microbiol 18(2):257–269CrossRefPubMedGoogle Scholar
  13. 13.
    Barbour AG, Travinsky B (2010) Evolution and distribution of the ospC Gene, a transferable serotype determinant of Borrelia burgdorferi. MBio 1(4). doi: 10.1128/mBio.00153-10. PMC: 2945197
  14. 14.
    Haven J, Vargas LC, Mongodin EF, Xue V, Hernandez Y, Pagan P, Fraser-Liggett CM, Schutzer SE, Luft BJ, Casjens SR, Qiu WG (2011) Pervasive recombination and sympatric genome diversification driven by frequency-dependent selection in Borrelia burgdorferi, the Lyme disease bacterium. Genetics 189(3):951–966. doi: 10.1534/genetics.111.130773. PMC: 3213364CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Nadelman RB, Hanincova K, Mukherjee P, Liveris D, Nowakowski J, McKenna D, Brisson D, Cooper D, Bittker S, Madison G, Holmgren D, Schwartz I, Wormser GP (2012) Differentiation of reinfection from relapse in recurrent Lyme disease. N Engl J Med 367(20):1883–1890. doi: 10.1056/NEJMoa1114362. PMC: 3526003CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wormser GP, Brisson D, Liveris D, Hanincova K, Sandigursky S, Nowakowski J, Nadelman RB, Ludin S, Schwartz I (2008) Borrelia burgdorferi genotype predicts the capacity for hematogenous dissemination during early Lyme disease. J Infect Dis 198(9):1358–1364CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Barbour AG, Heiland RA, Howe TR (1985) Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J Infect Dis 152(3):478–484CrossRefPubMedGoogle Scholar
  18. 18.
    Barbour AG (1988) Laboratory aspects of Lyme borreliosis. Clin Microbiol Rev 1(4):399–414CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Barbour AG (1988) Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. J Clin Microbiol 26(3):475–478PubMedPubMedCentralGoogle Scholar
  20. 20.
    Mathiesen DA, Oliver JH Jr, Kolbert CP, Tullson ED, Johnson BJ, Campbell GL, Mitchell PD, Reed KD, Telford SR 3rd, Anderson JF, Lane RS, Persing DH (1997) Genetic heterogeneity of Borrelia burgdorferi in the United States. J Infect Dis 175(1):98–107CrossRefPubMedGoogle Scholar
  21. 21.
    Lebech AM, Hansen K, Wilske B, Theisen M (1994) Taxonomic classification of 29 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis: a comparison of five different phenotypic and genotypic typing schemes. Med Microbiol Immunol 183(6):325–341CrossRefPubMedGoogle Scholar
  22. 22.
    Liveris D, Gazumyan A, Schwartz I (1995) Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis. J Clin Microbiol 33(3):589–595PubMedPubMedCentralGoogle Scholar
  23. 23.
    Gazumyan A, Schwartz JJ, Liveris D, Schwartz I (1994) Sequence analysis of the ribosomal RNA operon of the Lyme disease spirochete, Borrelia burgdorferi. Gene 146(1):57–65CrossRefPubMedGoogle Scholar
  24. 24.
    Fraser CM, Casjens S, Huang WM, Sutton GG, Clayton R, Lathigra R, White O, Ketchum KA, Dodson R, Hickey EK, Gwinn M, Dougherty B, Tomb JF, Fleischmann RD, Richardson D, Peterson J, Kerlavage AR, Quackenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams MD, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, Garland S, Fuji C, Cotton MD, Horst K, Roberts K, Hatch B, Smith HO, Venter JC (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390(6660):580–586CrossRefPubMedGoogle Scholar
  25. 25.
    Postic D, Assous MV, Grimont PA, Baranton G (1994) Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol 44(4):743–752CrossRefPubMedGoogle Scholar
  26. 26.
    Qiu WG, Bosler EM, Campbell JR, Ugine GD, Wang IN, Luft BJ, Dykhuizen DE (1997) A population genetic study of Borrelia burgdorferi sensu stricto from eastern Long Island, New York, suggested frequency-dependent selection, gene flow and host adaptation. Hereditas 127(3):203–216CrossRefPubMedGoogle Scholar
  27. 27.
    Qiu WG, Dykhuizen DE, Acosta MS, Luft BJ (2002) Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the Northeastern United States. Genetics 160(3):833–849PubMedPubMedCentralGoogle Scholar
  28. 28.
    Bunikis J, Tsao J, Garpmo U, Berglund J, Fish D, Barbour AG (2004) Typing of Borrelia relapsing fever group strains. Emerg Infect Dis 10(9):1661–1664. PMC: PMC3320305CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Porcella SF, Raffel SJ, Anderson DE Jr, Gilk SD, Bono JL, Schrumpf ME, Schwan TG (2005) Variable tick protein in two genomic groups of the relapsing fever spirochete Borrelia hermsii in western North America. Infect Immun 73(10):6647–6658. doi: 10.1128/IAI.73.10.6647-6658.2005. PMC: 1230938CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Cook VJ, Fedorova N, Macdonald WP, Lane RS, Barbour AG (2016) Unique strain of Borrelia miyamotoi in Ixodes pacificus ticks, California, USA. Emerg Infect Dis 22(12). doi: 10.3201/eid2212.152046
  31. 31.
    Barbour AG, Bunikis J, Travinsky B, Hoen AG, Diuk-Wasser MA, Fish D, Tsao JI (2009) Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. Am J Trop Med Hyg 81(6):1120–1131. doi: 10.4269/ajtmh.2009.09-0208. PMC: 2841027CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Cook V, Barbour AG (2015) Broad diversity of host responses of the white-footed mouse Peromyscus leucopus to Borrelia infection and antigens. Ticks Tick Borne Dis 6(5):549–558. doi: 10.1016/j.ttbdis.2015.04.009. PubMed PMC: 4504778CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Baum E, Randall AZ, Zeller M, Barbour AG (2013) Inferring epitopes of a polymorphic antigen amidst broadly cross-reactive antibodies using protein microarrays: a study of OspC proteins of Borrelia burgdorferi. PLoS One 8(6):e67445. doi: 10.1371/journal.pone.0067445. PMC: 3691210CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Microbiology & Molecular GeneticsUniversity of California IrvineIrvineUSA
  2. 2.Department of MedicineUniversity of California IrvineIrvineUSA
  3. 3.Department of Ecology & Evolutionary BiologyUniversity of California IrvineIrvineUSA

Personalised recommendations