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Infection

, Volume 44, Issue 1, pp 47–55 | Cite as

A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato

  • R. Venczel
  • L. Knoke
  • M. Pavlovic
  • E. Dzaferovic
  • T. Vaculova
  • C. Silaghi
  • E. Overzier
  • R. Konrad
  • S. Kolenčík
  • M. Derdakova
  • A. Sing
  • G. A. Schaub
  • G. Margos
  • V. FingerleEmail author
Original Paper

Abstract

Purpose

For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection.

Methods

Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 104 to 10−1) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment.

Results

The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 102 genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2 % for B. miyamotoi and 11.8 % for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples.

Conclusion

The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Keywords

Real-time PCR Borrelia burgdorferi sensu lato Borrelia miyamotoi 

Notes

Acknowledgments

The authors would like to acknowledge Cecilia Hizo-Teufel, Sylvia Stockmeier, Katja Meindl, Sabine Wolf, Jasmin Fräßdorf, A. Muminovic, AK Steinmann, Claudia Brefeld, and Simone Hanisch for excellent technical assistance. The work was partially funded by the Scientific Grant Agency of the Ministry of Education, science, research and sport of the Slovak Republic and the Slovak Academy of Sciences, Project VEGA No. 2/0108/13. This study was partially funded by EUgrant FP7-261504 EDENext and is cataloged by the EDENext Steering Committee (http:/www.edenext.eu/). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The work of CS was done under the frame of EurNegVec COST Action TD1303. V. Fingerle, G. Margos, and M. Derdakova are the members of ESGBOR.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

15010_2015_820_MOESM1_ESM.doc (490 kb)
Supplementary material 1 (DOC 490 kb)

References

  1. 1.
    Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diagnosis of lyme borreliosis. Clin Microbiol Rev. 2005;18:484–509.PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Amore G, Tomassone L, Grego E, Ragagli C, Bertolotti L, Nebbia P, et al. Borrelia lusitaniae in immature Ixodes ricinus (Acari: Ixodidae) feeding on common wall lizards in Tuscany, central Italy. J Med Entomol. 2007;44:303–7.CrossRefPubMedGoogle Scholar
  3. 3.
    Bunikis J, Tsao J, Garpmo U, Berglund J, Fish D, Barbour AG. Typing of Borrelia relapsing fever group strains. Emerg Infect Dis. 2004;10:1661–4.PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Cochez C, Heyman P, Heylen D, Fonville M, Hengeveld P, Takken W, et al. The presence of Borrelia miyamotoi, a relapsing fever spirochaete, in questing Ixodes ricinus in Belgium and in the Netherlands. Zoonoses Public Health. 2014.Google Scholar
  5. 5.
    Crowder CD, Carolan HE, Rounds MA, Honig V, Mothes B, Haag H, et al. Prevalence of Borrelia miyamotoi in Ixodes ticks in Europe and the United States. Emerg Infect Dis. 2014;20:1678–82.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    De Michelis S, Sewell HS, Collares-Pereira M, Santos-Reis M, Schouls LM, Benes V, et al. Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal. J Clin Microbiol. 2000;38:2128–33.PubMedCentralPubMedGoogle Scholar
  7. 7.
    Dsouli N, Younsi-Kabachii H, Postic D, Nouira S, Gern L, Bouattour A. Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi sensu lato (Spirochaetaceae) in Tunisia. J Med Entomol. 2006;43:737–42.CrossRefPubMedGoogle Scholar
  8. 8.
    Fraenkel CJ, Garpmo U, Berglund J. Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. J Clin Microbiol. 2002;40:3308–12.PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Fukunaga M, Koreki Y. The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species. FEMS Microbiol Lett. 1995;134:255–8.CrossRefPubMedGoogle Scholar
  10. 10.
    Geller J, Nazarova L, Katargina O, Järvekülg L, Fomenko N, Golovljova I. Detection and genetic characterization of relapsing fever spirochete Borrelia miyamotoi in Estonian ticks. PLoS One. 2012;7:e51914.PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Gugliotta JL, Goethert HK, Berardi VP, Telford SR 3rd. Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient. N Engl J Med. 2013;368:240–5.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Guy EC, Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J Clin Pathol. 1991;44:610–1.PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Hanincova K, Schafer SM, Etti S, Sewell HS, Taragelova V, Ziak D, et al. Association of Borrelia afzelii with rodents in Europe. Parasitology. 2003;126:11–20.CrossRefPubMedGoogle Scholar
  14. 14.
    Hovius JW, de Wever B, Sohne M, Brouwer MC, Coumou J, Wagemakers A, et al. A case of meningoencephalitis by the relapsing fever spirochaete Borrelia miyamotoi in Europe. Lancet. 2013;382:658.PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Krause PJ, Fish D, Narasimhan S, Barbour AG. Borrelia miyamotoi infection in nature and in humans. Clin Microbiol Infect. 2015. doi: 10.1016/j.cmi.2015.02.006.Google Scholar
  16. 16.
    Krause PJ, Narasimhan S, Wormser GP, Rollend L, Fikrig E, Lepore T, et al. Human Borrelia miyamotoi Infection in the United States. N Engl J Med. 2013;368:290–1.CrossRefGoogle Scholar
  17. 17.
    Kumar S, Tamura K, Nei M. Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 2004;5:150–63.CrossRefPubMedGoogle Scholar
  18. 18.
    Maraspin V, Ogrinc K, Ruzic-Sabljic E, Lotric-Furlan S, Strle F. Isolation of Borrelia burgdorferi sensu lato from blood of adult patients with borrelial lymphocytoma, Lyme neuroborreliosis, Lyme arthritis and acrodermatitis chronica atrophicans. Infection. 2011;39:35–40.CrossRefPubMedGoogle Scholar
  19. 19.
    Margos G, Stockmeier S, Hizo-Teufel C, Hepner S, Fish D, Dautel H, et al. Long-term in vitro cultivation of Borrelia miyamotoi. Ticks Tick Borne Dis. 2015;6:181–4.CrossRefPubMedGoogle Scholar
  20. 20.
    Margos G, Vollmer SA, Cornet M, Garnier M, Fingerle V, Wilske B, et al. A new Borrelia species defined by multilocus sequence analysis of housekeeping genes. Appl Environ Microbiol. 2009;75:5410–6.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Masuzawa T. Terrestrial distribution of the Lyme borreliosis agent Borrelia burgdorferi sensu lato in East Asia. Jpn J Infect Dis. 2004;57:229–35.PubMedGoogle Scholar
  22. 22.
    Ornstein K, Barbour AG. A reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector. Vector Borne Zoonotic Dis. 2006;6:103–12.CrossRefPubMedGoogle Scholar
  23. 23.
    Platonov AE, Karan LS, Kolyasnikova NM, Makhneva NA, Toporkova MG, Maleev VV, et al. Humans infected with relapsing fever spirochete Borrelia miyamotoi. Russia. Emerg Infect Dis. 2011;17:1816–23.CrossRefPubMedGoogle Scholar
  24. 24.
    Richter D, Schlee DB, Matuschka FR. Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent. Emerg Infect Dis. 2003;9:697–701.PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Sato K, Takano A, Konnai S, Nakao M, Ito T, Koyama K, et al. Human infections with Borrelia miyamotoi. Japan. Emerg Infect Dis. 2014;20:1391–3.CrossRefPubMedGoogle Scholar
  26. 26.
    Schwaiger M, Peter O, Cassinotti P. Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay. Clin Microbiol Infect. 2001;7:461–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Schwarz A, Honig V, Vavruskova Z, Grubhoffer L, Balczun C, Albring A, et al. Abundance of Ixodes ricinus and prevalence of Borrelia burgdorferi s.l. in the nature reserve Siebengebirge, Germany, in comparison to three former studies from 1978 onwards. Parasit Vectors. 2012;5:268.PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Scoles GA, Papero M, Beati L, Fish D. A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vector Borne Zoonotic Dis. 2001;1:21–34.CrossRefPubMedGoogle Scholar
  29. 29.
    Takano A, Toyomane K, Konnai S, Ohashi K, Nakao M, Ito T, et al. Tick surveillance for relapsing fever spirochete Borrelia miyamotoi in Hokkaido, Japan. PLoS One. 2014;9:e104532.PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Taylor KR, Takano A, Konnai S, Shimozuru M, Kawabata H, Tsubota T. Borrelia miyamotoi infections among wild rodents show age and month independence and correlation with Ixodes persulcatus larval attachment in Hokkaido. Japan. Vector Borne Zoonotic Dis. 2013;13:92–7.CrossRefPubMedGoogle Scholar
  31. 31.
    Tsao JI, Wootton JT, Bunikis J, Luna MG, Fish D, Barbour AG. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc Natl Acad Sci U S A. 2004;101:18159–64.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Ullmann AJ, Gabitzsch ES, Schulze TL, Zeidner NS, Piesman J. Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected ixodes nymphs in North America. J Med Entomol. 2005;42:1057–62.CrossRefPubMedGoogle Scholar
  33. 33.
    Vitorino LR, Margos G, Feil EJ, Collares-Pereira M, Ze-Ze L, Kurtenbach K. Fine-scale phylogeographic structure of Borrelia lusitaniae revealed by multilocus sequence typing. PLoS ONE. 2008;3:e4002.PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Yossepowitch O, Gottesman T, Schwartz-Harari O, Soroksky A, Dan M. Aseptic meningitis and adult respiratory distress syndrome caused by Borrelia persica. Infection. 2012;40:695–7.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • R. Venczel
    • 1
    • 2
  • L. Knoke
    • 3
  • M. Pavlovic
    • 2
  • E. Dzaferovic
    • 1
  • T. Vaculova
    • 4
  • C. Silaghi
    • 5
    • 7
  • E. Overzier
    • 5
    • 8
  • R. Konrad
    • 2
  • S. Kolenčík
    • 6
  • M. Derdakova
    • 4
  • A. Sing
    • 2
  • G. A. Schaub
    • 3
  • G. Margos
    • 1
    • 2
  • V. Fingerle
    • 1
    • 2
    Email author
  1. 1.German National Reference Centre for BorreliaOberschleissheimGermany
  2. 2.Bavarian Health and Food Safety AuthorityOberschleissheimGermany
  3. 3.Zoology/ParasitologyRuhr University BochumBochumGermany
  4. 4.Institute of ZoologySlovak Academy of SciencesBratislavaSlovakia
  5. 5.Comparative Tropical Medicine and ParasitologyLudwig-Maximilians-Universität München, MunichMunichGermany
  6. 6.University of Veterinary and Pharmaceutical SciencesBrnoCzech Republic
  7. 7.Swiss National Reference Center for Vector Entomology, Institute of Parasitology, Vetsuisse FacultyUniversity of ZurichZurichSwitzerland
  8. 8.Lehrstuhl für Bakteriologie und Mykologie, Veterinärwissenschaftliches DepartmentTierärztliche Fakultät der LMUMunichGermany

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