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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Nov;35(11):2752–2758. doi: 10.1128/jcm.35.11.2752-2758.1997

Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis.

D Roessler 1, U Hauser 1, B Wilske 1
PMCID: PMC230055  PMID: 9350727

Abstract

The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B. afzelii or B. garinii should be used solely, or in addition to B. burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.

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Selected References

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  1. Aguero-Rosenfeld M. E., Nowakowski J., McKenna D. F., Carbonaro C. A., Wormser G. P. Serodiagnosis in early Lyme disease. J Clin Microbiol. 1993 Dec;31(12):3090–3095. doi: 10.1128/jcm.31.12.3090-3095.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baranton G., Assous M., Postic D. Trois espèces bactériennes associées à la borréliose de Lyme. Conséquences cliniques et diagnostiques. Bull Acad Natl Med. 1992 Oct;176(7):1075–1086. [PubMed] [Google Scholar]
  3. Baranton G., Postic D., Saint Girons I., Boerlin P., Piffaretti J. C., Assous M., Grimont P. A. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol. 1992 Jul;42(3):378–383. doi: 10.1099/00207713-42-3-378. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burkert S., Rössler D., Münchhoff P., Wilske B. Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection. Med Microbiol Immunol. 1996 May;185(1):49–57. doi: 10.1007/s004300050014. [DOI] [PubMed] [Google Scholar]
  6. Dressler F., Ackermann R., Steere A. C. Antibody responses to the three genomic groups of Borrelia burgdorferi in European Lyme borreliosis. J Infect Dis. 1994 Feb;169(2):313–318. doi: 10.1093/infdis/169.2.313. [DOI] [PubMed] [Google Scholar]
  7. Dressler F., Whalen J. A., Reinhardt B. N., Steere A. C. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 1993 Feb;167(2):392–400. doi: 10.1093/infdis/167.2.392. [DOI] [PubMed] [Google Scholar]
  8. Engstrom S. M., Shoop E., Johnson R. C. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995 Feb;33(2):419–427. doi: 10.1128/jcm.33.2.419-427.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fawcett P. T., Rose C., Gibney K. M., Chase C. A., Kiehl B., Doughty R. A. Detection of antibodies to the recombinant P39 protein of Borrelia burgdorferi using enzyme immunoassay and immunoblotting. J Rheumatol. 1993 Apr;20(4):734–738. [PubMed] [Google Scholar]
  10. Fikrig E., Barthold S. W., Kantor F. S., Flavell R. A. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science. 1990 Oct 26;250(4980):553–556. doi: 10.1126/science.2237407. [DOI] [PubMed] [Google Scholar]
  11. Galfrè G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
  12. Ge Y., Charon N. W. An unexpected flaA homolog is present and expressed in Borrelia burgdorferi. J Bacteriol. 1997 Jan;179(2):552–556. doi: 10.1128/jb.179.2.552-556.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gilmore R. D., Jr, Kappel K. J., Dolan M. C., Burkot T. R., Johnson B. J. Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC. Infect Immun. 1996 Jun;64(6):2234–2239. doi: 10.1128/iai.64.6.2234-2239.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hauser U., Lehnert G., Lobentanzer R., Wilske B. Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato. J Clin Microbiol. 1997 Jun;35(6):1433–1444. doi: 10.1128/jcm.35.6.1433-1444.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hochuli E. Purification of recombinant proteins with metal chelate adsorbent. Genet Eng (N Y) 1990;12:87–98. doi: 10.1007/978-1-4613-0641-2_6. [DOI] [PubMed] [Google Scholar]
  16. Hughes C. A., Engstrom S. M., Coleman L. A., Kodner C. B., Johnson R. C. Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB. Infect Immun. 1993 Dec;61(12):5115–5122. doi: 10.1128/iai.61.12.5115-5122.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jauris-Heipke S., Fuchs R., Motz M., Preac-Mursic V., Schwab E., Soutschek E., Will G., Wilske B. Genetic heterogenity of the genes coding for the outer surface protein C (OspC) and the flagellin of Borrelia burgdorferi. Med Microbiol Immunol. 1993 Mar;182(1):37–50. doi: 10.1007/BF00195949. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Luft B. J., Steinman C. R., Neimark H. C., Muralidhar B., Rush T., Finkel M. F., Kunkel M., Dattwyler R. J. Invasion of the central nervous system by Borrelia burgdorferi in acute disseminated infection. JAMA. 1992 Mar 11;267(10):1364–1367. [PubMed] [Google Scholar]
  20. Ma B., Christen B., Leung D., Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol. 1992 Feb;30(2):370–376. doi: 10.1128/jcm.30.2.370-376.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marconi R. T., Samuels D. S., Landry R. K., Garon C. F. Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange. J Bacteriol. 1994 Aug;176(15):4572–4582. doi: 10.1128/jb.176.15.4572-4582.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mathiesen M. J., Hansen K., Axelsen N., Halkier-Sørensen L., Theisen M. Analysis of the human antibody response to outer surface protein C (OspC) of Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Med Microbiol Immunol. 1996 Nov;185(3):121–129. doi: 10.1007/s004300050021. [DOI] [PubMed] [Google Scholar]
  23. Mitchell P. D., Reed K. D., Aspeslet T. L., Vandermause M. F., Melski J. W. Comparison of four immunoserologic assays for detection of antibodies to Borrelia burgdorferi in patients with culture-positive erythema migrans. J Clin Microbiol. 1994 Aug;32(8):1958–1962. doi: 10.1128/jcm.32.8.1958-1962.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Norman G. L., Antig J. M., Bigaignon G., Hogrefe W. R. Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots). J Clin Microbiol. 1996 Jul;34(7):1732–1738. doi: 10.1128/jcm.34.7.1732-1738.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oksi J., Uksila J., Marjamäki M., Nikoskelainen J., Viljanen M. K. Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis. J Clin Microbiol. 1995 Sep;33(9):2260–2264. doi: 10.1128/jcm.33.9.2260-2264.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Preac-Mursic V., Wilske B., Patsouris E., Jauris S., Will G., Soutschek E., Rainhardt S., Lehnert G., Klockmann U., Mehraein P. Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection. Infection. 1992 Nov-Dec;20(6):342–349. doi: 10.1007/BF01710681. [DOI] [PubMed] [Google Scholar]
  27. Preac-Mursic V., Wilske B., Schierz G. European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Dec;263(1-2):112–118. doi: 10.1016/s0176-6724(86)80110-9. [DOI] [PubMed] [Google Scholar]
  28. Ramamoorthy R., Povinelli L., Philipp M T. Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi. Infect Immun. 1996 Apr;64(4):1259–1264. doi: 10.1128/iai.64.4.1259-1264.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rössler D., Eiffert H., Jauris-Heipke S., Lehnert G., Preac-Mursic V., Teepe J., Schlott T., Soutschek E., Wilske B. Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains. Med Microbiol Immunol. 1995 May;184(1):23–32. doi: 10.1007/BF00216786. [DOI] [PubMed] [Google Scholar]
  30. Schaible U. E., Kramer M. D., Eichmann K., Modolell M., Museteanu C., Simon M. M. Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc Natl Acad Sci U S A. 1990 May;87(10):3768–3772. doi: 10.1073/pnas.87.10.3768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schmitz J. L., Powell C. S., Folds J. D. Comparison of seven commercial kits for detection of antibodies to Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis. 1993 Jun;12(6):419–424. doi: 10.1007/BF01967435. [DOI] [PubMed] [Google Scholar]
  32. Schwan T. G., Schrumpf M. E., Gage K. L., Gilmore R. D., Jr Analysis of Leptospira spp., Leptonema illini, and Rickettsia rickettsii for the 39-kilodalton antigen (P39) of Borrelia burgdorferi. J Clin Microbiol. 1992 Mar;30(3):735–738. doi: 10.1128/jcm.30.3.735-738.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scriba M., Ebrahim J. S., Schlott T., Eiffert H. The 39-kilodalton protein of Borrelia burgdorferi: a target for bactericidal human monoclonal antibodies. Infect Immun. 1993 Oct;61(10):4523–4526. doi: 10.1128/iai.61.10.4523-4526.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simpson W. J., Cieplak W., Schrumpf M. E., Barbour A. G., Schwan T. G. Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbiol Lett. 1994 Jun 15;119(3):381–387. doi: 10.1111/j.1574-6968.1994.tb06917.x. [DOI] [PubMed] [Google Scholar]
  35. Simpson W. J., Schrumpf M. E., Schwan T. G. Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi. J Clin Microbiol. 1990 Jun;28(6):1329–1337. doi: 10.1128/jcm.28.6.1329-1337.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Skerra A., Pfitzinger I., Plückthun A. The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. Biotechnology (N Y) 1991 Mar;9(3):273–278. doi: 10.1038/nbt0391-273. [DOI] [PubMed] [Google Scholar]
  37. Sullivan T. J., Hechemy K. E., Harris H. L., Rudofsky U. H., Samsonoff W. A., Peterson A. J., Evans B. D., Balaban S. L. Monoclonal antibody to native P39 protein from Borrelia burgdorferi. J Clin Microbiol. 1994 Feb;32(2):423–429. doi: 10.1128/jcm.32.2.423-429.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Theisen M., Frederiksen B., Lebech A. M., Vuust J., Hansen K. Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen. J Clin Microbiol. 1993 Oct;31(10):2570–2576. doi: 10.1128/jcm.31.10.2570-2576.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weber K., Wilske B., Preac-Mursic V., Thurmayr R. Azithromycin versus penicillin V for the treatment of early Lyme borreliosis. Infection. 1993 Nov-Dec;21(6):367–372. doi: 10.1007/BF01728915. [DOI] [PubMed] [Google Scholar]
  40. Will G., Jauris-Heipke S., Schwab E., Busch U., Rössler D., Soutschek E., Wilske B., Preac-Mursic V. Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species. Med Microbiol Immunol. 1995 Aug;184(2):73–80. doi: 10.1007/BF00221390. [DOI] [PubMed] [Google Scholar]
  41. Wilske B., Busch U., Eiffert H., Fingerle V., Pfister H. W., Rössler D., Preac-Mursic V. Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany. Med Microbiol Immunol. 1996 Feb;184(4):195–201. doi: 10.1007/BF02456135. [DOI] [PubMed] [Google Scholar]
  42. Wilske B., Fingerle V., Preac-Mursic V., Jauris-Heipke S., Hofmann A., Loy H., Pfister H. W., Rössler D., Soutschek E. Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato. Med Microbiol Immunol. 1994 Feb;183(1):43–59. doi: 10.1007/BF00193630. [DOI] [PubMed] [Google Scholar]
  43. Wilske B., Luft B., Schubach W. H., Zumstein G., Jauris S., Preac-Mursic V., Kramer M. D. Molecular analysis of the outer surface protein A (OspA) of Borrelia burgdorferi for conserved and variable antibody binding domains. Med Microbiol Immunol. 1992;181(4):191–207. doi: 10.1007/BF00215765. [DOI] [PubMed] [Google Scholar]
  44. Wilske B., Preac-Mursic V., Göbel U. B., Graf B., Jauris S., Soutschek E., Schwab E., Zumstein G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol. 1993 Feb;31(2):340–350. doi: 10.1128/jcm.31.2.340-350.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wilske B., Preac-Mursic V., Jauris S., Hofmann A., Pradel I., Soutschek E., Schwab E., Will G., Wanner G. Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi. Infect Immun. 1993 May;61(5):2182–2191. doi: 10.1128/iai.61.5.2182-2191.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wilske B., Preac-Mursic V., Schierz G., Gueye W., Herzer P., Weber K. Immunochemische Analyse der Immunantwort bei Spätmanifestationen der Lyme Borreliose. Zentralbl Bakteriol Mikrobiol Hyg A. 1988 Mar;267(4):549–558. [PubMed] [Google Scholar]
  47. Zöller L., Burkard S., Schäfer H. Validity of western immunoblot band patterns in the serodiagnosis of Lyme borreliosis. J Clin Microbiol. 1991 Jan;29(1):174–182. doi: 10.1128/jcm.29.1.174-182.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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