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. 1995 Sep;33(9):2304–2315. doi: 10.1128/jcm.33.9.2304-2315.1995

Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.

R N Picken 1, Y Cheng 1, D Han 1, J A Nelson 1, A G Reddy 1, M K Hayden 1, M M Picken 1, F Strle 1, J K Bouseman 1, G M Trenholme 1
PMCID: PMC228401  PMID: 7494019

Abstract

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.

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Selected References

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  1. Anderson J. F., Duray P. H., Magnarelli L. A. Prevalence of Borrelia burgdorferi in white-footed mice and Ixodes dammini at Fort McCoy, Wis. J Clin Microbiol. 1987 Aug;25(8):1495–1497. doi: 10.1128/jcm.25.8.1495-1497.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson J. F., Magnarelli L. A., LeFebvre R. B., Andreadis T. G., McAninch J. B., Perng G. C., Johnson R. C. Antigenically variable Borrelia burgdorferi isolated from cottontail rabbits and Ixodes dentatus in rural and urban areas. J Clin Microbiol. 1989 Jan;27(1):13–20. doi: 10.1128/jcm.27.1.13-20.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson J. F., Magnarelli L. A., McAninch J. B. New Borrelia burgdorferi antigenic variant isolated from Ixodes dammini from upstate New York. J Clin Microbiol. 1988 Oct;26(10):2209–2212. doi: 10.1128/jcm.26.10.2209-2212.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Barbour A. G., Heiland R. A., Howe T. R. Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J Infect Dis. 1985 Sep;152(3):478–484. doi: 10.1093/infdis/152.3.478. [DOI] [PubMed] [Google Scholar]
  6. Barbour A. G. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984 Jul-Aug;57(4):521–525. [PMC free article] [PubMed] [Google Scholar]
  7. Barbour A. G., Schrumpf M. E. Polymorphisms of major surface proteins of Borrelia burgdorferi. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Dec;263(1-2):83–91. doi: 10.1016/s0176-6724(86)80107-9. [DOI] [PubMed] [Google Scholar]
  8. Barbour A. G., Tessier S. L., Hayes S. F. Variation in a major surface protein of Lyme disease spirochetes. Infect Immun. 1984 Jul;45(1):94–100. doi: 10.1128/iai.45.1.94-100.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Barbour A. G., Tessier S. L., Todd W. J. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect Immun. 1983 Aug;41(2):795–804. doi: 10.1128/iai.41.2.795-804.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Belfaiza J., Postic D., Bellenger E., Baranton G., Girons I. S. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis. J Clin Microbiol. 1993 Nov;31(11):2873–2877. doi: 10.1128/jcm.31.11.2873-2877.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Berger B. W., Johnson R. C., Kodner C., Coleman L. Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin. J Clin Microbiol. 1992 Feb;30(2):359–361. doi: 10.1128/jcm.30.2.359-361.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bissett M. L., Hill W. Characterization of Borrelia burgdorferi strains isolated from Ixodes pacificus ticks in California. J Clin Microbiol. 1987 Dec;25(12):2296–2301. doi: 10.1128/jcm.25.12.2296-2301.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bouseman J. K., Kitron U., Kirkpatrick C. E., Siegel J., Todd K. S., Jr Status of Ixodes dammini (Acari: Ixodidae) in Illinois. J Med Entomol. 1990 Jul;27(4):556–560. doi: 10.1093/jmedent/27.4.556. [DOI] [PubMed] [Google Scholar]
  14. Burgdorfer W., Barbour A. G., Hayes S. F., Benach J. L., Grunwaldt E., Davis J. P. Lyme disease-a tick-borne spirochetosis? Science. 1982 Jun 18;216(4552):1317–1319. doi: 10.1126/science.7043737. [DOI] [PubMed] [Google Scholar]
  15. Burgdorfer W., Lane R. S., Barbour A. G., Gresbrink R. A., Anderson J. R. The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am J Trop Med Hyg. 1985 Sep;34(5):925–930. doi: 10.4269/ajtmh.1985.34.925. [DOI] [PubMed] [Google Scholar]
  16. Callister S. M., Nelson J. A., Schell R. F., Jobe D. A., Bautz R., Agger W. A., Coggins J. Survey for Ixodes spp. and Borrelia burgdorferi in southeastern Wisconsin and northeastern Illinois. J Clin Microbiol. 1991 Feb;29(2):403–406. doi: 10.1128/jcm.29.2.403-406.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Canica M. M., Nato F., du Merle L., Mazie J. C., Baranton G., Postic D. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand J Infect Dis. 1993;25(4):441–448. doi: 10.3109/00365549309008525. [DOI] [PubMed] [Google Scholar]
  18. Davis J. P., Schell W. L., Amundson T. E., Godsey M. S., Jr, Spielman A., Burgdorfer W., Barbour A. G., LaVenture M., Kaslow R. A. Lyme disease in Wisconsin: epidemiologic, clinical, serologic, and entomologic findings. Yale J Biol Med. 1984 Jul-Aug;57(4):685–696. [PMC free article] [PubMed] [Google Scholar]
  19. Ferdows M. S., Barbour A. G. Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5969–5973. doi: 10.1073/pnas.86.15.5969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fuchs R., Jauris S., Lottspeich F., Preac-Mursic V., Wilske B., Soutschek E. Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa protein (pC) in Escherichia coli. Mol Microbiol. 1992 Feb;6(4):503–509. doi: 10.1111/j.1365-2958.1992.tb01495.x. [DOI] [PubMed] [Google Scholar]
  21. Hughes C. A., Kodner C. B., Johnson R. C. DNA analysis of Borrelia burgdorferi NCH-1, the first northcentral U.S. human Lyme disease isolate. J Clin Microbiol. 1992 Mar;30(3):698–703. doi: 10.1128/jcm.30.3.698-703.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hyde F. W., Johnson R. C. Genetic analysis of Borrelia. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Dec;263(1-2):119–122. doi: 10.1016/s0176-6724(86)80111-0. [DOI] [PubMed] [Google Scholar]
  23. Hyde F. W., Johnson R. C. Genetic relationship of lyme disease spirochetes to Borrelia, Treponema, and Leptospira spp. J Clin Microbiol. 1984 Aug;20(2):151–154. doi: 10.1128/jcm.20.2.151-154.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Kitron U., Jones C. J., Bouseman J. K. Spatial and temporal dispersion of immature Ixodes dammini on Peromyscus leucopus in northwestern Illinois. J Parasitol. 1991 Dec;77(6):945–949. [PubMed] [Google Scholar]
  26. Kuiper H., van Dam A. P., Spanjaard L., de Jongh B. M., Widjojokusumo A., Ramselaar T. C., Cairo I., Vos K., Dankert J. Isolation of Borrelia burgdorferi from biopsy specimens taken from healthy-looking skin of patients with Lyme borreliosis. J Clin Microbiol. 1994 Mar;32(3):715–720. doi: 10.1128/jcm.32.3.715-720.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kurashige S., Bissett M., Oshiro L. Characterization of a tick isolate of Borrelia burgdorferi that possesses a major low-molecular-weight surface protein. J Clin Microbiol. 1990 Jun;28(6):1362–1366. doi: 10.1128/jcm.28.6.1362-1366.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Lam T. T., Nguyen T. P., Fikrig E., Flavell R. A. A chromosomal Borrelia burgdorferi gene encodes a 22-kilodalton lipoprotein, P22, that is serologically recognized in Lyme disease. J Clin Microbiol. 1994 Apr;32(4):876–883. doi: 10.1128/jcm.32.4.876-883.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LeFebvre R. B., Probert W. S., Perng G. C. Characterization of a chromosomal gene and the antigen it expresses from the Lyme disease agent, Borrelia burgdorferi. J Clin Microbiol. 1993 Aug;31(8):2146–2151. doi: 10.1128/jcm.31.8.2146-2151.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liveris D., Gazumyan A., Schwartz I. Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis. J Clin Microbiol. 1995 Mar;33(3):589–595. doi: 10.1128/jcm.33.3.589-595.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Luft B. J., Jiang W., Munoz P., Dattwyler R. J., Gorevic P. D. Biochemical and immunological characterization of the surface proteins of Borrelia burgdorferi. Infect Immun. 1989 Nov;57(11):3637–3645. doi: 10.1128/iai.57.11.3637-3645.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marconi R. T., Garon C. F. Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol. 1992 Nov;30(11):2830–2834. doi: 10.1128/jcm.30.11.2830-2834.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marconi R. T., Konkel M. E., Garon C. F. Variability of osp genes and gene products among species of Lyme disease spirochetes. Infect Immun. 1993 Jun;61(6):2611–2617. doi: 10.1128/iai.61.6.2611-2617.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marconi R. T., Samuels D. S., Garon C. F. Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes. J Bacteriol. 1993 Feb;175(4):926–932. doi: 10.1128/jb.175.4.926-932.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nelson J. A., Bouseman J. K., Kitron U., Callister S. M., Harrison B., Bankowski M. J., Peeples M. E., Newton B. J., Anderson J. F. Isolation and characterization of Borrelia burgdorferi from Illinois Ixodes dammini. J Clin Microbiol. 1991 Aug;29(8):1732–1734. doi: 10.1128/jcm.29.8.1732-1734.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Padula S. J., Sampieri A., Dias F., Szczepanski A., Ryan R. W. Molecular characterization and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi. Infect Immun. 1993 Dec;61(12):5097–5105. doi: 10.1128/iai.61.12.5097-5105.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Picken R. N. Polymerase chain reaction primers and probes derived from flagellin gene sequences for specific detection of the agents of Lyme disease and North American relapsing fever. J Clin Microbiol. 1992 Jan;30(1):99–114. doi: 10.1128/jcm.30.1.99-114.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Postic D., Assous M. V., Grimont P. A., Baranton G. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol. 1994 Oct;44(4):743–752. doi: 10.1099/00207713-44-4-743. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Ralph D., Postic D., Baranton G., Pretzman C., McClelland M. Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes. FEMS Microbiol Lett. 1993 Aug 1;111(2-3):239–243. doi: 10.1111/j.1574-6968.1993.tb06392.x. [DOI] [PubMed] [Google Scholar]
  42. Samuels D. S., Marconi R. T., Garon C. F. Variation in the size of the ospA-containing linear plasmid, but not the linear chromosome, among the three Borrelia species associated with Lyme disease. J Gen Microbiol. 1993 Oct;139(10):2445–2449. doi: 10.1099/00221287-139-10-2445. [DOI] [PubMed] [Google Scholar]
  43. Schwan T. G., Schrumpf M. E., Karstens R. H., Clover J. R., Wong J., Daugherty M., Struthers M., Rosa P. A. Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California. J Clin Microbiol. 1993 Dec;31(12):3096–3108. doi: 10.1128/jcm.31.12.3096-3108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sigal L. H., Curran A. S. Lyme disease: a multifocal worldwide epidemic. Annu Rev Public Health. 1991;12:85–109. doi: 10.1146/annurev.pu.12.050191.000505. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Stanek G., Wewalka G., Groh V., Neumann R., Kristoferitsch W. Differences between Lyme disease and European arthropod-borne Borrelia infections. Lancet. 1985 Feb 16;1(8425):401–401. doi: 10.1016/s0140-6736(85)91424-2. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wallich R., Moter S. E., Simon M. M., Ebnet K., Heiberger A., Kramer M. D. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect Immun. 1990 Jun;58(6):1711–1719. doi: 10.1128/iai.58.6.1711-1719.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wallich R., Simon M. M., Hofmann H., Moter S. E., Schaible U. E., Kramer M. D. Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi. Infect Immun. 1993 Oct;61(10):4158–4166. doi: 10.1128/iai.61.10.4158-4166.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Welsh J., Pretzman C., Postic D., Saint Girons I., Baranton G., McClelland M. Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups. Int J Syst Bacteriol. 1992 Jul;42(3):370–377. doi: 10.1099/00207713-42-3-370. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Wilske B., Preac-Mursic V., Schierz G. Antigenic heterogeneity of European Borrelia burgdorferi strains isolated from patients and ticks. Lancet. 1985 May 11;1(8437):1099–1099. doi: 10.1016/s0140-6736(85)92396-7. [DOI] [PubMed] [Google Scholar]
  55. Wilske B., Preac-Mursic V., Schierz G., Busch K. V. Immunochemical and immunological analysis of European Borrelia burgdorferi strains. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Dec;263(1-2):92–102. doi: 10.1016/s0176-6724(86)80108-0. [DOI] [PubMed] [Google Scholar]
  56. Wilske B., Preac-Mursic V., Schierz G., Kühbeck R., Barbour A. G., Kramer M. Antigenic variability of Borrelia burgdorferi. Ann N Y Acad Sci. 1988;539:126–143. doi: 10.1111/j.1749-6632.1988.tb31846.x. [DOI] [PubMed] [Google Scholar]

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