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. 1996 Nov;62(11):4060–4065. doi: 10.1128/aem.62.11.4060-4065.1996

A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus.

F Kirstein 1, J S Gray 1
PMCID: PMC168227  PMID: 8899988

Abstract

The efficacy of the mitochondrially encoded cytochrome b gene as a molecular marker for the discrimination of the reservoir host species of the Lyme borreliosis spirochete, Borrelia burgdorferi sensu lato (s.l.), in its European vector Ixodes ricinus (Acari: Ixodidae) was determined. Degenerate PCR primers were designed which amplified orthologous regions of the cytochrome b gene in several animal species which act as B. burgdorferi s.l. reservoirs and hosts for I. ricinus. PCR products were amplified and characterized by hybridization and restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis of a 638-bp PCR product with HaeIII and DdeI revealed unique restriction fragment profiles, which allowed the taxonomic identification of animals to the genus level. A system was devised for the detection of the larval host blood meal from the remnants in unfed nymphal I. ricinus ticks by nested PCR amplification. An inverse correlation was demonstrated between amplicon size and successful PCR amplification of host DNA from the nymphal stage of the tick. The stability of the cytochrome b product as a marker for the identification of the larval host species in the nymphal instar was demonstrated up to 200 days after larval ingestion (approximately 165 days after molting) by reverse line blotting with a host-specific probe. This assay has the potential for the determination of the reservoir hosts of B. burgdorferi s.l. by using extracts from the same individual ticks for both the identification of the host species and the detection of the Lyme borreliosis spirochete.

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Selected References

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  1. Anderson S., de Bruijn M. H., Coulson A. R., Eperon I. C., Sanger F., Young I. G. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J Mol Biol. 1982 Apr 25;156(4):683–717. doi: 10.1016/0022-2836(82)90137-1. [DOI] [PubMed] [Google Scholar]
  2. Bibb M. J., Van Etten R. A., Wright C. T., Walberg M. W., Clayton D. A. Sequence and gene organization of mouse mitochondrial DNA. Cell. 1981 Oct;26(2 Pt 2):167–180. doi: 10.1016/0092-8674(81)90300-7. [DOI] [PubMed] [Google Scholar]
  3. Guy E. C., Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J Clin Pathol. 1991 Jul;44(7):610–611. doi: 10.1136/jcp.44.7.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem. 1985;54:1015–1069. doi: 10.1146/annurev.bi.54.070185.005055. [DOI] [PubMed] [Google Scholar]
  5. Irwin D. M., Kocher T. D., Wilson A. C. Evolution of the cytochrome b gene of mammals. J Mol Evol. 1991 Feb;32(2):128–144. doi: 10.1007/BF02515385. [DOI] [PubMed] [Google Scholar]
  6. Jaenson T. G. The epidemiology of lyme borreliosis. Parasitol Today. 1991 Feb;7(2):39–45. doi: 10.1016/0169-4758(91)90187-s. [DOI] [PubMed] [Google Scholar]
  7. Kaufhold A., Podbielski A., Baumgarten G., Blokpoel M., Top J., Schouls L. Rapid typing of group A streptococci by the use of DNA amplification and non-radioactive allele-specific oligonucleotide probes. FEMS Microbiol Lett. 1994 Jun 1;119(1-2):19–25. doi: 10.1111/j.1574-6968.1994.tb06861.x. [DOI] [PubMed] [Google Scholar]
  8. Päbo S., Gifford J. A., Wilson A. C. Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res. 1988 Oct 25;16(20):9775–9787. doi: 10.1093/nar/16.20.9775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Päbo S., Higuchi R. G., Wilson A. C. Ancient DNA and the polymerase chain reaction. The emerging field of molecular archaeology. J Biol Chem. 1989 Jun 15;264(17):9709–9712. [PubMed] [Google Scholar]
  10. Ribeiro J. M. The midgut hemolysin of Ixodes dammini (Acari:Ixodidae). J Parasitol. 1988 Aug;74(4):532–537. [PubMed] [Google Scholar]
  11. Rijpkema S. G., Molkenboer M. J., Schouls L. M., Jongejan F., Schellekens J. F. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes. J Clin Microbiol. 1995 Dec;33(12):3091–3095. doi: 10.1128/jcm.33.12.3091-3095.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Saiki R. K., Bugawan T. L., Horn G. T., Mullis K. B., Erlich H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature. 1986 Nov 13;324(6093):163–166. doi: 10.1038/324163a0. [DOI] [PubMed] [Google Scholar]
  13. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  14. Tobolewski J., Kaliszewski M. J., Colwell R. K., Oliver J. H., Jr Detection and identification of mammalian DNA from the gut of museum specimens of ticks. J Med Entomol. 1992 Nov;29(6):1049–1051. doi: 10.1093/jmedent/29.6.1049. [DOI] [PubMed] [Google Scholar]

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