Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Sep;34(9):2058–2065. doi: 10.1128/jcm.34.9.2058-2065.1996

Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA.

V Roux 1, P E Fournier 1, D Raoult 1
PMCID: PMC229190  PMID: 8862558

Abstract

Currently, the genotypic identification of the spotted fever group (SFG) rickettsiae is based on restriction fragment length polymorphism analysis of PCR-amplified genes coding for the enzyme citrate synthase and the surface proteins rOmpA and rOmpB. A set of useful restriction endonucleases was found following comparison of Rickettsia rickettsii and R. prowazekii sequences. However, by using three PCR amplifications and four enzyme digestions with this set, it was impossible to differentiate between all of the known serotypes of the SFG rickettsiae. We amplified by PCR and sequenced using an automated laser fluorescent DNA sequencer a fragment of the gene encoding the protein rOmpA from 21 serotypes of the SFG rickettsiae. A 632-bp amplification product was obtained for most of the strains, although no product could be obtained by using R. akari, R. australis, R. helvetica, and R. bellii DNAs. We found a characteristic sequence for all strains studied except the two isolates of R. massiliae, isolates GS and Mtul. Using the software package BISANCE, we determined the restriction map of this fragment and identified five potentially useful endonucleases, RsaI, AluI, PstI, XbaI, and AvaII. We confirmed the computer analysis-derived profiles by PCR-restriction fragment length polymorphism analysis. The combination of the profiles obtained after digestion of the PCR product by RsaI and PstI allowed for the differentiation of 16 strains. The use of AluI and XbaI allowed for the characterization of R. parkeri and strain HA-91, respectively. R. africae and strain S were differentiated by AvaII digestion. Thus, using a single PCR amplification, we were able to differentiate all of the SFG rickettsiae whose ompA gene was amplified by PCR.

Full Text

The Full Text of this article is available as a PDF (543.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anacker R. L., List R. H., Mann R. E., Hayes S. F., Thomas L. A. Characterization of monoclonal antibodies protecting mice against Rickettsia rickettsii. J Infect Dis. 1985 Jun;151(6):1052–1060. doi: 10.1093/infdis/151.6.1052. [DOI] [PubMed] [Google Scholar]
  2. Anacker R. L., Mann R. E., Gonzales C. Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae. J Clin Microbiol. 1987 Jan;25(1):167–171. doi: 10.1128/jcm.25.1.167-171.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson B. E., McDonald G. A., Jones D. C., Regnery R. L. A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences. Infect Immun. 1990 Sep;58(9):2760–2769. doi: 10.1128/iai.58.9.2760-2769.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson B. E., Tzianabos T. Comparative sequence analysis of a genus-common rickettsial antigen gene. J Bacteriol. 1989 Sep;171(9):5199–5201. doi: 10.1128/jb.171.9.5199-5201.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. BELL E. J., KOHLS G. M., STOENNER H. G., LACKMAN D. B. NONPATHOGENIC RICKETTSIAS RELATED TO THE SPOTTED FEVER GROUP ISOLATED FROM TICKS, DERMACENTOR VARIABILIS AND DERMACENTOR ANDERSONI FROM EASTERN MONTANA. J Immunol. 1963 May;90:770–781. [PubMed] [Google Scholar]
  6. BELL E. J., PICKENS E. G. A toxic substance associated with the rickettsias of the spotted fever group. J Immunol. 1953 May;70(5):461–472. [PubMed] [Google Scholar]
  7. BELL E. J., STOENNER H. G. Immunologic relationships among the spotted fever group of rickettsias determined by toxin neutralization tests in mice with convalescent animal serums. J Immunol. 1960 Feb;84:171–182. [PubMed] [Google Scholar]
  8. Babalis T., Tselentis Y., Roux V., Psaroulaki A., Raoult D. Isolation and identification of a rickettsial strain related to Rickettsia massiliae in Greek ticks. Am J Trop Med Hyg. 1994 Mar;50(3):365–372. doi: 10.4269/ajtmh.1994.50.365. [DOI] [PubMed] [Google Scholar]
  9. Baird R. W., Lloyd M., Stenos J., Ross B. C., Stewart R. S., Dwyer B. Characterization and comparison of Australian human spotted fever group rickettsiae. J Clin Microbiol. 1992 Nov;30(11):2896–2902. doi: 10.1128/jcm.30.11.2896-2902.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Beati L., Finidori J. P., Gilot B., Raoult D. Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains. J Clin Microbiol. 1992 Aug;30(8):1922–1930. doi: 10.1128/jcm.30.8.1922-1930.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burgdorfer W., Aeschlimann A., Peter O., Hayes S. F., Philip R. N. Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland. Acta Trop. 1979 Dec;36(4):357–367. [PubMed] [Google Scholar]
  12. Dessen P., Fondrat C., Valencien C., Mugnier C. BISANCE: a French service for access to biomolecular sequence databases. Comput Appl Biosci. 1990 Oct;6(4):355–356. doi: 10.1093/bioinformatics/6.4.355. [DOI] [PubMed] [Google Scholar]
  13. Eremeeva M. E., Beati L., Makarova V. A., Fetisova N. F., Tarasevich I. V., Balayeva N. M., Raoult D. Astrakhan fever rickettsiae: antigenic and genotypic analysis of isolates obtained from human and Rhipicephalus pumilio ticks. Am J Trop Med Hyg. 1994 Nov;51(5):697–706. doi: 10.4269/ajtmh.1994.51.697. [DOI] [PubMed] [Google Scholar]
  14. Eremeeva M., Balayeva N., Roux V., Ignatovich V., Kotsinjan M., Raoult D. Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia. J Clin Microbiol. 1995 Oct;33(10):2738–2744. doi: 10.1128/jcm.33.10.2738-2744.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eremeeva M., Yu X., Raoult D. Differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of PCR-amplified DNA. J Clin Microbiol. 1994 Mar;32(3):803–810. doi: 10.1128/jcm.32.3.803-810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. GIMENEZ D. F. STAINING RICKETTSIAE IN YOLK-SAC CULTURES. Stain Technol. 1964 May;39:135–140. doi: 10.3109/10520296409061219. [DOI] [PubMed] [Google Scholar]
  17. Gilmore R. D., Jr, Hackstadt T. DNA polymorphism in the conserved 190 kDa antigen gene repeat region among spotted fever group Rickettsiae. Biochim Biophys Acta. 1991 Jul 26;1097(1):77–80. doi: 10.1016/0925-4439(91)90027-7. [DOI] [PubMed] [Google Scholar]
  18. Goldwasser R. A., Steiman Y., Klingberg W., Swartz T. A., Klingberg M. A. The isolation of strains of rickettsiae of the spotted fever group in Israel and their differentiation from other members of the group by immunofluorescence methods. Scand J Infect Dis. 1974;6(1):53–62. doi: 10.3109/inf.1974.6.issue-1.10. [DOI] [PubMed] [Google Scholar]
  19. Higgins J. A., Radulovic S., Schriefer M. E., Azad A. F. Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas. J Clin Microbiol. 1996 Mar;34(3):671–674. doi: 10.1128/jcm.34.3.671-674.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kelly P. J., Beati L., Mason P. R., Matthewman L. A., Roux V., Raoult D. Rickettsia africae sp. nov., the etiological agent of African tick bite fever. Int J Syst Bacteriol. 1996 Apr;46(2):611–614. doi: 10.1099/00207713-46-2-611. [DOI] [PubMed] [Google Scholar]
  21. Marrero M., Raoult D. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture. Am J Trop Med Hyg. 1989 Feb;40(2):197–199. doi: 10.4269/ajtmh.1989.40.197. [DOI] [PubMed] [Google Scholar]
  22. McDonald G. A., Anacker R. L., Mann R. E., Milch L. J. Protection of guinea pigs from experimental Rocky Mountain spotted fever with a cloned antigen of Rickettsia rickettsii. J Infect Dis. 1988 Jul;158(1):228–231. doi: 10.1093/infdis/158.1.228. [DOI] [PubMed] [Google Scholar]
  23. Philip C. B., Hoogstraal H., Reiss-Gutfreund R., Clifford C. M. Evidence of rickettsial disease agents in ticks from Ethiopian cattle. Bull World Health Organ. 1966;35(2):127–131. [PMC free article] [PubMed] [Google Scholar]
  24. Philip R. N., Casper E. A., Burgdorfer W., Gerloff R. K., Hughes L. E., Bell E. J. Serologic typing of rickettsiae of the spotted fever group by microimmunofluorescence. J Immunol. 1978 Nov;121(5):1961–1968. [PubMed] [Google Scholar]
  25. Public Health Weekly Reports for NOVEMBER 8, 1946. Public Health Rep. 1946 Nov 8;61(45):1605–1640. [PMC free article] [PubMed] [Google Scholar]
  26. Regnery R. L., Spruill C. L., Plikaytis B. D. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol. 1991 Mar;173(5):1576–1589. doi: 10.1128/jb.173.5.1576-1589.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Robertson R. G., Wisseman C. L., Jr Tick-borne rickettsiae of the spotted fever group in West Pakistan. II. Serological classification of isolates from West Pakistan and Thailand: evidence for two new species. Am J Epidemiol. 1973 Jan;97(1):55–64. doi: 10.1093/oxfordjournals.aje.a121485. [DOI] [PubMed] [Google Scholar]
  28. Schriefer M. E., Sacci J. B., Jr, Dumler J. S., Bullen M. G., Azad A. F. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol. 1994 Apr;32(4):949–954. doi: 10.1128/jcm.32.4.949-954.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tamura A., Ohashi N., Urakami H., Miyamura S. Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Syst Bacteriol. 1995 Jul;45(3):589–591. doi: 10.1099/00207713-45-3-589. [DOI] [PubMed] [Google Scholar]
  30. Tarasevich I. V., Makarova V. A., Fetisova N. F., Stepanov A. V., Miskarova E. D., Balayeva N., Raoult D. Astrakhan fever, a spotted-fever rickettsiosis. Lancet. 1991 Jan 19;337(8734):172–173. doi: 10.1016/0140-6736(91)90833-b. [DOI] [PubMed] [Google Scholar]
  31. Uchida T., Uchiyama T., Kumano K., Walker D. H. Rickettsia japonica sp. nov., the etiological agent of spotted fever group rickettsiosis in Japan. Int J Syst Bacteriol. 1992 Apr;42(2):303–305. doi: 10.1099/00207713-42-2-303. [DOI] [PubMed] [Google Scholar]
  32. Werren J. H., Hurst G. D., Zhang W., Breeuwer J. A., Stouthamer R., Majerus M. E. Rickettsial relative associated with male killing in the ladybird beetle (Adalia bipunctata). J Bacteriol. 1994 Jan;176(2):388–394. doi: 10.1128/jb.176.2.388-394.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yan Y., Uchiyama T., Uchida T. Nucleotide sequence of polymerase chain reaction product amplified from Rickettsia japonica DNA using Rickettsia rickettsii 190-kilodalton surface antigen gene primers. Microbiol Immunol. 1994;38(11):865–869. doi: 10.1111/j.1348-0421.1994.tb02139.x. [DOI] [PubMed] [Google Scholar]
  34. Yu X., Jin Y., Fan M., Xu G., Liu Q., Raoult D. Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China. J Clin Microbiol. 1993 Jan;31(1):83–88. doi: 10.1128/jcm.31.1.83-88.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES