Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jul;178(14):4189–4199. doi: 10.1128/jb.178.14.4189-4199.1996

The phylogeny of intestinal porcine spirochetes (Serpulina species) based on sequence analysis of the 16S rRNA gene.

B Pettersson 1, C Fellström 1, A Andersson 1, M Uhlén 1, A Gunnarsson 1, K E Johansson 1
PMCID: PMC178177  PMID: 8763948

Abstract

Four type or reference strains and twenty-two field strains of intestinal spirochetes isolated from Swedish pig herds were subjected to phylogenetic analysis based on 16S rRNA sequences. Almost complete (>95%) 16S rRNA sequences were obtained by solid-phase DNA sequencing of in vitro-amplified rRNA genes. The genotypic patterns were compared with a previously proposed biochemical classification scheme, comprising beta-hemolysis, indole production, hippurate hydrolysis, and alpha-galactosidase, alpha-glucosidase, and beta-glucosidase activities. Comparison of the small-subunit rRNA sequences showed that the strains of the genus Serpulina were closely related. Phylogenetic trees were constructed, and three clusters were observed. This was also confirmed by signature nucleotide analysis of the serpulinas. The indole-producing strains, including the strains of S. hyodysenteriae and some weakly beta-hemolytic Serpulina strains, formed one cluster. A second cluster comprised weakly beta-hemolytic strains that showed beta-galactosidase activity but lacked indole production and hippurate-hydrolyzing capacity. The second cluster contained two subclusters with similar phenotypic profiles. A third cluster involved strains that possessed a hippurate-hydrolyzing capacity which was distinct from that of the former two clusters, because of 17 unique nucleotide positions of the 16S rRNA gene. Interestingly, the strains of this third cluster were found likely to have a 16S rRNA structure in the V2 region of the molecule different from that of the serpulinas belonging to the other clusters. As a consequence of these findings, we propose that the intestinal spirochetes of this phenotype (i.e., P43/6/78-like strains) should be regarded as a separate Serpulina species. Furthermore, this cluster was found to be by far the most homogeneous one. In conclusion, the biochemical classification of porcine intestinal spirochetes was comparable to that by phylogenetic analysis based on 16S rRNA sequences..

Full Text

The Full Text of this article is available as a PDF (366.7 KB).

Selected References

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

  1. Baum D. H., Joens L. A. Serotypes of beta-hemolytic Treponema hyodysenteriae. Infect Immun. 1979 Sep;25(3):792–796. doi: 10.1128/iai.25.3.792-796.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binek M., Szynkiewicz Z. M. Physiological properties and classification of strains of Treponema sp. isolated from pigs in Poland. Comp Immunol Microbiol Infect Dis. 1984;7(3-4):141–148. doi: 10.1016/0147-9571(84)90019-5. [DOI] [PubMed] [Google Scholar]
  3. Burrows M. R., Lemcke R. M. Identification of Treponema hyodysenteriae by a rapid slide agglutination test. Vet Rec. 1981 Feb 28;108(9):187–189. doi: 10.1136/vr.108.9.187. [DOI] [PubMed] [Google Scholar]
  4. Combs B., Hampson D. J., Mhoma J. R., Buddle J. R. Typing of Treponema hyodysenteriae by restriction endonuclease analysis. Vet Microbiol. 1989 Apr;19(4):351–359. doi: 10.1016/0378-1135(89)90100-4. [DOI] [PubMed] [Google Scholar]
  5. Dettori G., Amalfitano G., Polonelli L., Rossi A., Grillo R., Plaisant P. Electron microscopy studies of human intestinal spirochetes. Eur J Epidemiol. 1987 Jun;3(2):187–195. doi: 10.1007/BF00239758. [DOI] [PubMed] [Google Scholar]
  6. Duhamel G. E., Muniappa N., Mathiesen M. R., Johnson J. L., Toth J., Elder R. O., Doster A. R. Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis. J Clin Microbiol. 1995 Aug;33(8):2212–2215. doi: 10.1128/jcm.33.8.2212-2215.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dupont D. P., Duhamel G. E., Carlson M. P., Mathiesen M. R. Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper. Vet Microbiol. 1994 Jul;41(1-2):63–73. doi: 10.1016/0378-1135(94)90136-8. [DOI] [PubMed] [Google Scholar]
  8. Fellström C., Gunnarsson A. Phenotypical characterisation of intestinal spirochaetes isolated from pigs. Res Vet Sci. 1995 Jul;59(1):1–4. doi: 10.1016/0034-5288(95)90021-7. [DOI] [PubMed] [Google Scholar]
  9. Fellström C., Pettersson B., Uhlén M., Gunnarsson A., Johansson K. E. Phylogeny of Serpulina based on sequence analyses of the 16S rRNA gene and comparison with a scheme involving biochemical classification. Res Vet Sci. 1995 Jul;59(1):5–9. doi: 10.1016/0034-5288(95)90022-5. [DOI] [PubMed] [Google Scholar]
  10. Fitch W. M., Margoliash E. Construction of phylogenetic trees. Science. 1967 Jan 20;155(3760):279–284. doi: 10.1126/science.155.3760.279. [DOI] [PubMed] [Google Scholar]
  11. Fox G. E., Wisotzkey J. D., Jurtshuk P., Jr How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol. 1992 Jan;42(1):166–170. doi: 10.1099/00207713-42-1-166. [DOI] [PubMed] [Google Scholar]
  12. Gutell R. R. Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994. Nucleic Acids Res. 1994 Sep;22(17):3502–3507. doi: 10.1093/nar/22.17.3502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hampson D. J., Mhoma J. R., Combs B., Buddle J. R. Proposed revisions to the serological typing system for Treponema hyodysenteriae. Epidemiol Infect. 1989 Feb;102(1):75–84. doi: 10.1017/s0950268800029708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harris D. L., Glock R. D., Christensen C. R., Kinyon J. M. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction f the disease. Vet Med Small Anim Clin. 1972 Jan;67(1):61–64. [PubMed] [Google Scholar]
  15. Hookey J. V., Barrett S. P., Reed C. S., Barber P. Phylogeny of human intestinal spirochaetes inferred from 16S rDNA sequence comparisons. FEMS Microbiol Lett. 1994 Apr 15;117(3):345–349. doi: 10.1111/j.1574-6968.1994.tb06790.x. [DOI] [PubMed] [Google Scholar]
  16. Hovind-Hougen K., Birch-Andersen A., Henrik-Nielsen R., Orholm M., Pedersen J. O., Teglbjaerg P. S., Thaysen E. H. Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov. J Clin Microbiol. 1982 Dec;16(6):1127–1136. doi: 10.1128/jcm.16.6.1127-1136.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hovind-Hougen K., Høgh P., Birch-Andersen A. Morphological heterogeneity among spirochetes isolated from cases of swine dysentery. Zentralbl Bakteriol. 1990 Oct;274(1):1–15. doi: 10.1016/s0934-8840(11)80970-9. [DOI] [PubMed] [Google Scholar]
  18. Hultman T., Bergh S., Moks T., Uhlén M. Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. Biotechniques. 1991 Jan;10(1):84–93. [PubMed] [Google Scholar]
  19. Hultman T., Ståhl S., Hornes E., Uhlén M. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 1989 Jul 11;17(13):4937–4946. doi: 10.1093/nar/17.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jensen N. S., Casey T. A., Stanton T. B. Detection and identification of Treponema hyodysenteriae by using oligodeoxynucleotide probes complementary to 16S rRNA. J Clin Microbiol. 1990 Dec;28(12):2717–2721. doi: 10.1128/jcm.28.12.2717-2721.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jensen N. S., Stanton T. B. Comparison of Serpulina hyodysenteriae B78, the type strain of the species, with other S. hyodysenteriae strains using enteropathogenicity studies and restriction fragment length polymorphism analysis. Vet Microbiol. 1993 Sep;36(3-4):221–231. doi: 10.1016/0378-1135(93)90090-t. [DOI] [PubMed] [Google Scholar]
  22. Kinyon J. M., Harris D. L., Glock R. D. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect Immun. 1977 Feb;15(2):638–646. doi: 10.1128/iai.15.2.638-646.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koopman M. B., Käsbohrer A., Beckmann G., van der Zeijst B. A., Kusters J. G. Genetic similarity of intestinal spirochetes from humans and various animal species. J Clin Microbiol. 1993 Mar;31(3):711–716. doi: 10.1128/jcm.31.3.711-716.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee J. I., Hampson D. J., Combs B. G., Lymbery A. J. Genetic relationships between isolates of Serpulina (Treponema) hyodysenteriae, and comparison of methods for their subspecific differentiation. Vet Microbiol. 1993 Jan;34(1):35–46. doi: 10.1016/0378-1135(93)90005-r. [DOI] [PubMed] [Google Scholar]
  25. Lee J. I., Hampson D. J. Genetic characterisation of intestinal spirochaetes and their association with disease. J Med Microbiol. 1994 May;40(5):365–371. doi: 10.1099/00222615-40-5-365. [DOI] [PubMed] [Google Scholar]
  26. Lee J. I., Hampson D. J., Lymbery A. J., Harders S. J. The porcine intestinal spirochaetes: identification of new genetic groups. Vet Microbiol. 1993 Mar;34(3):273–285. doi: 10.1016/0378-1135(93)90017-2. [DOI] [PubMed] [Google Scholar]
  27. Lemcke R. M., Burrows M. R. A comparative study of spirochaetes from the porcine alimentary tract. J Hyg (Lond) 1981 Apr;86(2):173–182. doi: 10.1017/s0022172400068881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lussier G. Vibrionic Dysentery of Swine in Ontario-Part I : 1. Clinical Aspects and Pathology. Can Vet J. 1962 Aug;3(8):228–237. [PMC free article] [PubMed] [Google Scholar]
  29. Lymbery A. J., Hampson D. J., Hopkins R. M., Combs B., Mhoma J. R. Multilocus enzyme electrophoresis for identification and typing of Treponema hyodysenteriae and related spirochaetes. Vet Microbiol. 1990 Mar;22(1):89–99. doi: 10.1016/0378-1135(90)90127-h. [DOI] [PubMed] [Google Scholar]
  30. Maidak B. L., Larsen N., McCaughey M. J., Overbeek R., Olsen G. J., Fogel K., Blandy J., Woese C. R. The Ribosomal Database Project. Nucleic Acids Res. 1994 Sep;22(17):3485–3487. doi: 10.1093/nar/22.17.3485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mapother M. E., Joens L. A. New serotypes of Treponema hyodysenteriae. J Clin Microbiol. 1985 Aug;22(2):161–164. doi: 10.1128/jcm.22.2.161-164.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mhoma J. R., Hampson D. J., Robertson I. D. A serological survey to determine the prevalence of infection with Treponema hyodysenteriae in Western Australia. Aust Vet J. 1992 Apr;69(4):81–84. doi: 10.1111/j.1751-0813.1992.tb15555.x. [DOI] [PubMed] [Google Scholar]
  33. Miao R. M., Fieldsteel A. H., Harris D. L. Genetics of Treponema: characterization of Treponema hyodysenteriae and its relationship to Treponema pallidum. Infect Immun. 1978 Dec;22(3):736–739. doi: 10.1128/iai.22.3.736-739.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miao R., Fieldsteel A. H. Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes. J Bacteriol. 1978 Jan;133(1):101–107. doi: 10.1128/jb.133.1.101-107.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Muir S., Koopman M. B., Libby S. J., Joens L. A., Heffron F., Kusters J. G. Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene. Infect Immun. 1992 Feb;60(2):529–535. doi: 10.1128/iai.60.2.529-535.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Olsen G. J., Woese C. R. Ribosomal RNA: a key to phylogeny. FASEB J. 1993 Jan;7(1):113–123. doi: 10.1096/fasebj.7.1.8422957. [DOI] [PubMed] [Google Scholar]
  37. Park N. Y., Chung C. Y., McLaren A. J., Atyeo R. F., Hampson D. J. Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):225–229. doi: 10.1111/j.1574-6968.1995.tb07362.x. [DOI] [PubMed] [Google Scholar]
  38. Paster B. J., Dewhirst F. E., Weisburg W. G., Tordoff L. A., Fraser G. J., Hespell R. B., Stanton T. B., Zablen L., Mandelco L., Woese C. R. Phylogenetic analysis of the spirochetes. J Bacteriol. 1991 Oct;173(19):6101–6109. doi: 10.1128/jb.173.19.6101-6109.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pettersson B., Johansson K. E., Uhlén M. Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing. Appl Environ Microbiol. 1994 Jul;60(7):2456–2461. doi: 10.1128/aem.60.7.2456-2461.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pettersson B., Leitner T., Ronaghi M., Bölske G., Uhlen M., Johansson K. E. Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. J Bacteriol. 1996 Jul;178(14):4131–4142. doi: 10.1128/jb.178.14.4131-4142.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ramanathan M., Duhamel G. E., Mathiesen M. R., Messier S. Identification and partial characterization of a group of weakly beta-hemolytic intestinal spirochetes of swine distinct from Serpulina innocens isolate B256. Vet Microbiol. 1993 Oct;37(1-2):53–64. doi: 10.1016/0378-1135(93)90182-7. [DOI] [PubMed] [Google Scholar]
  42. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  43. Selander R. K., Caugant D. A., Ochman H., Musser J. M., Gilmour M. N., Whittam T. S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol. 1986 May;51(5):873–884. doi: 10.1128/aem.51.5.873-884.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spearman J. G., Nayar G., Sheridan M., Sheridan, Health H. S. Manitoba. Colitis associated with Treponema innocens in pigs. Can Vet J. 1988 Sep;29(9):747–747. [PMC free article] [PubMed] [Google Scholar]
  45. Stanton T. B., Jensen N. S., Casey T. A., Tordoff L. A., Dewhirst F. E., Paster B. J. Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov. Int J Syst Bacteriol. 1991 Jan;41(1):50–58. doi: 10.1099/00207713-41-1-50. [DOI] [PubMed] [Google Scholar]
  46. Stanton T. B. Proposal to change the genus designation Serpula to Serpulina gen. nov. containing the species Serpulina hyodysenteriae comb. nov. and Serpulina innocens comb. nov. Int J Syst Bacteriol. 1992 Jan;42(1):189–190. doi: 10.1099/00207713-42-1-189. [DOI] [PubMed] [Google Scholar]
  47. Taylor D. J., Alexander T. J. The production of dysentery in swine by feeding cultures containing a spirochaete. Br Vet J. 1971 Nov;127(11):58–61. doi: 10.1016/s0007-1935(17)37282-2. [DOI] [PubMed] [Google Scholar]
  48. Taylor D. J., Simmons J. R., Laird H. M. Production of diarrhoea and dysentery in pigs by feeding pure cultures of a spirochaete differing from Treponema hyodysenteriae. Vet Rec. 1980 Apr 12;106(15):326–332. doi: 10.1136/vr.106.15.326. [DOI] [PubMed] [Google Scholar]
  49. Wahlberg J., Holmberg A., Bergh S., Hultman T., Uhlén M. Automated magnetic preparation of DNA templates for solid phase sequencing. Electrophoresis. 1992 Aug;13(8):547–551. doi: 10.1002/elps.11501301112. [DOI] [PubMed] [Google Scholar]
  50. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Woese C. R., Magrum L. J., Gupta R., Siegel R. B., Stahl D. A., Kop J., Crawford N., Brosius J., Gutell R., Hogan J. J. Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence. Nucleic Acids Res. 1980 May 24;8(10):2275–2293. doi: 10.1093/nar/8.10.2275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Woese C. R., Winker S., Gutell R. R. Architecture of ribosomal RNA: constraints on the sequence of "tetra-loops". Proc Natl Acad Sci U S A. 1990 Nov;87(21):8467–8471. doi: 10.1073/pnas.87.21.8467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. ter Huurne A. A., van Houten M., Koopman M. B., van der Zeijst B. A., Gaastra W. Characterization of Dutch porcine Serpulina (Treponema) isolates by restriction endonuclease analysis and DNA hybridization. J Gen Microbiol. 1992 Sep;138(9):1929–1934. doi: 10.1099/00221287-138-9-1929. [DOI] [PubMed] [Google Scholar]

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

RESOURCES