Identification of porcine intestinal spirochetes isolated from Korea by NADH oxidase gene(nox) PCR-RFLP

NADH oxidase(nox) 유선자의 PCR- RFLP를 이용한 돼지 intestinal spirochetes 국내분리주의 동정

  • Kim, Tae-Jung (College of Veterinary Medicine, Chonnam National University) ;
  • Kim, Myoung-Hee (College of Veterinary Medicine, Chonnam National University) ;
  • Na, Young-Ran (College of Veterinary Medicine, Chonnam National University) ;
  • Jung, Suk-Chan (National Veterinary Research and Quarantine Service) ;
  • Lee, Jae-Il (College of Veterinary Medicine, Chonnam National University)
  • 김태중 (전남대학교 수의과대학) ;
  • 김명희 (전남대학교 수의과대학) ;
  • 나영란 (전남대학교 수의과대학) ;
  • 정석찬 (국립수의과학검역원) ;
  • 이재일 (전남대학교 수의과대학)
  • Accepted : 2004.10.22
  • Published : 2004.12.30

Abstract

In this study, we performed a PCR-RFLP analysis of NADH oxidase gene(nox) for the characterization of porcine intestinal spirochetes isolated from Korea by the comparison with Brachyspira hyodysenteriae and B. pilosicoli reference strains. Eleven strains including four reference strains, B. hyodysenteriae B204, B234, B169, B. pilosicoli P43/6/78 and seven Korean isolates were used. PCR products of 939 bp were amplified using nox-specific primers and digested with two restriction enzymes, Bfm I and Dpn II. In study using Bfm I, both strains showed no difference in fragmented size(197 and 741 bp). When use Dpn II, B. hyodysenteriae showed two bands(209 and 684 bp), however B. pilosicoli showed a single band of 896 bp. Our results indicate that nox-specific PCR-RFLP could be used as a typing method of Brachyspira species and as an epidemiological method for identifying spirochetes isolated from swine.

References

  1. Atyeo, R. F., Stanton, T. B., Jensen, N. S., Suriyaarachichi, D. S. and Hampson, D. J. Differentiation of Serpulina species by NADH oxidase gene (nox) sequence comparisons and nox-based polymerase chain reaction tests. Vet. Microbiol. 1999, 67, 47-60
  2. Barcellos, D. E. S. N., de Uzeda, M., Ikuta, N., Lunge, V. R., Fonseca, A. S., Kader, I. I. T. A. and Duhamel, G. E. Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA. Vet. Microbiol. 2000, 75, 189-198
  3. de Smet, K. A., Worth, D. E. and Barrett, S. P. Variation amongst human isolates of Brachyspira (Serpulina) pilosicoli based on biochemial characterization and 16S rRNA gene sequencing. Int. J. Syst. Bacteriol. 1998, 48, 1275-1263
  4. Dugourd, D., Jacques, M., Bigras-Poulin, M. and Harel, J. Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 by random amplification of polymorphic DNA analysis. Vet. Microbiol. 1996, 48, 305-314
  5. Duhamel, G. E. Colonic spirochetosis caused by Serpulina pilosicoli. Large Anim. Pract. 1998, 19, 14-2
  6. Elder, R. O., Duhamel, G. E., Schafer, R. W., Mathiesen, M. R. and Ramanathan, M. Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR. J. Clin. Microbiol. 1994, 32, 1497-1502
  7. Fellstrom, C., Pettersson, B., Thomson, J., Gunnarsson, A., Persson, M. and Johansson, K. E. Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR. J. Clin. Microbiol. 1997, 35, 462-467
  8. Fisher, L. N., Mathiesen, M. R. and Duhamel, G. E. Restriction fragment length polymorphism of the periplasmic flagellar flaA1 gene of Serpulina species. Clin. Diagn. Lab. Immunol. 1997, 4, 681-686
  9. Hampson, D. J. The Serpulina story, pp. 1-5. In C. Cargill and S. McOrist(ed.). Proceedings of the 16th IPVS Congress. Causal Productions Pty Ltd. Rundle Mall, 2000
  10. Kennedy, M. J., Rosnick, D. K., Ulrich, R. G. and Yancey, R. J. Association of Treponema hyodysenteriae with porcine intestinal mucosa. J. Gen. Microbiol. 1988, 134, 1565-1576
  11. La, T., Nyree, D. P. and Hampson, D. J. Development of a duplex PCR assay for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig feces. J. Clin. Microbiol. 2003, 41, 3372-3375
  12. Leser, T. D., Moller, K., Jensen, T. K. and Jorsal, S. E. Specific detection of Serpulina hyodysenteriae and potentially pathogenic weakly beta-haemolytic porcine intestinal spirochetes by polymerase chain reaction targeting 23S rDNA. Mol. Cell. Probes. 1997, 11, 363-372
  13. Pettersson, B., Fellstrom, C., Andersson, A., Uhlen, M., Gunarsson, A. and Johansson, K. E. The phylogeny of intestinal porcine spirochetes (Serpulina species) based on sequence analysis of the 16S rRNA gene. J. Bacteriol. 1996, 178, 4189-4199
  14. Rohde, J., Rothkamp, A. and Gerlach, G. F. Differentiation of porcine Brachyspira species by a novel nox PCR-based restriction fragment length polymorphism analysis. J. Clin. Microbiol. 2002, 40, 2598-2600
  15. Stanton, T. B. and Jensen, N. S. Purification and characterization of NADH oxidase from Serpulina hyodysenteriae. J. Bacteriol. 1993, 175, 2980-2987
  16. Stanton, T. B., Trott, D. J., Lee, J. I., McLaren, A. J., Hampson, D. J., Paster, B. J. and Jensen, N. S. Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons. FEMS. Microbiol. Lett. 1996, 136, 181-186