Journal of fish pathology (한국어병학회지)

The Korean Society of Fish Pathology (한국어병학회)
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Use of 16S-23S rRNA Intergenic Spacer Region for identification in the fish pathogenic Streptococcus iniae

16S-23S rRNA Intergenic Spacer Region을 이용한 어류 병원성Streptococcus iniae의 분자생물학적 동정

  • Published : 2004.08.30
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Abstract

This study was performed for the identification of Streptococcus sp. from cultured flounders (Paralichthys olivaceus) showing streptococcosis in the Jeju island. We isolated 10 strains of Streptococcus iniae from the cultured olive flounders with streptococcosis. Isolated strains were identified in S. iniae since they have formed the expected band through performing PCR assay using specific primers, Sin-1 (5'-CTAGAGTACACATGTACT(AGCT)AAG-3') and Sin-2 (5'-GGATTTTCCACTCCCATTAC-3'). In addition to 16S-23S rRNA intergenic spacers (ISR), operon structure of isolated strains showed that all strains had three 16S-23S rRNA ISR band patterns. The 16S-23S rRNA ISR sequence of isolated strains showed 96% sequence identity with S. iniae (GenBank accession number AF 048773). This paper is the first report that S. iniae is associated with streptococcosis of Olive flounder in Korea.

본 연구의 목적은 국내 어류 연쇄구균증 병원체 동정에 있어서 Streptococcus iniae에 대한 구체적인 국내 연구 보고가 없어, 기존에 수행되고 있는 동정방법을 근간으로 16S-23S intergenic spacer region (ISR)의 염기서열을 분석하여 보다 명확한 동정을 실시하고자 하였다. API 20 strep system에 의한 생화학적 성상을 분석해본 결과 정확한 종 동정은 이루어지지 않았지만 기존의 연구에서 보고된 API 20 strep system에 의한 S. iniae의 생화학적 성상과 높은 유사성을 보이는 10균주를 분리할 수 있었다. S. iniae 16S rRNA gene 서열 유래 종 특이적 primer Sin-1 5'-CTAGAGTACACATGTACT(AGCT)AAG-3'와 Sin-2 5'-GGATTTTC CACTCCCATTAC-3'에 의한 PCR-assay 결과 시험균주 10 균주 모두 동일하게 약 300bp의 증폭산물이 관찰되었고, 비 특이적인 반응은 관찰되지 않았다. 시험균주 모두 3개의 16S-23S ISR operon 구조가 관찰되었으나 S. iniae 표준균주 (KCTC 3657)의 경우 단일 구조가 관찰되었다. 16S-23S intergenic spacer region (ISR)의 염기서열을 분석해본 결과 기존에 보고된 S. iniae (Genbank accession number AF 048773)와 96%의 상동성을 보였으며 전체서열에서 상동성을 보이는 근연종이나 근연속은 검색되지 않아 최종적으로 S. iniae로 동정하였다.

Keywords

References

  1. Aoki, T., Park, C-I., Yamashita, H. and Hirono, I.: Species-specific polymerase chain reaction primers for Lactococcus garvieae. J. Fish Disease, 23:1-6, 2000 https://doi.org/10.1046/j.1365-2761.2000.00207.x
  2. Austin, A. and Austin, D. A. : Bacterial fish pathogens, 3rd ed., Springer. London, 1999
  3. Bercovier, H., Ghittino, C., Eldar, A.: Immunization with bacterial antigens: infections with streptococci and related organisms. Dev. Biol. Stand. 90:153-60, 1997
  4. Berridge, B., Fuller, J. D., Azavedo, J., Low, D. E., Bercovier, H. and Frelier, P. F.: Development of specific nested oligonucleotide PCR primers for the Streptococcus iniae 16S-23S ribosomal DNA intergenic spacer. J. Clin. Microbiol. 36:2778-2781, 1998
  5. Colorni, A., Diamant, A., Eldar, A., Kvitt, A., Zlotkin, A., : Streptococcus iniae infections in Red Sea Cage-cultured and wild fishes. Dis. Aquat. Org., 49:165-170, 2002 https://doi.org/10.3354/dao049165
  6. Jensen, M. A., Webster, J. A. and Straus. N., : Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol., 59:945-952, 1993
  7. Lee, D. C., Lee, J. I., Park, C. I. and Park, S. I. : The study on the causal agent of Streptococcosis(Lactococcus garvieae), isolated from cultured marine fishes. J. Fish Pathol., 14:71-80, 2001. (in korean)
  8. Nagpal, M. L., Fox, K. F., and Fox, A. : Utility of 16S-23S rRNA spacer region methodology: how similar are intergenic spacer regions within a genome and between strains for closely related organisms?. J. Microbiol. Methods, 33:211-219, 1998 https://doi.org/10.1016/S0167-7012(98)00054-2
  9. Nagatsugawa, T.: A streptococcal disease of cultured flounder. Fish Pathol., 17:281-285, 1983 https://doi.org/10.3147/jsfp.17.281
  10. Nguyen, H. T. and Kanai K.: Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment. J. Appl. Microbiol. 86:769-776, 1999 https://doi.org/10.1046/j.1365-2672.1999.00724.x
  11. Nguyen, H. T., Yoshikoshi, K., and Kanai K.: Ecological investigation of Streptococcus iniae in cultured Japanese flounder (Paralichthys olivaceus) using selective isolation procedures. Aquaculture 205:7-17, 2002 https://doi.org/10.1016/S0044-8486(01)00667-6
  12. Song, J. K., Kim, J. H., Kim, E. H., : Comparison of RAPD profiles and phenotypical characters of streptococcal strains. J. Fish Pathol., 16:51-59, 2003. (in korean)
  13. Zlotkin, A., Hershko H, Eldar A., : Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl. Environ. Microbiol., 64:4065-4067, 1998 b