Journal of fish pathology (한국어병학회지)

The Korean Society of Fish Pathology (한국어병학회)
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Differentiations between the viable but nonculturable (VBNC) or dead state of Edwardsiella tarda by ethidium monoazide (EMA) treatment-PCR

Ethidium monoazide (EMA) - PCR 법을 이용한 비배양성 생존 상태(VBNC)의 Edwardsiella tarda 검출

  • Kang, Nam I (Department of Aqualife Medicine, Chonnam National University) ;
  • Kim, Eunheui (Department of Aqualife Medicine, Chonnam National University)
  • 강남이 (전남대학교 수산생명의학과) ;
  • 김은희 (전남대학교 수산생명의학과)
  • Received : 2018.10.20
  • Accepted : 2018.10.30
  • Published : 2018.12.31
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Abstract

Edwardsiella tarda predominantly causes edwardsiellosis in fish at high temperature, but is rarely isolated from water when water temperature is low. However, E. tarda is viable but nonculturable (VBNC) in low water temperature, but it can be revived when water temperature rises and cause disease to fish. Therefore, in order to prevent disease, it is very important to identify pathogens that are in the VBNC state in environmental water. In this study, E. tarda cells in the VBNC state were detected by the ethidium monoazide (EMA)-PCR method using the low-temperature oligotrophic sea water microcosm obtained by inoculation of E. tarda at a concentration of $10^8CFU/ml$. In order to distinguish between live and dead bacteria in E. tarda, each sample was treated with EMA at different concentrations, photoactivated with a 500 W halogen lamp, and PCR was performed with E. tarda specific primer. At the concentration of $10^7CFU/ml$ bacterium, DNA amplification was observed only in the live cells when treated with $60{\mu}g/ml$ of EMA, and smaller amounts of live cells could be distinguished from dead cells by adjusting the EMA concentration. In addition, the VBNC cells of E. tarda in the oligotrophic low temperature seawater microcosm were estimated to be in the range of $10^4{\sim}10^5CFU/ml$ by EMA-PCR. Therefore, it is possible to detect VBNC cells that will act as potential pathogens in environmental water using EMA-PCR method, and quantitative confirmation using concentration change is also possible.

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References

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