Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Quantification of White Spot Syndrome Virus (WSSV) in Seawaters Using Real-Time PCR and Correlation Analyses between WSSV and Environmental Parameters

Real-Time PCR을 이용한 해수 존재 흰반점 바이러스의 정량 및 양식 환경인자와의 상관관계 분석

  • Song, Jae-Ho (Division of Biology and Ocean Sciences, Inha University) ;
  • Choo, Yoe-Jin (Division of Biology and Ocean Sciences, Inha University) ;
  • Cho, Jang-Cheon (Division of Biology and Ocean Sciences, Inha University)
  • 송재호 (인하대학교 자연과학대학 생명해양과학부) ;
  • 추여진 (인하대학교 자연과학대학 생명해양과학부) ;
  • 조장천 (인하대학교 자연과학대학 생명해양과학부)
  • Published : 2008.03.31
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Abstract

White Spot Syndrome Virus (WSSV) is one of the most virulent viral agents in the penaeid shrimp culture industry. In this study, WSSV in a Fenneropenaeus chinensis shrimp farm and an adjacent seawater were concentrated using a membrane filtration and quantified using the quantitative real-time PCR (QRT-PCR) method with newly designed primers and Taqman probe. Sensitivity of primers and probe was proven by WSSV standard curve assay in QRT-PCR. In order to demonstrate the relationship between WSSV and environmental parameters, physicochemical and biological parameters of the farm and influent seawaters were monitored from June to September, 2007. The abundance of WSSV ranged 3,814-121,546 copies per 1 liter of seawater, which was correlated with fecal enterococci ($r^2=0.9$, p=0.02), chlorophyll ${\alpha}$ ($r^2=0.8$, p=0.03) and $BOD_5$ ($r^2=0.8$, p=0.07). Subsequently, it is concluded that the QRT-PCR method using Taqman probe established in this study was efficient to clarify the quantification of WSSV in seawaters. Statistical analyses of environmental parameters obtained in this study also showed that the abundance of WSSV was correlated with several biological parameters rather than physicochemical parameters.

흰반점 바이러스(white spot syndrome virus, WSSV)는 양식산 새우에 감염하여 대량폐사를 일으키는 전염성이 매우 강한 병원성 바이러스이다. 본 연구에서는 강화도에 위치한 대하(Fenneropenaeus chinensis) 양식장의 양식수와 양식장으로 유입되는 해수에서 WSSV를 막여과법을 이용하여 농축하였으며, 새롭게 디자인한 primer와 Taqman probe를 사용하여 정량 실시간 PCR (quantitative real-time PCR, QRT-PCR)을 적용하여 WSSV를 정량하였다. 농도표준을 사용한 QRT-PCR 결과, 제작된 primer와 probe를 이용하여 WSSV가 정확하고 민감하게 검출됨을 확인하였다. 해수에 존재하는 WSSV와 물리화학적, 생물학적 환경요인간의 상관관계를 도출하기 위하여 양식수와 해수 유입수에서 대하 양식기간인 2007년 6월부터 9월까지 총 8회에 거쳐 다양한 환경요인을 분석하였다. 양식수 1L에 존재하는 WSSV의 양은 3,814-121,545 copy였으며, 이는 분원성 enterococci ($r^2=0.9$, p=0.02), 엽록소${\alpha}$ ($r^2=0.8$, p=0.03), 생화학적 산소요구량($r^2=0.8$, p=0.07)과 상관관계를 나타내었다. 결론적으로 본 연구에서 정립된 WSSV의 농축법 및 QRT-PCR 방법은 해수에 존재하는 WSSV를 정량하는데 효과적이었으며, 해수에 존재하는 WSSV의 양은 물리화학적 환경요인보다 생물학적 환경요인과 밀접한 관련을 보였다.

Keywords

References

  1. 해양수산부. 2005. 해양환경공정시험방법
  2. APHA. 1995. Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association, New York, N.Y., USA
  3. Azam, F. 1974. Silicic acid uptake in diatoms studied with $[^{68}Ge]$germanic acid as a tracer. Planta 121, 205-212 https://doi.org/10.1007/BF00389321
  4. Chang, C.F., M.S. Su, H.Y. Chen, and I.C. Liao. 2003. Dietary ${\beta}$-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish Shellfish Immunol. 15, 297-310 https://doi.org/10.1016/S1050-4648(02)00167-5
  5. Chang, P.S., H.C. Chen, and Y.C. Wang. 1998. Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization. Aquaculture 164, 233-242 https://doi.org/10.1016/S0044-8486(98)00189-6
  6. Choo, Y.-J. 2005. Ph. D. thesis. Prevalence of enteric viruses in oysters and oyster-harvesting environments. Seoul Nat'l University, Seoul
  7. Chou, H.Y., C.Y. Huang, C.H. Wang, H.C. Chiang, and C.F. Lo. 1995. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Org. 23, 165-173 https://doi.org/10.3354/dao023165
  8. Darley, W.M. and B.E. Volcani. 1969. Role of silicon in diatom metabolism, A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca fusiformis Reimann and Lewin. Exp. Cell Res. 58, 334-342 https://doi.org/10.1016/0014-4827(69)90514-X
  9. Dhar, A.K., M.M. Roux, and K.R. Klimpel. 2001. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry. J. Clin. Microbiol. 39, 2835-2845 https://doi.org/10.1128/JCM.39.8.2835-2845.2001
  10. Durand, S.V. and D.V. Lightner. 2002. Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis. 25, 381-389 https://doi.org/10.1046/j.1365-2761.2002.00367.x
  11. Flegel, T.W. 1997. Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13, 433-442 https://doi.org/10.1023/A:1018580301578
  12. Francki, R.I.B., C.M. Fauquet, D.L. Knudson, and F. Brown. 1991. Classifiation and nomenclature of viruses: Fifth report of the international committee on taxonomy of viruses, p. 1-450. Springer-Verlag, New York, N.Y., USA
  13. Hameed, A.S.S., V. Parameswaran, S.S. Musthaq, R. Sudhakaran, G. Balasubramanian, and K. Yoganandhan. 2005. A simple PCR procedure to detect white spot syndrome virus (WSSV) of shrimp, Penaeus monodon (Fabricious). Aquacult. Int. 13, 441-450 https://doi.org/10.1007/s10499-005-7898-4
  14. Hennig, O.L. and E.R. Andreatta. 1998. Effect of temperature in an intensive nursery system for Penaeus paulensis (Perez Farfante, 1967). Aquaculture 164, 167-172 https://doi.org/10.1016/S0044-8486(98)00184-7
  15. Kalff, J. and E. Bentzen, 1984. A method for the analysis of total nitrogen in natural waters. Can. J. Fish. Aquat. Sci. 41, 815-819 https://doi.org/10.1139/f84-096
  16. Katayama, H., A. Shimasaki, and S. Ohgaki. 2002. Development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater. Appl. Environ. Microbiol. 68, 1033-1039 https://doi.org/10.1128/AEM.68.3.1033-1039.2002
  17. Kim, Y.J. and M.J. OH. 2004. Characteristics of white spot syndrome virus (WSSV) infected shrimp (Penaeus chinensis). Bull. Fish. Sci. Inst., Yosu Nat'l Univ. 13, 16-22
  18. Le Moullac, G. and P. Haffner. 2000. Environmental factors affecting immune responses in Crustacea, Aquaculture 191, 121-131 https://doi.org/10.1016/S0044-8486(00)00422-1
  19. Liu, B., Z.M. Yu, X.X. Song, and Y.Q. Guan. 2007. Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae. J. Invertebr. Pathol. 95, 87-92 https://doi.org/10.1016/j.jip.2007.01.007
  20. Liu, B., Z.M. Yu, X.X. Song, Y.Q. Guan, X.F. Jian, and J.F. He. 2006. The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis. Aquaculture 253, 163-170 https://doi.org/10.1016/j.aquaculture.2005.08.022
  21. Lo, C.F., C.H. Ho, S.E. Peng, C.H. Chen, H.C. Hsu, Y.L. Chiu, C.F. Chang, K.F. Liu, M.S. Su, C.H. Wang, and G.H. Kou. 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Org. 27, 215-225 https://doi.org/10.3354/dao027215
  22. Lorenzen, K., J. Struve, and V.J. Cowan. 1997. Impact of farming intensity and water management on nitrogen dynamics in intensive pond culture: A mathematical model applied to Thai commercial shrimp farms. Aquaculture Res. 28, 493-507 https://doi.org/10.1111/j.1365-2109.1997.tb01068.x
  23. Lu, Y., L.M. Tapay,and P.C. Loh. 1996. Development of a nitrocellulose-enzyme immunoassay for the detection of yellow-head virus from penaeid shrimp. J. Fish Dis. 19, 9-13 https://doi.org/10.1111/j.1365-2761.1996.tb00114.x
  24. Rosenberg, R. 2000 World shrimp farming 2000. Shrimp News International 13, 1-324
  25. Tapay, L.M., E.C.B. Nadala, and P.C. Loh. 1999. A polymerase chain reaction protocol for the detection of various geographical isolates of White Spot Virus. J. Virol. Methods 82, 39-43 https://doi.org/10.1016/S0166-0934(99)00081-6
  26. Van Hulten, M.C.W., M.F. Tsai, C.A. Schipper, C.F. Lo, G.H. Kou, and J.M. Vlak. 2000. Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J. Gen. Virol. 81, 307-316 https://doi.org/10.1099/0022-1317-81-2-307
  27. Van Hulten, M.C.W., J. Witteveldt, S. Peters, N. Kloosterboer, R. Tarchini, M. Fiers, H. Sandbrink, R.K. Lankhorst, and J.M. Vlak. 2001. The white spot syndrome virus DNA genome sequence. Virology 286, 7-22 https://doi.org/10.1006/viro.2001.1002
  28. Verghese, B., E.V. Radhakrishnan, and A. Padhi. 2007. Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758). Fish Shellfish Immunol. 23, 928-936 https://doi.org/10.1016/j.fsi.2007.01.021
  29. Wang, Y.C., C.F. Lo, P.S. Chang, and G.H. Kou. 1998. Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture 164, 221-231 https://doi.org/10.1016/S0044-8486(98)00188-4
  30. Wongteerasupaya, C., J.E. Vickers, S. Sriurairatana, G.L. Nash, A. Akarajamorn, V. Boonsaeng, S. Panyim, A. Tassanakajon, B. Withyachumnarnkul, and T.W Flegel. 1995. A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis. Aquat. Org. 21, 69-77 https://doi.org/10.3354/dao021069
  31. Yang, B., X.L. Song, J. Huang, C.Y. Shi, Q.H. Liu, and L. Liu. 2006. A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic mecrosis virus in penaeid shrimp. J. Fish Dis. 29, 301-305 https://doi.org/10.1111/j.1365-2761.2006.00713.x
  32. Yang, F., J. He, X.H. Lin, Q. Li, D. Pan, X.B. Zhang, and X. Xu. 2001. Complete genome sequence of the shrimp white spot bacilliform virus. J. Virol. 75, 11811-11820 https://doi.org/10.1128/JVI.75.23.11811-11820.2001