Journal of fish pathology (한국어병학회지)

The Korean Society of Fish Pathology (한국어병학회)
권호 표지

Pathogenicity and PCR detection of Vibrio tapetis in Manila clams, Ruditapes philippinarum

양식 바지락, Ruditapes philippinarum에 대한 Vibrio tapetis의 병원성과 PCR법에 의한 진단

  • Park, Sung-Woo (Dept. of Aquatic Life Medicine. Kunsan National University) ;
  • Lee, Kyung-Hee (Fisheries Management Division, Mokpo Regional Maritime Affairs and Fisheries Office)
  • Published : 20050400
Download PDF
⟨ Previous Next ⟩

Abstract

Pathogenicity of Vibrio tapetis, the causative bacterium of 'brown ring disease (BRD)' was evaluated in Manila clams (Ruditapes philippinarumi by artificially 0.1 $m\ell$ infection of $1.0\times10^5$cells and $1.0\times10^8$ cells at 20 $^{\circ}C$. A PCR assay based on 16S rRNA to detect the bacteria in clam tissues was established. Accumulative mortality of clams infected with $1.0\times10^7$cells and $1.0\times10^4$ cells per an individual of the bacteria was 67.5% and 7.5%, respectively. However, the deposit of brown pigment in the inner shells by accumulation of chonchiolin was not found. The bacteria were not be able to re-isolate from the infected clams by the conventional agar plate method but were easily detected by PCR assay established in this experiment. In clams artificially infected with 10 species of Vibrio, a 414bp for V. tapetis was detected in PCR assay. The specific band in the clams infected with $1.0\times10^4$cells per an individual of V. tapetis was detected only in gills one day after the infection but never be found in any tissues including gills three days after the infection. In the case of clams infected with $1.0\times10^8$cells per an individual of V. tapetis the specific band was detected in gills and intestine one day after the infection, in all tissues three days after the infection, and then in gills and adductor muscle nine days after the infection. The PCR assay was applied to detect V. tapetis in manila clam, surf clam (Mactra veneriformis), oyster (Crassostrea gigas) and Thomas' rapa whelk (Rapana venosa) taken from Taean and Gochang from April to July 2004. The infection rates were detected to 23.1% and 9.4% in the oyster and surf clam, while manila clam and Thomas' rapa whelk were not found.

'Brown ring disease (BRD)'의 원인균인 Vibrio tapetis (NCIMB 13622)의 바지락(Ruditapes philippinarum)에서의 병원성을 조사하고, 바지락 조직 내의 균의 존재를 PCR법으로 진단하였다. 실온에 12시간 노출시킨 바지락 개체당 1.0$\time$$10^7 cells과 1.0$\time$$10^4 cells의 V. tapetis를 멸균주사기를 사용하여 인위감염 시켰을 때의 누적폐사율은 각각 67.5%와 7.5%로 나타났다. 폐사개체에서 BRD의 특징적인 증상인 패각내면의 chonchiolin 침착에 의해 형성되는 갈색반점은 관찰되지 않았으나, PCR법에 의해 균의 존재를 확인할 수 있었다.10종의 Vibrio균을 대상으로 PCR법의 검출 특이성을 조사한 결과 V. tapetis에 대해서만 414 bp의 특이밴드가 검출되었다. 또 V. tapetis를 개체당 1.0$\time$$10^4 cells과 1.0$\time$$10^8 cells 접종시킨 다음 사육하면서 경시적으로 균의 동태를 조사한 결과 개체당 1.0$\time$$10^4 cells을 접종하였을 때는 1일 후에는 아가미에서만 특이밴드가 검출되었고, 3일 후에는 모든 조직에서 검출되지 않았다. 한편 개체당 1.0$\time$$10^8 cells을 접종하였을 때 1일 후에는 아가미와 소화관, 3일 후에는 모든 조직에서 검출되었으며, 5일과 7일 후에는 외투막과 발, 9일 후에는 아가미와 발에서 검출되어, 검출장기로는 아가미가 가장 효과적인 것으로 판명되었다. PCR법으로 태안과 고창 지역의 바지락, 동죽, 굴 및 피뿔고둥의 조직내 V. tapetis의 존재를 검사한 결과 바지락과 피뿔고둥에서는 전혀 검출되지 않은 반면 굴과 동죽에서 각각 23.1%, 와 9.4% 검출되어 바지락 이외의 다른 패류의 진단에도 유용한 방법으로 판단되었다.

Keywords

References

  1. Allam, B., Ashton-Alcox, K. A. and Ford, S. E.: Haemocyte parameters associated with resistance to brown ring disease in Rudi-tapes spp. clams. Dev. & Comp. Immunol., 25:365-375,2001 https://doi.org/10.1016/S0145-305X(00)00072-0
  2. Allam, B. Paillard, C. and Ford, S. E.: Pathogenicity of Vibrio tapetis, the etiologic agent brown ring disease in clams. Dis. Aquat. Org., 48: 221-231,2002 https://doi.org/10.3354/dao048221
  3. Arzul, I., Renault, T., Lipar, C. and Davison, A. J.: Evidence for interspecies transmission of oyster herpesvirus in marine bivalves. J. Gen. Virol., 82: 865-870, 2001 https://doi.org/10.1099/0022-1317-82-4-865
  4. Atmar, R. R., Neill, F H., Woodley, C. M., Nanger, R., Fout, G. S., Burkhardt, W., Leja, L. and McGovern, E. R.: Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR. Appl. Environ. Microbiol., 62: 254-258,1996
  5. Borrego, J. J., Castro, D., Luque, A., Paillare C, Maes, P., Garcia, M. T. and Ventosa, A.: Vibrio tapetis nov., the causative agent of the brown ring disease affecting cultured clams. Intl. J. Sys. Bacterid., 46:480-484,1996 https://doi.org/10.1099/00207713-46-2-480
  6. Castro, D., Martinez-Manzanares, E., Luque, A., Fouz, B., Morinigo, M, A., Borrego, J. J. and Toanzo, A. E.: Characterization of strains related to brown ring disease outbreaksin southwestern Spain. Dis. Aquat. Org., 14: 229-236,1992 https://doi.org/10.3354/dao014229
  7. Castro, D., Luque, A., Santamaria, J. A., Maes, P., Marinez-Manzanares, E. and Borrego J. J.: Development of immunological techniques for the detection of the potential causative agent of the brown ring disease. Aquaculture, 132:97-104, 1995 https://doi.org/10.1016/0044-8486(94)00377-Z
  8. Castro, D., Santamaria. J. A., Luque, A., Marinez-Manzanares, E. and Borrego, J. J.: Antigenic characterization of the etiological agent of the brown ring disease affecting manila clams. Syst. Appl. Microbiol., 19, 231-239, 1996
  9. Farley, C. A.: Michinia nesoni (Haplosporidia) disease syndrome in the American oyster Cras-sostrea virginica. J. Protozool., 15: 585-599, 1968 https://doi.org/10.1111/j.1550-7408.1968.tb02176.x
  10. Itoh, N., Tun, K. L., Komiyama, H., Ueki, N. and Ogawa, K.: An ovarian infection in the Iwagaki oyster, Crassostrea nippona, with the protozoan parasites Martelliodes chungmuensis. J. Fish Dis., 27: 311-314, 2004 https://doi.org/10.1111/j.1365-2761.2004.00540.x
  11. Kawamato, K.: Gonadal development of cultured scallops, Patinopecten yessoensis (Jay) off Rumoi (Hokkaido). Scient. Rep. Hokkaido Fish. Expl.Stn., 31: 9-13,1985
  12. Kellner-Cousin, K., Gall, Gl L., Despres, B., Kaghad, M., Legoux, P. and Shire, D.: Genomic DNA cloning of rickettsia-like organisms(RLO) of Saint-Jacques scallop Pecten maximus: evaluation of prokaryote diagnosis by hybridization with a non-iso-topically labelled probe and by polymerase chain reaction. Dis. Aquat. Org., 15: 145-152,1993 https://doi.org/10.3354/dao015145
  13. Le Roux, F. Audemard, C., Barnaud A. and Berthe, F.: DNA probes as potential tools for the detection of Marteilia refringens. Mar. Biotechnol., 1: 588-597,1999 https://doi.org/10.1007/PL00011814
  14. Lipart, C. and Renault T.: Herps-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes. J. Virol. Method. 101: 1-10,2002 https://doi.org/10.1016/S0166-0934(01)00413-X
  15. Machado, J., Coimbra, J. Castilho, F. and Sa, C: Effect of Diflubenzuron on ell formation of the freshwater clam, Anadonta cygnea. Archs. Envrion. Contam. Toxicol., 19:, 35-39, 1990
  16. Malouf, R., Keck, R., Maurer, D. and Epifanio, C: Occurrence of gas-bubble disease in three species of bivalve molluscs. J. Fish. Res. Bd. Can., 29: 588-589,1972 https://doi.org/10.1139/f72-098
  17. Mori, K.: Seasonal variation in physiological activity of scallops under culture in the coastal waters of Sannriku district, Japan, and a physiological approach of a possible cause of their mass mortality. Bull. Mar. Biol. Stn. Asamushi Tohoku Univ.. 15: 59-79,1975
  18. Noel, R., Nicolas, J. L., Boulo, V., Mialhe, E. and Roch P.: Development of colony-blot Elisa assay using monoclonal antibodies to identify Vibrio Pl responsible for 'brown ring disease': in the clam Tapes philippinarum. Aquaculture, 146: 171-178, 1996 https://doi.org/10.1016/S0044-8486(96)01385-3
  19. Novoa, B., Ordas, M. C, and Figueras, A.: Hyper-nospores detected by RFTM in clam (Ruditapes decussatus) tissues belong to two different protozoan organisms, Perkinsus atlanticus and Perkinsus-like organism. Aquaculture, 209: 11-18, 2002 https://doi.org/10.1016/S0044-8486(01)00803-1
  20. Paillard, C., Alcox, K., and Ford, S. E.: Changes in hemolymph and extrapallial fluid parameters in the American oyster diseases Cras-sostrea virginica affected by the juvenile oyster disease. Aquat. Living Resour., 9: 145-158, 1996
  21. Paillard, C. and Maes, P.: The brown ring disease in the Manila clams, Ruditapes philippinarum. 1. Ultrastructural alternations of the perios-tracal laminia. J. Invert. Pathol., 65: 95-101, 1995a
  22. Paillard, C. and Maes, P.: The brown ring disease in the Manila clams, Ruditapes philippinarum. 2. Microscopic study of the brown ring syndrome. J. Invert. Pathol., 65: 101-110, 1995b https://doi.org/10.1006/jipa.1995.1016
  23. Paillard, C. Maes, P. and Oubella, R.: Brown ring disease in clams. Annu. Rev. Fish Dis., 4: 219-240,1994 https://doi.org/10.1016/0959-8030(94)90030-2
  24. Raghukumar, C. and Lande, V.: Shell disease of rock oyster, Crassostrea cucullata. Dis. Aquat. Org., 4: 77-41,1988 https://doi.org/10.3354/dao004077
  25. Renault, X, Le Deuff, R.-M., Lipart, C. and Delsert, C: Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France. J. Virol. Method., 88: 41-50, 2000 https://doi.org/10.1016/S0166-0934(00)00175-0
  26. Stokes, N. A., Siddall, M. E. and Burreson, E. M.: Detection of Haplosporidium nelsoni (Hal-posporidia: Haplosporidiidae) in oysters by PCR amplification. Dis. Aquat. Org., 23: 145-152,1995 https://doi.org/10.3354/dao023145