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A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity

  • Invertebrate Microbiology
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Abstract

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.

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References

  1. Fleury PG, Goyard E, Mazurie J, Claude S, Bouget JF, Langlade A, Le Coguic Y (2001) The assessing of Pacific oyster (Crassostrea gigas) rearing performances by the IFREMER/REMORA network: method and first results (1993–98) in Brittany (France). Hydrobiologia 465:195–208

    Article  Google Scholar 

  2. Saulnier D, De Decker S, Haffner P (2009) Real-time PCR assay for rapid detection and quantification of Vibrio aestuarianus in oyster and seawater: a useful tool for epidemiologic studies. J Microbiol Methods 77:191–197

    Article  PubMed  CAS  Google Scholar 

  3. Garnier M, Labreuche Y, Garcia C, Robert M, Nicolas JL (2007) Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas. Microb Ecol 53:187–196

    Article  PubMed  CAS  Google Scholar 

  4. Le Roux F, Gay M, Lambert C, Waechter M, Poubalanne S, Chollet B, Nicolas JL, Berthe F (2002) Comparative analysis of Vibrio splendidus-related strains isolated during Crassostrea gigas mortality events. Aquat Living Resour 15:251–258

    Article  Google Scholar 

  5. Gay M, Renault T, Pons AM, Le Roux F (2004) Two Vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations. Dis Aquat Organ 62:65–74

    Article  PubMed  Google Scholar 

  6. Soletchnik P, Ropert M, Mazurie J, Fleury PG, Le Coz F (2007) Relationships between oyster mortality patterns and environmental data from monitoring databases along the coasts of France. Aquaculture 271:384–400

    Article  Google Scholar 

  7. Goulletquer P, Soletchnik P, Le Moine O, Razet D, Geairon P, Faury N (1998) Summer mortality of the Pacific cupped oyster Crassostrea gigas in the Bay of Marennes-Oleron (France). CM—Int Counc Explor Sea, Cascais (Portugal) 16–19:20

    Google Scholar 

  8. Samain JF, McCombie H (2008) Summer mortality of Pacific oyster Crassostrea gigas. The Morest Project, Editions Quae, 379p

    Google Scholar 

  9. Tubiash HS, Chanley PE, Leifson E (1965) Bacillary Necrosis, a disease of larval and juvenile bivalve mollusks. J Bacteriol 90:1036–1044

    PubMed  CAS  Google Scholar 

  10. Estes R, Friedman C, Elston R, Herwig R (2004) Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae. Dis Aquat Organ 58:223–230

    Article  PubMed  Google Scholar 

  11. Elston RA, Hasegawa H, Humphrey KL, Polyak IK (2008) Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management. Dis Aquat Organ 82:119–134

    Article  PubMed  Google Scholar 

  12. Inamura H, Nakai T, Muroga K (1985) An extracellular protease produced by Vibrio anguillarum. Bulletin of the Japanese Society of Scientific Fisheries 51:1915–1920

    CAS  Google Scholar 

  13. Lee KK, Liu PC, Chuang WH (2002) Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish. Mar Biotechnol 4:267–277

    Article  PubMed  CAS  Google Scholar 

  14. Lee KK, Chen YL, Liu PC (1999) Hemostasis of Tiger prawn Penaeus monodon affected by Vibrio harveyi, extracellular products, and a toxic cysteine protease. Blood Cells Mol Dis 25:180–192

    Article  PubMed  CAS  Google Scholar 

  15. Lee KK, Yu SR, Liu PC (1997) Alkaline serine protease is an exotoxin of Vibrio alginolyticus in Kuruma prawn, Penaeus japonicus. Curr Microbiol 34:110–117

    Article  PubMed  Google Scholar 

  16. Nottage AS, Birkbeck TH (1987) Production of proteinase during experimental infection of Ostrea edulis L. larvae with Vibrio alginolyticus NCMB 1339 and the antigenic relationship between proteinases produced by marine vibrios pathogenic for fish and shellfish. J Fish Dis 10:265–273

    Article  CAS  Google Scholar 

  17. Travis J, Potempa J, Maeda H (1995) Are bacterial proteinases pathogenic factors? Trends Microbiol 3:405–407

    Article  PubMed  CAS  Google Scholar 

  18. Hase CC, Finkelstein RA (1993) Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev 57:823–837

    PubMed  CAS  Google Scholar 

  19. Miyoshi SI, Shinoda S (2000) Microbial metalloproteases and pathogenesis. Microbs Infect 2:91–98

    Article  CAS  Google Scholar 

  20. Labreuche Y, Soudant P, Goncalves M, Lambert C, Nicolas JL (2006) Effects of extracellular products from the pathogenic Vibrio aestuarianus strain 01/32 on lethality and cellular immune responses of the oyster Crassostrea gigas. Dev Comp Immunol 30:367–379

    Article  PubMed  CAS  Google Scholar 

  21. Le Roux F, Binesse J, Saulnier D, Mazel D (2007) Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector. Appl Environ Microbiol 73:777–784

    Article  PubMed  CAS  Google Scholar 

  22. Delston R, Kothary M, Shangraw K, Tall B (2003) Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii. Can J Microbiol 49:525–529

    Article  PubMed  CAS  Google Scholar 

  23. Binesse J, Delsert C, Saulnier D, Champomier-Verges MC, Zagorec M, Munier-Lehmann H, Mazel D, Le Roux F (2008) The metalloprotease Vsm is the main toxic factor for Vibrio splendidus extracellular products. Appl Environ Microbiol 74:7108–7117

    Article  PubMed  CAS  Google Scholar 

  24. Hasegawa H, Lind EJ, Boin MA, Hase CC (2008) The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for Pacific oyster (Crassostrea gigas) larvae. Appl Environ Microbiol 74:4101–4110

    Article  PubMed  CAS  Google Scholar 

  25. Gay M, Berthe FC, Le Roux F (2004) Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas. Dis Aquat Organ 59:49–56

    Article  PubMed  Google Scholar 

  26. Lambert C, Nicolas JL (1998) Specific inhibition of chemiluminescent activity by pathogenic vibrios in hemocytes of two marine bivalves: Pecten maximus and Crassostrea gigas. J Invertebr Pathol 71:53–63

    Article  PubMed  CAS  Google Scholar 

  27. Yamamoto S, Harayama S (1995) PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61:1104–1109

    PubMed  CAS  Google Scholar 

  28. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57:2259–2261

    Article  PubMed  CAS  Google Scholar 

  29. Hall T (1999) BioitEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  30. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  31. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  32. Teo JWP, Zhang LH, Poh CL (2003) Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6. Gene 303:147

    Article  PubMed  CAS  Google Scholar 

  33. Le Roux F, Gay M, Lambert C, Nicolas JL, Gouy M, Berthe F (2004) Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences. Dis Aquat Organ 58:143–150

    Article  PubMed  Google Scholar 

  34. Faury N, Saulnier D, Thompson FL, Gay M, Swings J, Le Roux F (2004) Vibrio crassostreae sp. nov., isolated from the haemolymph of oysters (Crassostrea gigas). Int J Syst Evol Microbiol 54:2137–2140

    Article  PubMed  CAS  Google Scholar 

  35. Thompson JR, Pacocha S, Pharino C, Klepac-Ceraj V, Hunt DE, Benoit J, Sarma-Rupavtarm R, Distel DL, Polz MF (2005) Genotypic diversity within a natural coastal bacterioplankton population. Science 307:1311–1313

    Article  PubMed  CAS  Google Scholar 

  36. Sawabe T, Kita-Tsukamoto K, Thompson FL (2007) Inferring the evolutionary history of vibrios by means of multilocus sequence analysis. J Bacteriol 189:7932–7936

    Article  PubMed  CAS  Google Scholar 

  37. Guisande JA, Montes M, Farto R, Armada SP, Perez MJ, Nieto TP (2004) A set of tests for the phenotypic identification of culturable bacteria associated with Galician bivalve mollusc production. J Shellfish R 3:599–609

    Google Scholar 

  38. Weiner RM, Segall AM, Colwell RR (1985) Characterization of a marine bacterium associated with Crassostrea virginica (the Eastern oyster). Appl Environ Microbiol 49:83–90

    PubMed  CAS  Google Scholar 

  39. Colwell RR, Liston J (1960) Microbiology of shellfish. Bacteriological study of the natural flora of Pacific oysters (Crassostrea gigas). Appl Microbiol 8:104–109

    PubMed  CAS  Google Scholar 

  40. Macián M, Garay E, González-Candelas F, Pujalte M, Aznar R (2000) Ribotyping of Vibrio populations associated with cultured oysters (Ostrea edulis). Syst Appl Microbiol 23:409–417

    PubMed  Google Scholar 

  41. Olafsen JA, Mikkelsen HV, Giæver HM, Høvik Hansen G (1993) Indigenous bacteria in hemolymph and tissues of marine bivalves at low temperatures. Appl Environ Microbiol 59:1848–1854

    PubMed  Google Scholar 

  42. Ortigosa M, Garay E, Pujalte MJ (1994) Numerical taxonomy of Vibrionaceae isolated from oysters and seawater along an annual cycle. Syst Appl Microbiol 17:216–225

    Google Scholar 

  43. Pujalte MJ, Ortigosa M, Macián MC, Garay E (1999) Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. Int Microbiol 2:259–266

    PubMed  CAS  Google Scholar 

  44. Labreuche Y (2006) Caractérisation de la virulence d'une souche de Vibrio aestuarianus, pathogène de l'huître creuse Crassostrea gigas. Université de Bretagne Occidentale, Brest, France, PhD, 277p

    Google Scholar 

  45. Labreuche Y, Lambert C, Soudant P, Boulo V, Huvet A, Nicolas JL (2006) Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01/32. Microbs Infect 8:2715–2724

    Article  CAS  Google Scholar 

  46. Nicolas JL, Corre S, Gauthier G, Robert R, Ansquer D (1996) Bacterial problems associated with scallop Pecten maximus larval culture. Dis Aquat Organ 27:67–76

    Article  Google Scholar 

  47. Gomez-Leon J, Villamil L, Lemos ML, Novoa B, Figueras A (2005) Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl Environ Microbiol 71:98–104

    Article  PubMed  CAS  Google Scholar 

  48. Travers MA, Le Goïc N, Huchette S, Koken M, Paillard C (2008) Summer immune depression associated with increased susceptibility of the European abalone, Haliotis tuberculata to Vibrio harveyi infection. Fish Shellfish Immunol 25:800–808

    Article  PubMed  CAS  Google Scholar 

  49. Nicolas JL, Basuyaux O, Mazurié J, Thébault A (2002) Vibrio carchariae, a pathogen of the abalone Haliotis tuberculata. Dis Aquat Organ 50:35–43

    Article  PubMed  CAS  Google Scholar 

  50. Chang AK, Kim HY, Park JE, Acharya P, Park IS, Yoon SM, You HJ, Hahm KS, Park JK, Lee JS (2005) Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis. J Bacteriol 187:6909–6916

    Article  PubMed  CAS  Google Scholar 

  51. Denkin SM, Nelson DR (2004) Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl Environ Microbiol 70:4193–4204

    Article  PubMed  CAS  Google Scholar 

  52. Takahashi KG, Nakamura A, Mori K (2000) Inhibitory effects of ovoglobulins on bacillary necrosis in larvae of the Pacific oyster, Crassostrea gigas. J Invertebr Pathol 75:212–217

    Article  PubMed  CAS  Google Scholar 

  53. Watanabe H, Miyoshi SI, Kawase T, Tomochika KI, Shinoda S (2004) High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans. Microb Pathog 36:117–123

    Article  PubMed  CAS  Google Scholar 

  54. Kim C-M, Kang S-M, Jeon H-J, Shin S-H (2007) Production of Vibrio vulnificus metalloprotease VvpE begins during the early growth phase: usefulness of gelatin-zymography. J Microbiol Methods 70:96–102

    Article  PubMed  CAS  Google Scholar 

  55. Milton DL, Norqvist A, Wolf-Watz H (1992) Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol 174:7235–7244

    PubMed  CAS  Google Scholar 

  56. Le Roux F, Mohamed Z, Chakroun N, Binesse J, Saulnier D, Bouchier C, Zidane N, Ma L, Rusniok C, Lajus A, Buchrieser C, Médigue C, Polz MF, Mazel D (2009) Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity. Environ Microbiol 11:1959–1970

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Helen McCombie for her help with English editing.

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Correspondence to Denis Saulnier.

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Saulnier, D., De Decker, S., Haffner, P. et al. A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity. Microb Ecol 59, 787–798 (2010). https://doi.org/10.1007/s00248-009-9620-y

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