Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China.

During the winter-spring from 2004 to 2006 in northeastern China cultured Japanese sea cucumber Apostichopus japonicus suffered from a serious disease. Clinical signs included swollen mouth, skin ulceration and massive mortality. Clinical samples taken during this period were studied. Thirty-one bacterial samples were isolated from diseased sea cucumbers and identified through biochemical tests, 16S rRNA gene sequence analysis and PCR amplification, followed by pathogenicity determination. The results showed that the 31 isolates belonged to the genera Vibrio (64.5%), Shewanella (12.9%), Serratia (12.9%), Pseudoalteromonas (6.4%) and Flavobacterium (3.2 %). The 3 prominent strains were Vibrio splendidus (41.9%), Shewanella (12.9%) and Serratia odorifera biogroup I (12.9%). Pathogenicity tests demonstrated that 13 out of 31 isolates were pathogenic, including 8 strains of V splendidus, 3 strains of Shewanella sp. and 2 strains of Pseudoalteromonas tetraodonis. The pathogenic V splendidus showed the highest frequency of appearance. Median lethal dose (LD50) values (14 d) of V splendidus, Shewanella sp. and P. tetraodonis were 1.74 x 10(7), 7.76 x 10(6), 7.24 x 10(7) CFU g(-1) body weight of sea cucumber, respectively. The virulences differed by species: Shewanella sp. > V splendidus> P. tetraodonis. This is the first report of Shewanella sp. virulence in sea cucumber.

[1]  Xianggang Gao,et al.  Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. , 2009 .

[2]  M. Garnier,et al.  Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas. , 2008, Systematic and applied microbiology.

[3]  Q. Qin,et al.  Development of a SYBR Green I real‐time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood , 2007, Journal of applied microbiology.

[4]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[5]  L. Smythe,et al.  Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene , 2006, BMC Microbiology.

[6]  O. Koren,et al.  Bacteria Associated with Mucus and Tissues of the Coral Oculina patagonica in Summer and Winter , 2006, Applied and Environmental Microbiology.

[7]  Xiang Xiao,et al.  Microbial Diversity at a Deep-Sea Station of the Pacific Nodule Province , 2005, Biodiversity & Conservation.

[8]  F. Thompson,et al.  Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas). , 2005, International journal of systematic and evolutionary microbiology.

[9]  H. Holt,et al.  Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. , 2005, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[10]  Chanathip Pharino,et al.  Genotypic Diversity Within a Natural Coastal Bacterioplankton Population , 2005, Science.

[11]  M. Jangoux,et al.  The skin ulceration disease in cultivated juveniles of Holothuria scabra (Holothuroidea, Echinodermata) , 2004 .

[12]  T. Oh,et al.  Shewanella marisflavi sp. nov. and Shewanella aquimarina sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. , 2004, International journal of systematic and evolutionary microbiology.

[13]  J. J. Borrego,et al.  Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution , 2004 .

[14]  R. Kong,et al.  Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences. , 2002, Marine pollution bulletin.

[15]  L. Gram,et al.  Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces , 2001, Applied and Environmental Microbiology.

[16]  M. Pujalte,et al.  Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. , 1999, International microbiology : the official journal of the Spanish Society for Microbiology.

[17]  C. Lambert,et al.  Pathogenicity of Vibrio splendidus strains associated with turbot larvae, Scophthalmus maximus , 1999, Journal of applied microbiology.

[18]  B. Austin,et al.  Bacterial fish pathogens: disease in farmed and wild fish. , 1999 .

[19]  J. J. Borrego,et al.  Survey of bacterial pathologies affecting farmed gilt-head sea bream (Sparus aurata L.) in southwestern Spain from 1990 to 1996 , 1998 .

[20]  Eleonor V. Alapide-Tendencia,et al.  Isolation of Vibrio spp. from Penaeus monodon (Fabricius) with red disease syndrome , 1997 .

[21]  D. Martin,et al.  Ocular infection caused by three unusual marine organisms. , 1997, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[22]  L. Gram,et al.  Shewanella alga bacteremia in two patients with lower leg ulcers. , 1996, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[23]  A. Brink,et al.  Shewanella (Pseudomonas) putrefaciens bacteremia. , 1995, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  M. Matsuo,et al.  Alteromonas atlantica sp. nov. and Alteromonas carrageenovora sp. nov., Bacteria That Decompose Algal Polysaccharides , 1992 .

[25]  T. Ezaki,et al.  Isolation and characterization of Shewanella alga from human clinical specimens and emendation of the description of S. alga Simidu et al., 1990, 335. , 1992, International journal of systematic bacteriology.

[26]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[27]  J. Plumb.  Methods for the Microbiological Examination of Fish and Shellfish , 1990 .

[28]  K. Kersters,et al.  Bergey's Manual of Systematic Bacteriology , 1989 .

[29]  T. P. Nieto,et al.  Virulence properties and enterotoxin production of Aeromonas strains isolated from fish , 1988, Infection and immunity.

[30]  T. P. Nieto,et al.  Comparison between the bacterial flora associated with fingerling rainbow trout cultured in two different hatcheries in the North-West of Spain , 1984 .

[31]  R. Colwell,et al.  Haemagglutinating, haemolytic and cytotoxic activities of Vibrio anguillarum and related vibrios isolated from striped bass on the Atlantic Coast , 1983 .

[32]  L. Reed,et al.  A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS , 1938 .

[33]  Zhang Chun-yun,et al.  Isolation and identification of causative pathogen for skin ulcerative syndrome in Apostichopus japonicus , 2006 .

[34]  Yingeng Wang,et al.  Natural resources, culture and problems of sea cucumber worldwide , 2004 .

[35]  J. Jofre,et al.  Identification and Typing of Vibrio anguillarum: A Comparison of Different Methods , 1995 .

[36]  A. Blanch,et al.  A set of keys for biochemical identification of environmental Vibrio species. , 1994, The Journal of applied bacteriology.

[37]  J. J. Borrego,et al.  Characterization of strains related to brown ring disease outbreaks in southwestern Spain , 1992 .

[38]  G. Tortora,et al.  Pseudomonas putrefaciens bacteremia associated with shellfish contact. , 1990, The American journal of medicine.

[39]  L. D. Peña,et al.  Studies on the chemical control of luminous bacteria Vibrio harveyi and V. splendidus isolated from diseased Penaeus monodon larvae and rearing water , 1990 .