Multiplex nested‐polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.

[1]  R. Kusuda,et al.  Etiological Studies on Bacterial Pseudotuberculosis in Cultured Yellowtail with Pasteurella Piscicida as the Causative Agent-I , 1972 .

[2]  C. López,et al.  Vibrio alginolyticus infection in cobia (Rachycentron canadum) cultured in Taiwan , 2001 .

[3]  Douglas R. Call,et al.  Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray , 2004, Journal of Clinical Microbiology.

[4]  R. Colwell,et al.  Aeromonas hydrophila: ecology and toxigenicity of isolates from an estuary. , 1981, The Journal of applied bacteriology.

[5]  N. Takada,et al.  Structural variation in the 16S-23S rRNA intergenic spacers of Vibrio parahaemolyticus. , 2000, FEMS microbiology letters.

[6]  L. Domínguez,et al.  Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish , 2004, Applied and Environmental Microbiology.

[7]  Yutaka Fukuda,et al.  Pasteurella piscicida Infection in Cultured Juvenile Japanese Flounder , 1996 .

[8]  E. Capkin,et al.  Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens. , 2008, Veterinary microbiology.

[9]  D. Call,et al.  Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays , 2004, Applied and Environmental Microbiology.

[10]  P. Taylor,et al.  Optimization of Nested Polymerase Chain Reaction Assays for Identification of Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum , 2002 .

[11]  D. Bruno,et al.  Evaluation and development of diagnostic methods for Renibacterium salmoninarum causing bacterial kidney disease (BKD) in the UK , 2007 .

[12]  Alicia E. Toranzo,et al.  Pasteurellosis in cultured gilthead seabream (Sparus aurata): first report in Spain. , 1991 .

[13]  D. Call,et al.  Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays , 2004, Applied and Environmental Microbiology.

[14]  G. Fox,et al.  How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. , 1992, International journal of systematic bacteriology.

[15]  J. Romalde,et al.  First occurrence of pasteurellosis in Portugal affecting cultured gilthead seabream (Sparus aurata [Pagrus aurata]). , 1996 .

[16]  Toshio Nakatsugawa,et al.  Edwardsiella tarda Isolated from Cultured Young Flounder , 1983 .

[17]  A. Candan,et al.  Pasteurellosis in cultured sea bass (Dicentrarchus labrax) in Turkey , 1996 .

[18]  T. Aoki,et al.  Drug Resistance and Transferable R Plasmids in Edwardsiella tarda from Fish Culture Ponds (魚病に関する国際セミナ , 1981 .

[19]  Satoru Matsuoka,et al.  Pasteurella piscicida Infection in Hatchery-Reared Juvenile Striped Jack , 1992 .

[20]  R. Christen,et al.  Small-subunit rRNA sequences and whole DNA relatedness concur for the reassignment of Pasteurella piscicida (Snieszko et al.) Janssen and Surgalla to the genus Photobacterium as Photobacterium damsela subsp. piscicida comb. nov. , 1995, International journal of systematic bacteriology.

[21]  G. Bullock,et al.  PASTEURELLA SP. FROM AN EPIZOOTIC OF WHITE PERCH (ROCCUS AMERICANUS) IN CHESAPEAKE BAY TIDEWATER AREAS , 1964, Journal of bacteriology.

[22]  Kunio Suzuki,et al.  Real-time PCR for quantification of viable Renibacterium salmoninarum in chum salmon Oncorhynchus keta. , 2007, Diseases of aquatic organisms.

[23]  José Luis Balcázar,et al.  Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers. , 2007, Journal of medical microbiology.

[24]  Masao Kimura,et al.  Studies of a bacterial tuberculoidosis of the yellowtail-I Symptomatolgy and histopathology , 1970 .

[25]  J. Romalde,et al.  Phenotypic and pathobiological characteristics of Pasteurella piscicida , 1996 .

[26]  Y. Jo,et al.  Studies on Streptococcal Infection in Pond-Cultured Fishes-I , 1981 .

[27]  C. R. Osorio,et al.  Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae. , 2000, Diseases of aquatic organisms.

[28]  K. Kawai,et al.  Streptococcus sp. Isolated from an Epizootic of Cultured Eels , 1978 .

[29]  Ramesh P. Perera,et al.  Streptococcus iniae Associated with Mortality of Tilapia nilotica × T. aurea Hybrids , 1994 .

[30]  J. A. Guijarro,et al.  Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR , 2002, Applied and Environmental Microbiology.

[31]  S. Egusa,et al.  Some Bacterial Diseases of Freshwater Fishes in Japan , 1976 .

[32]  M. Collins,et al.  16S rRNA Gene Sequence Analysis ofPhotobacterium damselae and Nested PCR Method for Rapid Detection of the Causative Agent of Fish Pasteurellosis , 1999, Applied and Environmental Microbiology.

[33]  H. Wakabayashi,et al.  Pathogenic Activities of Aeromonas hydrophila biovar hydrophila(Chester) POPOFF and VERON,1976 to Fishes (魚病に関する国際セミナ-) , 1981 .

[34]  K. Hatai,et al.  Multiplex PCR for Detection of Lactococcus garvieae, Streptococcus iniae and S. dysgalactiae in Cultured Yellowtail , 2006 .

[35]  Leigh M. Schmidtke,et al.  Lactococcus garvieae strains isolated from rainbow trout and yellowtail in Australia, South Africa and Japan differentiated by repetitive sequence markers , 2003 .

[36]  W A Janssen,et al.  Morphology, Physiology, and Serology of a Pasteurella Species Pathogenic for White Perch (Roccus americanus) , 1968, Journal of bacteriology.

[37]  A. Eldar,et al.  Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases. , 1999, Diseases of aquatic organisms.

[38]  A. E. Toranzo,et al.  Characterization of Vibrio damsela strains isolated from turbot Scophthalmus maximus in Spain , 1992 .

[39]  K. Dybvig,et al.  Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila. , 2007, Diseases of aquatic organisms.

[40]  Lulla Cp,et al.  Multiplex PCR assay for the detection of pathogenic Aeromonas hydrophila , 2005 .