Isolation and Identification of Vibrio parahaemolyticus from Cultured Marsupenaeus japonicus

Key words: 
• Marsupenaeus japonicus /
• matrix-assisted laser desorption / ionization time of flight mass spectrometry /
• 16S rRNA /
• Vibrio parahaemolyticus /
• chemo-sensitivity test

Abstract: Two strains（ MJBX-1 and MJBX-2） of bacteria were isolated from the hepatopancreas,stomach and intestines of infected Marsupenaeus japonicus,and used for immersion challenge of V. parahaemolyticus to study their infectivity. These isolates induced 100% mortality of V. parahaemolyticus immersion challenged at the concentration of 10⁷ cfu·mL^-1,and 90% mortality at 10⁶cfu·mL^-1. Subsequent bacterial isolation from the challenged V. parahaemolyticus showed the same bacterial colony type as in the field samples. These isolates were gram-negative,arcshaped,rhabditiform or filamentous,without spore-forming,but mostly with an obvious arc shape. They formed transparent or translucent round colonies on the LB solid medium and green colonies on the TCBS medium. In the meantime,they could grow at the culture medium containing 2%,4%,6% or 8% of Na CL,but not 0% or 10% of Na CL. The isolated strains were preliminarily identified by using the matrix assisted laser desorption/ionization time of flight mass spectrometry（ MALDI-TOF-MS） through comparison of the mass spectrometry between the isolated strains and the standard strain. Moreover,their sequences were analyzed by using the 16 S rRNA to produce phylogenetic tree. The MALDI-TOF-MS and the 16 S rRNA analysis showed that the isolated strains MJBX-1 and MJBX-2 belonged to Vibrio parahaemolyticus. The antibiotic sensitivity test showed that MJBX-1 and MJBX-2 were sensitive to Florfenicol, Norfloxacin, Macrodantin, Gentamicin, Streptomycin and Kanamycin, but not to Amoxicillin and Tetracycline. This study will provide scientific reference for accurate,quick identification and control of the disease of V. parahaemolyticus caused by V. parahaemolyticus.