The Typing Of Fish Pathogen Vibrio Harveyi In South China And The Research Of Its Prevention Strategies

Epidemiologic investigations of several marine cultured fishes (including Epinephelus coioides、Lutjanus erythopterus、Epinephelus fuscoguttatus and E. fuscoguttatus♀×E. lanceolatus♂) were carried out in southern China from May2011to May2013. Sixty-three strains of vibrio were isolated from the liver,spleen,kidney and skin ulcers of the diseased fishes, within whichforty-three strains were V. harveyi, seven strains were V. alginolyticus, five strains were V. parahaemolyticus, two strains were V. rotiferianus, one strain was V. Natriegens and five strains were un-identified Vibrios. When the water salinity were at30,18Vibrio harveyi, a Vibrio alginolyticus, a Vibrio rotiferianus and an unidentified Vibrio strain were pathogenic to Epinephelus coioides. Within these pathogenic vibrios, escept for4virulent strains (GDH11385、GDH11387、GDH12388、 GDH13389), all the rest were low virulent strains (zero death within24h of infection). LD50values of GDH11385, GDH11387, GDH12388were4.06x102CFU/g,2.65×103CFU/g,1.06×103CFU/g,1.30×103CFU/g,Using molecular biological, phenotypic and spectral methods, the isolated strains were thoroughly identified first of all,63Vibrios were identified using16S rDNA analysis, the results showed that43strains had99%homology with Vibrio harveyi. These43stains can be divided into four categories, with Xicun Harbour,Puqian Town occupying one category each. strains from other areas were scattered in the four categories; secondly, house-keeping genes topA and mreB were used to further characterize these43Vibrios, the results also showed that these strains43strains had96%～99%homology with Vibrio harveyi, and they can be divided into three categories, Xicun Harbour was divided into a class of their own, strains in other areas were scattered in three categories. Using physiological and biochemical phenotype determination. House-keeping genes rpoA and pyrH showed that all four strains had high high homology (99%) with Vibrio harveyi. These43Vibrios were made cluster analysis, and results showed that the43strains can be divided into four categories, with Xicun Harbour was divided into a class of their own, strains in other areas were scattered in four categories. Finally, the antimicrobial susceptibility tests of the43Vibrios showed that most of the strains were showed antimicrobial susceptibility only to five kinds of antibiotics:doxycycline, cephalothin, Cefobid/sulbactam, ofloxacin and rifampin.. Cluster analysis showed that the43strains can be divided into four categories, Each category includes some strains from each area, with Xincun Harbour was scattered in four categories.Thus, epidemics caused by the Vibrio harveyi in southern China has become one of the major marine fish bacterial disease, only a few of the important chemical drugs for the treatment of human disease were able to control the pathogen, posing higher disease control risk.Haemolyticus and exoprotein of the43stains of Vibrio harveyi were measured using blood plate and milk-sea solid medium method. Results showed that both virulent and non-virulent strains showed zone of hemolysis; escept for virulent stains GDH11385, GDH11387, GXH11001, GXH11003, GXH12001, GXH11004and non-virulent stains GXH11002, GXH12002, GXH13001, GXH13002, GXH13004, WCH11D222, WCH12D251, all the other stains had no proteolysis except GXH13005and WCH12D251. Thus, the virulence of the43strains showed no correlation with their haemolyticus or exoprotein.The effect of salinity on the virulence of GDH11385(the most virulent strain) were determined by immersion method.The test period was15d. When the salinity were35,30,25and20, the fish mortality were100%; When salinity was15, the mortality rate was33.3%; When the salinity were10or5, the mortality rate was0. Meanwhile, the survival of GDH11385in water of different salinities were also determined.In the water of5,10,15salinity, the survival of Vibrio harveyi were greatly influenced. Within4d, the concentration of Vibrio dropped from107CFU/mL to105CFU/mL;in the water of20, the growth of Vibrio has also been adversely affected to a certain extent, Within4d, Vibrio concentrations decreased from107CFU/mL to106CFU/mL. Thus, the salinity of water showed a significant effect on the virulence of Vibrio harveyi.. Our results provide a reliable basis for the future use of desalination breeding patterns against Vibrio harveyi diseases.Select a nontoxic strain GXH13002which had the closest relationship with GDH11385as immune strains for Epinephelus coioides,15d later, when the final concentration of GDH11385in the water was103CFU/ml and104CFU/ml, mortality rate after immunization is0, the mortality of unvaccinated group was (20.9±4.2)%,(33.4±8.4)%; When the final concentration was105CFU/ml, mortality rate immunohistochemistry was (37.5±4.2)%, the earliest death appeared at7th d, the mortality of unvaccinated was (62.5±4.2)%, and the earliest time of death advance to2th d; When the final concentration was106CFU/ml, mortality rate immunohistochemistry was100%, the earliest death appeared at4th d, mortality rate of unvaccinated was also100%, but the earliest time of death advance to24h. Thus, immunization with avirulent strain can significantly improve the immunity of marine cultured Fishes against Vibrio harveyi, which opens up a new path for the future bacterial pathogen immune protection strategy.Therefore, Vibrio harveyi separated from fishes in the South China sea have high toxicity, strong drug resistance, and can infection a variety of hosts. In the current circumstances within whichthe pathogenic mechanism of Vibrio harveyi were not clear and the chemical control is not ideal, desalination of culture waterand immunization with closed-related avirulent strain could be effective means in the prevention and treatment of Vibrio harveyi diseases.