Isolation And Identification Of Pathogenic Bacteria In The Flounder Along With The Preparation And Application Of Polyclonal Antibodies

Infected marine fish samples（such as turbot, flounder, C. semilaevi et al.） werecollected from the coastal area of Shandong Province in the year2009and2010.Thenthe pathogens were isolated, purified, identified and analysised by the drug sensitivetests. By the results we can know the type of pathogens and give reasonable advice forfish diseases in aquaculture; virulences of five pathogens—Vibrio anguillarum, Vibrioharveyi,Vibrio parahaemolyticus, Aeromonas salmonicida and Shewanellaputrefaciens were evaluated，antisera against them were prepared，and the indirectELISA method which could detect these pathogens in a quick way were established.Identification of bacteria mainly adopted the16S rDNA method as well asbacterial fatty acid identification and Biolog method. Bacteria isolated from thediseased fish were mainly of Pseudoalteromonas sp. and Vibrio sp.,including thecommon pathogenic bacteria—V. harveyi, V. fischeri, V. splendidus and V.ichthyoenteri.Drug sensitive tests used the paper disk method. The results showed that of the37bacteria isolated in2009,100%、100%and97.3%had drug resistance to PenicillinG、Lincomycin and Tetracycline respectively, while83.78%、86.49%and100%weresentitive to Azithromycin、Rifampicin and Polymyxin B Sulfate respectively. SoPolymyxin B Sulfate may be efficient in aquaculture. Of the43strains isolated in2010,100%、97.67%、97.67%and95.34%had drug resistance to Penicillin G、Lincomycin、Tetracycline and Cephaloridine Ⅵ respectively, while79.07%and95.35%were sensitive to Norfloxacin and Chloramphenicol.Virulence evaluation of pathogenic bacteria was carried out by the determinationof LD₅₀to Turbots. The results showed that LD₅₀values of V. anguillarum, V. harveyi, V. parahaemolyticus, Aeromonas salmonicida and Shewanella putrefaciens were7.00×10²、2.17×10⁶、3.89×10⁶、3.16×10⁶、3.73×10⁷respectively, which indicated thatthese five bacteria were all pathogenic.Making use of the prepared polyclonal antibodies, the indirect ELISA methodwas established for detecting the pathogenic bacteria in a quick way. The researchshowed that the titers of antisera against V. anguillarum, V. parahaemolyticus, V.harveyi, Aeromonas salmonicida and Shewanella putrefaciens were1:2048000、1:1024000、1:16000、1:16000and1:128000respectively. The sensitivity of the serawas tested，and the lowest concentration of five pathogens that could be detected were10⁴、10⁵、10⁷、10⁸、10⁶CFU/mL respectively. Obvious cross reactions existed amongthree vibrios. In the application of this method, one bacterium from the flouder andblack rockfish samples in the area of Jiaonan（Shandong Province） were positive forShewanella putrefaciens and two for V. harveyi; one bacterium from the C. semilaeviin the area of Wuli was positive for both V. parahaemolyticus and V. harveyi.