The Identification Of Bacterial Pathogen From Turbot(Scophthalmus Maximus) And Clone Of Hemolysin Gene From Vibrio Harveyi

Nine strains were isolated from diseased frabot(Scophthalmus maximus) which are then named after VA'1,VA*2,VA-3,VH-1,VI-1,ET-1,EN-1,MA-1 and PA-l.They are identified by morphological and biochemical characteristics, the result is that VA-l,VA-2 and VA-3 are Vibrio anguillarum,V}i-\ is Vibrio harveyi,VH-\ is Vibrio ichthyoenteri,ET-l is Edwardsiella tarda, EN-1 is Enterococcus sp.,MA-l is Marinomonas sp.,PA-l is Alteromonas sp..The 16S-rDNA genes of nine strains are amplified,cloned ,sequeneed and then aligned.The alignment result shows VA-l,VA-2 and VA-3 are closest to Vibrio anguillarum, VH-1 is closest to Vibrio harveyi, VI-1 is closest to Vibrio ichthyoenteri, ET-1 is closest to Edwardsiella tarda, EN-1 is closest to Enterococcus sp., PA-1 is closest to Alteromonas sp.,but MA-1 is not closest to Marinomonas sp.-From the results of morphological and biochemical characteristics identification and 16SrDNA genes alignment, it is concluded that VA-l,VA-2 and VA-3 are Vibrio anguillarum,VH-\ is Vibrio harveyi, VH-1 is Vibrio ichthyoenteri,ET-\ is Edwardsiella tarda, EN-1 is Enterococcus sp., PA-1 is Alteromonas sp.,otherwise MA-1 need further identification.The pathogenicity of nine strains to turbot is studied by intraperitoneal injection , VA-1 > VA-2s VA-3 are highly pathogenic, VH-1 and EN-1 have weak pathogenicity, VI-1 has no pathogenicity to turbot and is highly pathogenic to flunder,while MA-1 and PA-1 have no pathogenicity.28 kinds of antibiotics are used in the susceptible test.VA-1 is resistant to 16 kinds of antibiotics, mid-sensitive to 5 kinds of antibiotics and sensitive to 7 kinds ofantibiotics. VA-2 is resistant to 14 kinds of antibiotics,mid-sensitive to 8 kinds of antibiotics and sensitive to 6 kinds of antibiotics. VA-3 is resistant to 12 kinds of antibiotics, mid-sensitive to 7 kinds of antibiotics and sensitive to 9 kinds of antibiotics. VH-1 is resistant to 25 kinds of antibiotics, mid-sensitive to 2 kinds of antibiotics and sensitive to 1 kinds of antibiotic. VI-1 is resistant to 12 kinds of antibiotics, mid-sensitive to 3 kinds of antibiotics and sensitive to 13 kinds of antibiotics.ET-1 is resistant to 12 kinds of antibiotics,mid-sensitive to 7 kinds of a antibiotics and sensitive to 9 kinds of antibiotics.EN-1 is resistant to 22 kinds of antibiotics, mid-sensitive to 4 kinds of antibiotics and sensitive to 2 kinds of antibiotics. MA-1 is resistant to 16 kinds of antibiotics, mid-sensitive to 4 kinds of antibiotics and sensitive to 4 kinds of antibiotics. PA-1 is resistant to 27 kinds of antibiotics, mid-sensitive to 1 kinds of antibioticThe hemolysin gene fragment of Vibrio harveyi VH-1 is amplified by PCR.The nucleotide sequence is determined.The fragment includes the whole ORF of the gene which consists of 1257 base pairs. The hemolysin nucleotide sequence of VH-1 shares 99.7% and 98.4% similarity with that of vhhA and vhhB respectively which are both the hemolysin gene of Vibrio harveyi VIB 645 .The identical percent of nucleotide sequence of hemolysin between VH-1 and Vibrio parahaemolyticus, Vibrio hollisae, Vibrio anguillarum, Vibrio cholerae, Vibrio fluvialis, Vibrio mimicus is 74.6%,24.6 %,20.1%,22.5%,20.8%,20.5%,21.7%.