| Ulcerous disease is the most common disease of unknown etiology of Southern catfish(Silurus meridionalis) and channel catfish (Ictalurus punctatus) during 2012-2014 in Sichuan Province. China. Four Gram-negative.arc-s haped. single polar flagellum brevibacterium (SCCF03-SCCF06) were isolated from the liver, spleen and kidney of the diseased catfish from different farms. Bacterium colonies were smooth and shiny. circular, entire and low convex after incubation at 28℃ for 48 h on TSA, while colonies on TCBS were of green color. And they were Gram-negative rods characteristically curved or comma shape bacteria with single polar flagellum. The four isolates were primarily identified to be Vibrio, sp according to thier morphology, physiological and biochemical characteristics. According to the 16S rDNA sequence of the four strains had a high homology with those of the V.cholerae, while the gyrB sequence were similar to the V. mimicus. A phylogenetic tree was constructed based on the 16S rDNA sequences of the four isolated strains, together with V.cholerae and V. mimicus formed a tight cluster with high sequence similarities, while the gyrB sequences formed a tight cluster with V. mimicus. And furthermore, resulting duplex PCR products from all the four strains which based on dna.1 sequences, a 177 bp segments were amplified, which confirmed the isolated strains were V. mimicus. The results of sensitivity to antibacterial drugs showed that both the four strains were sensitive to streptomycin, amikacin, norfloxacin, florfenicol and ofloxacin, but resistant to roxithromycin and penicillin. Histopathologic observation showed that V. mimicus infection could lead to serious pathological changes in multiple tissues and organs, which were hemorrhages, degeneration, necrosis and inflammatory cell infiltration, especially in muscle, liver, spleen and kidney.We screened the V. mimicus isolates for the presence of the ctxA, sot, ace, tcpA genes, toxR. ompU and vmh genes. The vmh and ompU gene were detected in all of the V. mimicus isolates, whereas none possessed the ctxA. zot. ace, tcpA or toxR genes. Vmh gene of V. mimicus SCCF06 strain was amplified by PCR. cloned, sequenced and analyzed by bioinformatics software. The full length of vmh gene of SCCF06 contained a 2235bp open reading frame, encoding a protein comprising 744 amino acid residues with a predicted molecular mass and theoretical iso-electric point were 85.25 kDa kDa and 5.04, respectively. The polypeptide with higher content of Ser (8.6%). Ala (7.9%) and Leu (7.7%) while lower content of Met or Cys (0.9%). Multiple sequence alignment indicated that the nucleotide sequence and amino acid sequence of vmh gene displayed 95%～97% and 96%～97% of homology respectively, compared with those of different reference strains published in GenBank. The conserved domains analysis suggested the vmh amino acid sequence contained Hemolysin_N, Leukocidin, RICIN and Jaclin.like superfamily conserved domains. Besides, the polypeptide contained 24 antigenic determinants,40 B-cell epitopes,50 flexible regions and 21 surface probability regions, and the hydrophobic regions of the polypeptide were more than the hydrophilic regions, suggesting the peptides was a hydrop hobic protein. The signal peptide and transmembrane region prediction revealed there was a signal peptide with 25 amino acid residues at the N-terminus of the polypeptide and no transmembrane region. According to the prediction of modification sites, the nucleoprotein may contain 44 phosphorylation sites,9 N-glycosylation sites and 8 O-glycosylation sites. |
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