คุณลักษณะของเชื้อ Vibrio alginolyticus ที่แยกได้จากสิ่งแวดล้อมและปลาที่เป็นโรค

Abstract

In this study, primers specific to the gyrB gene of Vibrio alginolyticus were developed. A total of 160 strains of V. alginolyticus were isolated from clinical (10), diseased fish (20) and environmental (130) samples using CHROMagar Vibrio and biochemical tests. PCR using gyrB, collagenase, toxR and ompk primers were performed on clinical strains. Nine out of 10 strains showed a positive result using gyrB and collagenase primers. Five out of 10 were positive using tox primers. Ompk primers failed to identify V. alginolyticus isolated from clinical samples. Only 139 and 134 out of 160 strains were positive by PCR using gyB and collagenase primers, respectively. Five environmental strains positive for gyrB gene but negative for collagenase gene were confirmed by 16S rRNA sequencing. The sequences were more than 99% homology to V. alginolyticus. Therefore, PCR using gyrB primers showed higher specificity and sensitivity than collagenase, toxR and ompk primers. These primers could be used to identify V. alginolyticus isolated from various sources. Virulence test of V. alginolyticus was performed on Galleria mellonella larvae. Eleven and 33 strains of V. alginolyticus isolated from diseased fish and environmental samples, respectively were selected for virulence test. They were classified into three virulence types. The high virulence strains were observed only in those isolated from diseased fish (5/11, 45.45%) with the death rates of 66.7 to 100%. The low virulence strains of V. alginolyticus were observed in both isolated from diseased fish (1/11, 9.10%) and environments (13/33, 39.39%). The nonvirulence strains were found in both diseased fish (5/11, 45.45%) and environments (20/33, 60.61%). This indicates that V. alginolyticus can be both true and opportunistic pathogens in fish. The differentially expressed proteins of the highest virulence strain KF2.4 and the nonvirulence strain W6.2 were observed using two-dimensional gel electrophoresis. At least 20 proteins were upregulated in the virulence strain KF2.4. These proteins may be the virulence factors which involved in the pathogenicity of the bacteria and need further identification and characterization. V. alginolyticus virulence factor determination will be the useful information for further development of pathogenic identification method and vaccine development against pathogenic V. alginolyticus.