The Studies Of Adhesion Of Vibrio Fluvialis To Epinephelus Awoara

Vibrio fluvialis is a Gram-negative bacterium that is considered to be one of the foodborne pathogenic bacteria and has been implicated in outbreaks and sporadic cases of diarrhea. In this paper, we investigate the characteristics and mechanisms involved in the adherence of bacteria to the skin mucus for a better understanding of the bacterial pathogencity. Otherwise, we also study the adherence of V.fluvialis in the digestive tract through the construction of EGFP-V.fluvialis.Adhesion of Vibrio fluvialis to skin mucus of Epinephelus awoara had been investigated by using [methyl-~3H] thymidine as the isotope tracer. The results show that the bacterial adhesion of V. fluvialis incubated in TSB is extremely remarkably stronger than that of V. fluvialis incubated on TSA. The adhesion quantity of V. fluvialis to the skin mucus of E. awoara increases with the densities of bacterial suspension, and the bacterial adhesion measures up to the saturation kinetics. After treating the bacteria with antibody against O-antigen of V. fluvialis prepared from rabbit, the number of adhering bacteria reduces by 52%. V. fluvialis at different growth stages show different adhesion ability. At its early growth stage, the number of adhering bacteria increases with the incubating time and reach its peak at 24 h, and then the bacterial adhesion quantity drops sharply as the incubating time exceed 24 h. The results indicate that V. fluvialis adhere to the skin mucus of E. awoara strongly by specific adhesion, this bacterial adhesion is influenced by culture conditions and nutrition level remarkably. These results are helpful for better understanding of the characteristic of epidemiology and pathogenesis of V. fluvialis.Use the same labeling method which is described above to investigate the bacterial adhesion of the pathogen to the mucus after different treatment. The results showed that: adhesion rate of V. fluvialis to the skin mucus was remarkably enhanced after heating the mucus at 40, 60 and 80℃for 10 min, while the bacterial adhesion was extremely remarkably inhibited by heating the mucus at 100℃for 10 min; no remarkably effect on the bacterial adhesion was found after treating the mucus with protease K and trypsin, whereas the bacterial adhesion was extremely remarkably enhanced by treating the mucus with periodic acid; among 8 carbohydrates, glucose, mannose, mannitol and maltose could enhance the bacterial adhesion extremely remarkably, and other 3 carbohydrates enhanced the bacterial adhesion remarkably, while galactose could not enhance the bacterial adhesion remarkably; surface extract of V. fluvialis with high concentration could inhibit the bacterial adhesion remarkably, whereas the surface extract with relative low concentration enhanced the bacterial adhesion; 3 protein peaks were obtained by fractionating the mucus with a Sephadex G-100 column, and the strong adhesion was found to the first peak. The results indicated that: pathogenic V. fluvialis could adhere to the skin mucus of E. awoara strongly; there were specific adhesive receptors within the mucus; the adhesive receptors were likely to be macromolecules with relative stability to heating and protease.The expression of EGFP-V. fluvialis was constructed by using pEGFP-C1 plasmid as template. Full-length of EGFP cDNA was amplified by polymerase chain reaction. The products were transformed into E. coli Top10. The ecombinant plasmid was extracted for further identification and sequenced. EGFP cDNA fragment and linearized pET32a fragment were purified by low melting point agarose electrophoresis and ligated with Ligafast DNA ligation system. The resultant ligation product was transformed into E. coli BL21. Then pET32a-EGFP was extracted, purified and transformed into V. fluvialis. EGFP-V. fluvialis can express efficiently EGFP after continuous cultured for 10 generations without antibiotics.EGFP-V. fluvialis were introduced into the digestive tract of E. awoara by intragastic injection method for the investigations of bacterial colonization to the digestive tract. The results showed that labelled bacterium could be detected at 3h in machcontents, at 12h in forgut, at 24h in hindgut. No bacterium was detected at 72h and 96h in machcontents and hindgut, respectively. The results indicated that forgut and midgut were the main location of colonization. The results showed that the alkaline protease activites in forgut and midgut are higher than that in hindgut. The lipase activities in hindgut were higher than those in forgut and midgut. The amylase activity in forgut is higher than higher than those in midgut and hindgut.