间接ELISA法研究嗜水气单胞菌对鳗鲡表皮黏液的黏附特性

摘要

采用间接 ELISA 法研究菌浓度、孵育时间、温度、pH、阳离子及碳源等因子对嗜水气单胞菌(Aeromonas hydrophila)黏附鳗鲡(Anguilla anguilla)表皮黏液的影响。结果表明,改良后的间接 ELISA 法的检测灵敏度约为9.92×104 CFU,细菌的黏附量随菌浓度的升高而逐渐增大并符合饱和黏附动力学方程： y=0.135ln(x)-0.936(R2=0.986)；嗜水气单胞菌黏附鳗鲡表皮黏液的最佳条件为：温度20~28℃, pH 6.2~6.6, NaCl、MgCl2质量浓度分别为15~25 g/L和3 g/L,孵育时间为150 min。碳源对嗜水气单胞菌的黏附作用有不同程度的影响,葡萄糖和麦芽糖能显著提高嗜水气单胞菌的黏附量(P<0.05),果糖则显著降低嗜水气单胞菌的黏附量(P<0.05)。以上结果说明,嗜水气单胞菌对鳗鲡表皮黏液具有较强的黏附作用,且其黏附作用具有可控性。%Adhesion to the host mucus, which is closely related to the virulence of pathogenic bacteria, is the first crucial step in most infections. In this paper, the adhesion characteristics of Aeromonas hydrophila to eel(Anguilla anguilla) skin mucus was investigated by indirect ELISA. First, we confirmed that OD492 can be used as an index to reflect the number of adhering bacteria. Second, we confirmed that the indirect ELISA method can detect bac-teria with a sensitivity of 9.92×104CFU. The number of adhering A. hydrophila on eel mucus rose with increased bacterial density;a saturation kinetics equation y=0.135 n(x)?0.936(R2=0.986) was obtained by plotting the number of adhering bacteria against cell density. The factors affecting bacterial adhesion were then investigated. The re-sults revealed that the number of adhering A. hydrophila increased with incubation time and reached a peak after 150 min. Temperature was one of the major factors affecting bacterial adhesion, with peak adhesion occurring at 20?28℃. pH also influenced bacterial adhesion. In a weak acid with a pH of 6.2?6.6, the adhesion ability of A. hydrophila was remarkably enhanced. The optimum concentrations of Na+and Mg2+ for A. hydrophila adhesion were 1.5?2.5 g/L and 3 g/L, respectively. In summary, the optimum conditions for the adhesion of A. hydrophila to eel skin mucuswere 20?28℃, pH 6.2?6.6, 1.5?2.5 g/L NaCl, 0.3 g/L MgCl2, and 150 min incubation time. In ad-dition, we found that bacterial adhesion was influenced by carbohydrates. Seven kinds of carbohydrates were investigated and the results indicate that glucose and maltose significantly enhance A. hydrophila adhesion (P<0.05), while fructose inhibited it (P<0.05). Understanding the mechanisms of A. hydrophila adhesion will help to further elucidate bacterial pathogenicity and help control fish disease.