Influence of environmental factors on the adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea

研究了不同环境条件下溶藻弧菌对大黄鱼肠粘液的粘附作用。采用细菌计数法测定溶藻弧菌的粘附作用。粘附的细菌经SYBR GreenⅠ染色后在荧光显微镜下观察,用数码相机拍照后在电脑上计数。试验结果表明,溶藻弧菌对大黄鱼肠粘液的粘附量随菌浓度的升高而升高,并在1~1.5h内趋于饱和;粘附作用在15~30℃、pH偏酸时较强;盐度在5~35范围内对前肠粘液的粘附作用影响不明显,后肠粘液的粘附作用在此范围内随盐度增大而加强,在盐度为0时,溶藻弧菌对前、后肠粘液都无粘附作用;56℃热处理5min及60℃处理1h均能大幅减弱溶藻弧菌对两种肠粘液的粘附作用,表明溶藻弧菌表面的某些热敏结构在粘附作用中起着重要作用。根据以上结果,可以认为溶藻弧菌能够很好地粘附于大黄鱼肠粘液层,其粘附作用受温度、盐度、pH值等环境因子影响很大,溶藻弧菌表面的某些热敏结构可能在粘附过程中起着重要作用。研究结果表明,溶藻弧菌对大黄鱼肠粘液的粘附作用是可控制的,这对于鱼类养殖疾病的控制有一定的参考价值。The mucus layer is the most outer defence barrier of fish.The bacterial attachment to mucus layer is a critical step in the host colonization and subsequent occurrence of infection.Adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea has been investigated in order to understand the characteristics and mechanisms involved in the adherence of bacteria onto fish mucus so that preventive measures can be devised to reduce infection.In vitro adhesion assay was carried out by bacterial counting.The adhesive bacteria were stained with SYBR Green Ⅰ,observed under fluorescent microscope,counted on computer display after taking pictures.The results showed that the adhesive quantity of V.alginolyticus increases with bacterial concentrations and reached equilibrium after being incubated 1-1.5 h;the higher adhesive quantity was achieved at 15-30 ℃ and acid condition;adhesion of V.alginolyticus to the mucus of foregut was not obvious variety with salinity(5-35),but adhesive quantity to the hindgut mucus enhances while salinity increases;V.alginolyticus could not adhere to intestinal mucus without salinity;adhesive quantity reduced remarkably after heat treatment of the germ(56 ℃ 5 min,60 ℃ 1 h),which indicated that heat sensitive structure of V.alginolyticus might play an important role in adhesion.The conclusions of this paper showed V.alginolyticus could adhere to intestinal mucus of P.crocea facilely in seawater;this adhesion was remarkably affected by environmental factors,such as temperature,pH and salinity;V.alginolyticus adhered to the intestinal mucus of P.crocea by specific adhesion,some heat-sensitive structures on V.alginolyticus surface played a crucial role in adherence of the bacteria to gill mucus.The results indicated that the bacterial adhesion of V.alginolyticus to intestinal mucus of P.crocea was governable.The results obtained are useful for the epidemic control in fish culture.国家863计划项目(2002AA639600);; 福建省自然科学基金(B0410022);; 福建省青年创新基金(2002J037

Characteristics of adhesion of Vibrio alginolyticus to the skin mucus of Pseudosciaena crocea

采用3H-TdR同位素示踪方法研究了溶藻弧菌对大黄鱼表皮黏液的黏附机制.试验结果表明溶藻弧菌经营养饥饿、热处理、抗体处理、蛋白酶及高碘酸处理后黏附作用明显下降;在8种碳水化合物的干扰下溶藻弧菌的黏附作用会有一定程度的变化,其中葡萄糖、果糖、甘露糖能明显促进溶藻弧菌的黏附作用;溶藻弧菌对大黄鱼表皮黏液中较大分子量的物质有较强的亲和力.这些结果表明溶藻弧菌对大黄鱼表皮黏液存在特异性黏附作用,这种黏附与细菌活力、其表面的蛋白类、糖类等物质以及黏液的化学组成都有密切关系.Characteristics of adhesion of Vibrio alginolyticus to the skin mucus of Pseudosciaena crocea had been investigated by isotope tracer.The results showed that: adhesion of V.alginolyticus was inhibited evidently by starvation,heat treatment,antibody,protease,periodic acid;bacterial adhesion was influenced by 8 carbohydrates,among them,glucose,fructose,mannose could enhance the adhesion of V.alginolyticus;V.alginolyticus has a strong affinity to higher molecular mass component of skin mucus.The results indicated that V.alginolyticus adhered to the skin mucus of P.crocea by specific adhesion,this adhesion had closely relation to superficial protein,saccharide and activity of the bacteria as well as the chemical component of skin mucus.国家863计划项目(2002AA639600);; 福建省自然科学基金(B0410022

ENVIRONMENTAL INFLUENCE ON ADHESION OF VIBRIO ALGINOLYTICUS TO SKIN MUCUS OF PSEUDOSCIAENA CROCEA

采用3H-TdR同位素示踪方法研究了环境因子对溶藻弧菌对大黄鱼表皮粘液粘附作用的影响。试验结果表明,溶藻弧菌能很好地粘附于大黄鱼表皮粘液,其粘附量在菌浓度不超过6.52×108cfu/ml情况下随菌浓度的升高而升高;粘附量在25℃下孵育180min趋于饱和,在180min以内与孵育时间呈正相关关系;粘附作用在温度25—30℃、pH值偏酸、盐度35条件下较强;在无Na+(盐度为0)时,无粘附作用;Ca2+能显著加强溶藻弧菌的粘附作用,而Mg2+作用不明显。这些结果表明,溶藻弧菌对大黄鱼表皮粘液有较强的粘附作用,其粘附作用受温度、盐度、pH值等环境因子的影响。Fish pathogens can colonize and infect the host by attaching bacteria onto host's mucus layer. Study on the attachment and adhesion of a pathogen is important to prevent the host from infection. In this paper, we study the case of pathogen Vibrio alginolyticus and host Pseudosciaena crocea (large yellow croaker) as an example for better understanding of adhesion of the pathogen to the skin mucus of the host. The mucus samples are extracted from P. crocea with protein concentration adjusted at 1mg/ml, using microtiter plate and methyl-3H thymidine as the isotope tracer. Our results show that the quantity of V. alginolyticus adhesion increases with bacteria concentrations at the equilibrium 180min post-incubation. However, quantity does not reach an equilibrium point when the bacterial concentration ranges from 0.044 to 1.11 (OD550) 90min after incubation at 25℃; incubation temperature is one of the main factors that affect the bacterial adhesion. V. alginolyticus shows stronger adhesion in warm condition reaching the maximum at 25℃. On the other aspect, pH value affects the bacteria adhe-sion remarkably. The adhesion is remarkably stronger at pH 3---5 than at pH 7---9, peaked at pH 6. The adhesion fluctuates in seawater with different salinities optimized at 3.5%. No specific adhesion of V. alginolyticus to skin mucus is exhibited in Na+-free solution. Existence of Ca2+ but Mg2+ can obviously promotes the adhesion. All the results suggest that the pathogenic V. alginolyticus can adhere to the skin mucus of P. crocea easily in seawater; however, environmental factors of temperature, pH and salinity can influence the adhesion effectively. The optimal condition is 25-30℃, pH about 6, and 3.5% in salinity.国家863计划项目资助,2001AA635070号、2002AA639600号;; 福建省自然科学基金资助,B0410022号