Vaccination of Silver Sea Bream (Sparus sarba) against Vibrio alginolyticus: Protective Evaluation of Different Vaccinating Modalities

In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.p.) injections exhibited the best protection, and the RPS values were 100 or 85 upon higher or lower doses of pathogenic V. alginolyticus challenges. Both oral vaccination and a combination of injection/immersion trial were also effective, which achieved relatively high protection (the RPS values ranged from 45 to 64.3). However, two hyperosmotic immersions could not confer satisfactory protection, especially when fish were exposed to the severe pathogenic bacteria challenge. Marked elevations of serum agglutinating antibody titer were detected in all immunized fish. Macrophage phagocytosis was enhanced significantly, especially in the fish immunized by formalin- and phenol-killed bacterins through various administration routes. Both adaptive (specific antibody) and innate (phagocytic activity) immunity elicited by different immunization strategies were in parallel with the degree of protection offered by each of them. Although all vaccination trials had no significant effect on the serum hematocrit and hemoglobin levels, the circulating lymphocyte counts were significantly elevated in the fish immunized with LPS, formalin- and phenol-killed bacterins. Serum cortisol levels appeared to be reduced in all immunized fish except the trial of hyperosmotic immersion, which indicated the stressful impact on vaccinated fish.     

Some Ozone Applications in Seafood

The use of ozonized seawater to reduce and eliminate bacterial pathogens in mariculture facilities and to extend shelf life of marine food products is demonstrated. Consequent benefits of this treatment are also discussed. Laboratory and pilot experiments were conducted using ozone gas to reduce disease-producing Vibrio sp. bacteria at a shrimp (Litopenaeus vannameii) hatchery in Ecuador, South America. Pacific Ocean seawater was treated in a 1,540 L capacity fiberglass contact tower (5–7 min retention) with an ozone oxidant residual of 0.07 mg/L. Prior to ozone treatment, Vibrio determined by TCBS plating was too numerous to count, causing shrimp to die of disease (30 tanks of 13,000 L each). After treatment, Vibrio counts and shrimp disease were eliminated, ozonized seawater decreased the time required for normal molting of shrimp and the total growth cycle was reduced by three days versus control water. From June 1991 until September 1992, survival rates of larval shrimp were robust, routine antibiotic addition was reduced, and one additional growth cycle was realized. Ozonized ice (fresh water) was prepared in the Milford Laboratory CT, USA; Gloucester Food Tech Lab, MA, USA; and a field station (brine water) for sockeye salmon (Oncorhynchus nerka) in Homer, AK. USA. In these studies, squid (Loligo pealei) and commercially captured salmon demonstrated a reduction in spoilage and extension of shelf life of 3 to 5 days' time using ozonized ice. Bacteria associated with commercial ice-producing machines were reduced by 4 logs using ozone treatment. In addition, no flavor aberration was noted using ozonized ice.     

Decoupled Roles for the Atypical,Bifurcated Binding Pocket of the ybfF Hydrolase

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate‐binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc‐ybfF) by using a 21‐member library of fluorogenic ester substrates. We assigned the roles of the two substrate‐binding clefts in controlling the substrate selectivity and folded stability of Vc‐ybfF by comprehensive substitution analysis. The overall substrate preference of Vc‐ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc‐ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc‐ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc‐ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.     

PCR法制备地高辛标记探针斑点杂交检测鳗弧菌

以鳗弧菌(Vibrio anguillarum)toxR基因内366~571 bp之间的基因序列为靶序列,采用PCR法制备对鳗弧菌特异性的地高辛标记DNA探针,探针长度为206 bp。选取2株鳗弧菌和8株与鳗弧菌亲缘关系非常接近的常见水产动物致病弧菌,通过斑点杂交法对制备的探针进行特异性检测,结果显示该探针对鳗弧菌杂交阳性,而与弧菌属的其它8株细菌均无交叉反应。这表明该地高辛标记探针具有很强的特异性,它能够很好地对鳗弧菌感染进行检测。     

生食三文鱼片副溶血性弧菌污染的定量风险评估研究

目的评估上海市生食三文鱼片副溶血性弧菌污染的风险。方法按WHO/FAO危害识别、危害特征描述、暴露评估和风险特征描述4个步骤进行了副溶血性弧菌风险评估,危害识别、危害特征描述资料收集自公开发表的科学文献、报告,暴露评估、风险特征描述应用了监测、膳食调查、生长模型,调整因子、Beta-Poisson剂量反应模型、蒙特卡洛模拟及统计方法。结果在上海市生食被副溶血性弧菌污染的三文鱼片致病的发病概率1—2月、3—5月、6—8月、9—11月分别为8.00×10-7、6.23×10-7、4.14×10-6、8.71×10-6,敏感度分析显示6—11月生食三文鱼中的副溶血性弧菌的污染量对发病概率影响最大。结论上海市生食三文鱼片副溶血性弧菌污染存在比较小的健康风险,夏、秋季控制生食三文鱼片中的副溶血性弧菌的污染量是减少健康风险的关键。     

响应面法优化溶藻弧菌的培养条件

通过单因素实验确定pH、温度、盐度三个因素下溶藻弧菌的最优培养条件。根据Box-Behnken中心组合实验原理进行响应面分析,以菌液的OD600为响应值,确定影响溶藻弧菌培养的各因素水平。结果表明,最优培养条件为pH7.62、温度34.55℃、盐度3.22%;菌体生长过程中pH和温度、温度和盐度对V.a ATCC17749生长的交互作用显著。