杂色蛤中副溶血弧菌的定量风险评估

基于国际食品法典委员会关于微生物定量风险评估的理论框架,研究了从水产品批发市场到各零售点（主要为超市）杂色蛤中副溶血弧菌的动态变化情况,对因食用杂色蛤感染副溶血弧菌而引发疾病的风险进行预测,预测出每年、每人因消费生杂色蛤而导致由副溶血弧菌引发的食源性疾病的可能平均值被估计为1.65×10-8.同时对杂色蛤的另一种食用模式——烧烤,也进行了分析研究,确定因其所导致的风险十分小,可以不作为评估的对象.最后根据研究的结果提出针对政府监管部门的管理以及商业运营者和消费者的建议,这些风险管理措施、控制程序和安全食用规范的实施,均可大大降低因食用杂色蛤而感染副溶血弧菌并引发疾病的风险.     

PCR-核酸薄膜层析法检测创伤弧菌

采用自行建立和优化的PCR-核酸薄膜层析检测体系,对创伤弧菌(Vibriovulnificus)进行检测。采用创伤弧菌的vvhA基因为目的片段设计特异性引物,建成可快速检测创伤弧菌的PCR-核酸薄膜层析检测法,并进行了特异性和灵敏度试验。结果表明,所建立起的检测法灵敏度为3.6×102 cfu·mL-1,比PCR-琼脂糖凝胶电泳检测方法高一个数量级。创伤弧菌的PCR-核酸薄膜层析检测方法具有较高灵敏度和良好特异性,操作简单,检测速度快,同时又保护了操作人员的身体健康,具有广阔的推广前景。     

中国明对虾凝血栓蛋白基因全长cDNA的克隆及其表达特征

获得了中国明对虾凝血栓蛋白(TSP)基因全长cDNA,该基因由2868个碱基组成,具有一个2763bp的开放阅读框,编码920个氨基酸,其中包含21个氨基酸组成的信号肽.中国明对虾TSP由四类不同的结构域组成,从N端开始顺序为几丁质结合结构域,EGF-like结构域,Type Ⅲ repeat和TSP carboxyl-terminal domain(TSP-C结构域).结构域分析发现,中国明对虾的N端不具有TSP N-terminal domain(TSPN结构域),但有一个几丁质结合结构域,其C端的Type Ⅲ和TSP-C结构域非常保守.该基因在弧菌感染后的对虾淋巴器官和肝胰脏中的表达量显著增加,并具有不同的时空表达趋势,提示中国明对虾TSP基因在免疫反应中具有重要作用.     

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.     

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.     

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.     

Toxicity to Daphnia magna and Vibrio fischeri of Kraft bleach plant effluents treated by catalytic wet-air oxidation

Two Kraft-pulp bleaching effluents from a sequence of treatments which include chlorine dioxide and caustic soda were treated by catalytic wet-air oxidation (CWAO) at T=463 K in trickle-bed and batch-recycle reactors packed with either TiO2 extrudates or Ru(3 wt%)/TiO2 catalyst. Chemical analyses (TOC removal, color, HPLC) and bioassays (48-h and 30-min acute toxicity tests using Daphnia magna and Vibrio fischeri, respectively) were used to get information about the toxicity impact of the starting effluents and of the treated solutions. Under the operating conditions, complex organic compounds are mostly oxidized into carbon dioxide and water, along with short-chain carboxylic acids. Bioassays were found as a complement to chemical analyses for ensuring the toxicological impact on the ecosystem. In spite of a large decrease of TOC, the solutions of end products were all more toxic to Daphnia magna than the starting effluents by factors ranging from 2 to 33. This observation is attributed to the synergistic effects of acetic acid and salts present in the solutions. On the other hand, toxicity reduction with respect to Vibrio fischeri was achieved: detoxification factors greater than unity were measured for end-product solutions treated in the presence of the Ru(3 wt%)/TiO2 catalyst, suggesting the absence of cumulative effect for this bacteria, or a lower sensitivity to the organic acids and salts. Bleach plant effluents treated by the CWAO process over the Ru/TiO2 catalyst were completely biodegradable.     

发光细菌DH132的筛选、鉴定及其在氯霉素生物毒性测定中的应用

利用药敏纸片法,从本实验室保存的发光细菌中筛选出一株对氯霉素敏感的细菌DH132。通过生理生化特征鉴定并利用16SrDNA序列分析的方法,鉴定细菌DH132为哈维氏弧菌。通过在细菌培养液中添加不同浓度的氯霉素溶液,研究了氯霉素浓度与细菌发光强度抑制率之间的关系。结果表明,当氯霉素浓度为80ng/mL和160ng/mL时,对细菌发光的抑制率分别达到10%和50%。氯霉素浓度在0~60ng/mL,60~140ng/mL,140ng/mL~200ng/mL和200~1000ng/mL之间,细菌发光抑制率与氯霉素浓度分别具有很好的线性关系。相对应的回归方程分别为y=0.0627x-0.0233;y=0.3599x-18.276;y=0.7527x-70.219;y=0.0195x 75.964,回归曲线R2值分别为0.9805,0.9918,0.9502和0.9731。