灿烂弧菌和创伤弧菌的不同保存方法比较

比较了琼脂斜面、穿刺石蜡保存法和甘油冷冻冻存法保存灿烂弧菌和创伤弧菌的效果。结果表明,甘油冷冻冻存法和穿刺石蜡保存法的保存期显著比琼脂斜面法长,保存12月后各保存方法细菌的毒力都有不同程度的下降,甘油冷冻冻存法显著好于其它2种保存方法。     

Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.     

鳗弧菌L-18株的限铁条件和铁调节外膜蛋白的免疫效果研究

利用Western-blot技术,对鳗弧菌L-18株的OMPs和IROMPs的免疫反应性进行了分析,并通过免疫牙鲆比较二者免疫保护力的差异,以期从IROMPs方向探索开发鳗弧菌新型鱼用疫苗,并为研究其免疫原理提供重要的科学依据。结果表明,培养基中加入不同浓度的联铁剂,鳗弧菌L-18株的生长和铁载体的表达出现规律性变化,其中加入100~130μmol·L-1 2,2’-联吡啶为最适合限铁条件,此时铁载体的表达量最高且生长受抑制程度较小。在此条件下,菌株表达出74.3ku和77.4ku的IROMPs,其中77.4ku蛋白具有免疫反应性,为重要抗原。用全菌灭活疫苗、OMPs、IROMPs分别腹腔注射免疫牙鲆7周后攻毒, IROMPs疫苗的相对免疫保护力达到75.9%,远高于OMPs疫苗的51.8%。相对于OMPs,IROMPs的抗体效价显著提高,接近全菌疫苗组的水平,而血清杀菌活力没有显著变化。 关键词：鳗弧菌;铁调节外膜蛋白;牙鲆     

Surface-exposed expression of Edwardsiella tarda EseB in live attenuated Vibrio anguillarum based on novel surface display systems

Live, attenuated Vibrio anguillarum strains can serve as vectors for the delivery of heterologous antigens for development of multivalent recombinant vaccines. Based on the outer membrane anchoring elements of V. anguillarum , we have previously constructed several efficient surface display systems Lpp-Omporf1, Lpp-OmpU, Lpp-Omp26La, Wza-Omporf1, Wza-OmpU and Wza-Omp26La. In this study, with these constructed surface display systems, a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of an attenuated V. anguillarum strain to get multivalent vaccine candidates. Further immune protection evaluation in zebra fish ( Danio rerio ) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger full protection against V. anguillarum infection and early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.     

凡纳滨对虾ARMC8基因的克隆及表达

为了研究含Armadillo重复蛋白8在凡纳滨对虾抗病感染过程中所起的免疫作用,本实验采用RACE-PCR技术首次克隆得到凡纳滨对虾ARMC8基因(LvARMC8,Gen Bank注册号:KX058562)的c DNA序列全长,并利用在线软件进行生物信息学分析;采用实时荧光定量PCR(Rt-PCR)技术对LvARMC8基因在凡纳滨对虾不同组织及感染白斑综合征病毒和副溶血弧菌过程中的表达变化特征进行分析。结果显示,LvARMC8基因全长为2917bp,其中开放阅读框长2046 bp,编码681个氨基酸,5′非编码区长49 bp,3′非编码区长822 bp。预测分析显示LvARMC8编码的蛋白质含有6个ARM结构域。同源性分析发现,LvARMC8基因与内华达古白蚁ARMC8基因的相似度最高,为71%。系统进化分析结果显示,LvARMC8和多种无脊椎动物ARMC8聚为一支,其中与昆虫类动物赤拟谷盗、致倦库蚊和柑橘凤蝶的亲缘关系最近。Rt-PCR分析发现,LvARMC8基因在凡纳滨对虾多个组织中均能检测出,在表皮中表达量最高,眼柄中表达量最低。WSSV感染后12 h,LvARMC8基因在凡纳滨对虾血液中的表达量显著降低,但48 h后显著升高,72 h达到最高水平。除副溶血弧菌感染后24 h外的时间点,LvARMC8基因在凡纳滨对虾血液中的表达量均显著升高。研究表明,LvARMC8基因可能参与凡纳滨对虾抗病免疫应答途径。     

养殖大菱鲆肠道中大菱鲆弧菌的分离鉴定及药敏试验

2014年11月,大连地区某大菱鲆养殖场发生病害并伴有死亡,主要症状为吻部下颌明显出血,腹腔积水,肝脏充血,肠道出血有白便。从患病大菱鲆肠道中分离出1株可在硫代硫酸盐—柠檬酸盐—胆盐—蔗糖琼脂培养基上生长的菌株HZ-C1,人工回接感染试验证明其对健康大菱鲆具有较强致病性。通过形态学观察、16SrDNA序列同源性分析及生理生化试验确定菌株HZ-C1为大菱鲆弧菌。药敏检测结果显示,大菱鲆弧菌HZ-C1对氯霉素、丁胺卡那及头孢曲松等11种抗生素敏感,但对青霉素、四环素及诺氟沙星等15种抗生素耐药。本研究为大连地区养殖大菱鲆细菌性病害防治提供了一定病原学依据。     

两种弧菌对菲律宾蛤仔代谢途径影响的比较研究

采用基于核磁共振波谱技术的代谢组学技术,探索菲律宾蛤仔在受到鳗弧菌和灿烂弧菌感染的代谢物变化特征,构建菲律宾蛤仔对弧菌感染后的代谢网络调控图谱,并比较两种弧菌毒性效应的差异。试验结果表明,3种代谢物葡萄糖、谷氨酸、苏氨酸在两种弧菌感染时均发生了变化,表征鳗弧菌感染的代谢物为牛磺酸、精氨酸、酪氨酸、苯丙氨酸、赖氨酸、亮氨酸、异亮氨酸、缬氨酸;而表征灿烂弧菌污染的代谢物为甜菜碱、二甲基甘氨酸、胆碱、谷氨酸、亚牛磺酸。     

Impact of farm management on expression of early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) on penaeid shrimp farms in Thailand

Asian shrimp farming industry has experienced massive production losses due to a disease caused by toxins of Vibrio bacteria, known as early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) for the last 5 years. The disease can cause up to 100% cumulative pond mortality within a week. The objective of this study was to identify factors associated with AHPND occurrence on shrimp farms. A case–control study was carried out on shrimp farms in four provinces of Thailand. Factors related to farm characteristics, farm management, pond and water preparation, feed management, post‐larvae (PL) shrimp and stock management were evaluated. Multivariable logistic regression analysis identified factors affecting AHPND occurrence at the pond level. Chlorine treatment, reservoir availability, use of predator fish in the water preparation, culture of multiple shrimp species in one farm and increased PL stocking density contributed to an increased risk of AHPND infection, while delayed first day of feeding, polyculture and water ageing were likely to promote outbreak protection. Additionally, the source of PL was found to be associated with AHPND occurrence in shrimp ponds, which requires further study at the hatchery level. Identification of these factors will facilitate the development of effective control strategies for AHPND on shrimp farms.     

Soybean molasses as an organic carbon source in the farming of Litopenaeus vannamei (Boone, 1931) in a biofloc system

Soybean molasses was evaluated as a partial replacement for sugarcane molasses as a carbon source for biofloc development in the superintensive culture of Pacific white shrimp (Litopenaeus vannamei). A 50‐day study was conducted with juvenile (3.2 g) shrimp stocked in 16 800 L tanks at a stocking density of 250 shrimp m^?3. Control of total ammonia concentration was performed by the addition of combined mixtures of soybean and sugarcane molasses to the culture water. Three different molasses treatments were evaluated using different soybean‐to‐sugarcane molasses ratios: 15–85%, 38–62% and 60–40% respectively. The control group was treated only with sugarcane molasses. Water quality, chlorophyll a concentration, heterotrophic bacterial load, Vibrio spp. concentration and zootechnical indexes were all evaluated. Total ammonia concentration was controlled by heterotrophic and chemotrophic pathways. Biofloc formation, as quantified by measuring the total suspended solids, was not altered. The Vibrio spp. concentration showed a significant reduction in treatments with soybean‐to‐sugarcane molasses ratios of 38–62% and 60–40%. All combined mixtures of soybean and sugarcane molasses could maintain water quality and productivity in the superintensive culture of L. vannamei using the biofloc system. Thus, the potential use of a residue from agroindustry as a carbon source in a biofloc culture is demonstrated.     

哈维氏弧菌Y91301菌株培养工艺的研究

用大黄鱼分离的哈维氏弧菌Y91301进行试验,研究接种浓度、培养瓶装液量、起始pH值、培养时间、培养温度和培养液配方等因素对菌株生长的影响,确定静置培养时最适条件为:接种母液浓度1×108～1×109cfu ml,接种量6%～8%(相对培养瓶装液量),装液量40～50ml 250ml,培养液初始pH值为6 0～6 5,培养温度28℃;使用Zobell2216E培养配方更适合大量培养和生产的需要;旋转培养能够获得更高的生物量。