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。     

Experimental infection of Mytilus edulis by two Vibrio splendidus-related strains: Determination of pathogenicity level of strains and influence of the origin and annual cycle of mussels on their sensitivity

This study aimed at assessing the pathogenicity of two Vibrio splendidus-related species and evaluating the influence of the origin and annual life cycle of mussels on their sensitivity during a bacterial challenge. Thus, in vivo infection assays were made with Vibrio crassostreae 7T4_12 and Vibrio splendidus 3G1_6, over, respectively, thirteen and 9 months, on adult blue mussels from five recruitment areas in France. Two bacterial concentrations were tested: one consistent with the loads of Vibrio spp. in environment and mussel tissues (~10⁵ CFU/ml) and another one much higher (~10⁸ CFU/ml). The tested environmental concentration has no pathogenic effect whatever the time of year, the strain used and the origin of mussels. However, at the highest concentration, a pathogenic effect was observed only at specific moments, and one of the origins appeared to be more resistant. The physiological state of mussels—depending on the time of year—seemed significant in mussels’ sensitivity, as their recruitment origin. This study is the first to test the pathogenicity of V. splendidus-related strains at concentrations close to what is found in the wild, over the annual cycle of mussels, and considering their origin.     

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。     

刺参感染细菌后免疫相关因子的表达研究

刺参是一种重要的极具经济价值的养殖棘皮动物.在应对免疫应激或环境压力时,NF-κB/Rel转录因子,作为关键调控子,在调控免疫相关基因的表达过程中发挥着重要作用.作为刺参体内天然免疫系统中的关键酶,溶菌酶在防御刺参病原菌入侵过程中也发挥了重要作用.该研究对刺参感染病原菌后NF-κB/Rel和溶菌酶mRNA的表达情况进行了研究.感染细菌后,Aj-rel mRNA的转录水平显著升高,在10 min时达到峰值,是对照组的3.26倍(P＜0.05).Aj-p105基因的表达水平也呈现了上升的趋势,在240 min时达到峰值,是对照组的6.12倍(P＜0.05).Aj-p50 mRNA的表达水平呈现了下降的趋势,在10 min时达到峰值,是对照组的17.80倍(P＜0.05).溶菌酶mRNA的表达水平分别在10 min和240 min时达到峰值,分别是对照组的1.54倍(P＜0.05)和2.48倍(P＜0.05).该研究将为进一步了解棘皮动物的免疫防御机制提供参考,为疾病控制提供启示.     

灿烂弧菌灭活菌苗对美国红鱼免疫效果的研究

制备灿烂弧菌(Vibrio splendidus)福尔马林灭活菌苗,通过浸泡、注射和口服3种方式接种美国红鱼(Sci-aenops ocellatus),分别在接种免疫后第7、14、21、28、35天采鱼血,检测其血清抗体效价、溶菌酶活力和白细胞吞噬活性;第28天用1.0×108CFU/mL的灿烂弧菌悬液进行攻毒试验,检测菌苗的免疫效果。结果表明,各免疫组血清抗体效价在第21天达到峰值,且浸泡组和注射组的血清抗体效价高于口服组;在35d实验期内,各免疫组的血清溶菌酶活力有明显提高,各浸泡组和注射组于免疫接种后14d显著高于对照组(P<0.05);除浸泡3组外,其余各免疫组的白细胞吞噬百分比(PP)和吞噬指数(PI)在免疫接种后14d均较对照组有显著差异(P<0.05);各组受免鱼对人工攻毒均具有保护作用,以注射2组的免疫保护率最高,达77.8%。     

刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析

microRNA参与基因的转录后调控,在真核生物的生长发育、细胞分化和免疫防御等过程中发挥重要作用。刺参(Apostichopus japonicus)病害问题已成为产业发展的主要限制因素之一,而其病害发生的分子机制尚待进一步完善。本研究以刺参重大疾病“腐皮综合征”的重要致病原灿烂弧菌(Vibrios splendidus)为侵染菌株,通过人工侵染实验制备患病刺参样本,采用miRNA-seq技术对侵染组(PT16S)和对照组(PT10H)各3头刺参的体壁组织进行miRNA测序,通过相关生物信息学软件对miRNAs进行鉴定和分析,筛选差异表达miRNAs (DEmiRNAs)并预测其靶基因,构建关键调控途径的miRNA-mRNA调控网络。结果显示,PT10H组平均得到5 902 588条有效序列,194个已知miRNA和19个新的miRNA;PT16S组平均得到5 053 529条有效序列,182个已知miRNA和42个新的miRNA。对2组鉴定到的miRNA进行差异表达分析,共筛选到2个上调和11个下调的具有显著差异的DEmiRNAs (P≤0.05),上调的DEmiRNAs靶基因预测结合到3010个靶基因,注释到585个GO terms及24条信号通路(P≤0.05),下调的DEmiRNAs靶基因预测到19 072个靶基因,注释到514个GO terms以及22条信号通路(P≤0.05)。对筛选到的DEmiRNAs进行实时荧光定量PCR (qRT-PCR)验证,显示miRNA-seq与qRT-PCR的一致率达到70%。根据KEGG分析结果构建泛素介导的蛋白水解途径和Notch信号通路的miRNA-mRNA调控网络,结果显示,13个DEmiRNAs分别靶向结合134个与泛素介导的蛋白水解相关的mRNAs和109个与Notch信号通路相关的mRNAs,Aja-miR-184、Aja-miR-2478和Aja-miR-9277p等DEmiRNAs可能参与对Notch信号通路和对泛素介导的蛋白水解的调控。相关研究结果将为刺参疾病发生调控网络建立和机制解析提供依据。     

基于gyrB基因的SYBR Green I实时定量PCR检测病原灿烂弧菌

灿烂弧菌Vibrio splendidus是多数海水养殖动物的主要致病菌,对养殖业危害较大。本研究根据灿烂弧菌gyrB基因的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测灿烂弧菌的方法。构建含gyrB基因的重组质粒作为标准品,进行SYBR Green I实时定量PCR,在Tm为62℃时,扩增产物的熔解曲线仅有一个单特异峰,扩增所得标准曲线为y=-3.338x+37.67,相关系数为0.999,扩增效率为0.99,最低能检测到20个拷贝。实验结果表明,该检测技术具有较高的特异性、敏感性和重复性,对灿烂弧菌病的快速诊断和流行病学调查有重要意义。     

Effects of dietary supplementation of A3α‐peptidoglycan on the growth,immune response and defence of sea cucumber Apostichopus japonicus

To determine the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the growth, immune response and disease resistance of sea cucumber Apostichopus japonicus, a 70‐day feeding trial was conducted in this study. A total of 216 sea cucumbers were fed with four practical diets prepared from a commercial feed with different contents (0, 1.5, 2.5 and 4.0 g kg^?1) of A3α‐PG. The specific growth rate (SGR), total coelomocyte count (TCC), phagocytotic activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extracellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), peroxidase (POD) and superoxide dismutase (SOD), were measured at the end of the feeding trial. Finally, the animals were administered a 16‐day Vibrio splendidus challenge via intraperitoneal injection to test the potency of A3α‐PG on disease resistance. Compared with the control (0 g kg^?1 A3α‐PG), a significant increase (P < 0.05) in SGR was observed in the groups fed with 1.5 and 2.5 g kg^?1 A3a‐PG. The TCC, ranging from 7.25 × 10⁶ to 1.05 × 10⁷ cells mL^?1, was not significantly affected (P > 0.05) by A3α‐PG,. Coelomocyte phagocytotic activities in all of the A3α‐PG‐supplemented groups were significantly activated (P < 0.05), but no significant difference (P > 0.05) was observed. Sea cucumbers fed with 1.5 and 2.5 g kg^?1 A3α‐PG exhibited significant activation (P < 0.05) of EC/IC‐ACP, EC/IC‐ALP, and EC/IC‐POD activities. A significant increase in EC‐SOD activities (P < 0.05) was exhibited by all groups with A3α‐PG supplementation. The challenge test showed that animals fed with diets containing 2.5 and 4.0 g kg^?1 A3α‐PG had significantly lower cumulative mortalities compared with the control 16 days after exposure. All of the results presented here show that A3α‐PG can positively enhance the growth, immune response and disease resistance of sea cucumber, suggesting that dietary supplementation of A3α‐PG has potential applications in the health management of economic species of sea cucumber.     

单原子层氮化碳在可见光下对灿烂弧菌的灭菌效果研究

为了在可见光下实现对海水中灿烂弧菌Vibrio splendidus的光催化灭菌,通过煅烧和超声剥离相结合的方法制备了一种新型的非金属单原子层氮化碳(SL g-C3N4)光催化剂,在可见光下对海水中灿烂弧菌进行了光催化灭菌研究。结果表明:制备的SL g-C3N4厚度约为0.5 nm,其吸收边带位于420 nm左右;当SL g-C3N4用量为20 mg/L,海水中灿烂弧菌的初始浓度为1×107cfu/m L时,经过90 min可见光照射,与对照组相比海水中灿烂弧菌的数量降低了约2.3个lg单位。研究表明,SL g-C3N4在可见光下能够实现光催化杀灭海水中的灿烂弧菌。     

麻保沙星对主要海洋致病性弧菌的体外抗菌活性及抗菌后效应

采用牛津杯法和试管二倍稀释法分别测定麻保沙星对溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(V.harveyi)、费氏弧菌(V.fischeri)及灿烂弧菌(V.splendidus)的敏感性、最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并与盐酸二氟沙星、盐酸恩诺沙星、诺氟沙星及氟苯尼考进行比较;采用菌落计数法测定麻保沙星对溶藻弧菌、哈维氏弧菌和灿烂弧菌的抗菌后效应(PAE)。结果表明,麻保沙星对4种弧菌的体外抗菌活性与氟苯尼考相近,高于盐酸二氟沙星、盐酸恩诺沙星和诺氟沙星;麻保沙星在1×MIC、2×MIC和4×MIC浓度时,对溶藻弧菌、哈维氏弧菌和灿烂弧菌的PAE值分别为:0.49h、0.87h和1.17h;0.64h、0.98h和1.22h;0.75h、1.02h和1.25h,表明麻保沙星对溶藻弧菌、哈维氏弧菌和灿烂弧菌均有不同程度的抗菌后效应,且PAE值与药物浓度呈正相关。