南宁市售文蛤中弧菌的分离鉴定

【目的】对从南宁市售文蛤体内分离获得的待测菌株进行鉴定,为掌握了解广西文蛤产品中弧菌科细菌的带菌情况提供参考依据。【方法】用硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基（TCBS）从南宁海鲜市场的文蛤中分离获得4株细菌,观察其菌落形态,对分离菌株进行生理生化特性鉴定及16SrRNA基因序列进行克隆、测序,运用DNASTAR中的MegAlign软件进行序列分析,并构建系统进化树。【结果】TCBS培养基30℃培养24h后,菌株No．8和No．9生长良好,形成直径2mm左右、圆形、隆起的菌落;菌株No．10、No．11生长不良,部分形成直径1mm左右、圆形、隆起的黄色菌落。菌株生化鉴定结果表明,菌株No．8、No．9在无盐蛋白胨水中不生长,且不能分解葡萄糖;而菌株No．10、No．11可在无盐蛋白胨水中生长,且能分解葡萄糖并产气。16SrRNA序列系统进化树显示,菌株No．8与副溶血弧菌聚集为一个分支,No．9与河流弧菌聚集为一个分支,No．10、No．11则分别与斑点气单胞菌、嗜水气单胞菌聚集为一个分支。综合表型和分子特征可知,菌株No．8为副溶血弧菌,No．9为河流弧菌,No．10为斑点气单胞菌、No．11为嗜水气单胞菌。细菌药敏结果显示,4株分离菌株对四环素、环丙沙星、先锋噻肟、先锋必均表现为高度敏感。【结论】南宁市售文蛤产品主要是污染了弧菌属及气单胞菌属细菌,生产上可选用四环素、环丙沙星、先锋噻肟、先锋必等药物进行防治。     

高位虾池水体细菌和弧菌的数量变化及影响因素

研究了位于广东湛江东海岛北寮村和庵里村的4个凡纳滨对虾（Penaeus vannamei）高位池养殖过程水体中的可培养异养细菌和弧菌的数量变化及其影响因素.结果表明：1）北寮村虾池BL1和BL2的可培养异养细菌数量变化均呈现中间高两头低、后期多于初期的单峰变化趋势;BL1的弧菌数量变化趋势和可培养异养细菌的变化趋势相似,而BL2则是前期稍微升高而后下降,后期又呈升高态势,峰值出现在养殖末期;水源地BL3的可培养异养细菌数量变化为初期平缓,后期明显多于初期,大体呈上升态势;而BL3弧菌数量变化趋势则比较平缓.2）庵里村发病的虾池AL1的可培养异养细菌和弧菌数量呈现出单峰变化趋势,在养殖中途爆发病害时,可培养异养细菌数量高出未发病的正常池2个数量级;庵里村虾池AL2的可培养异养细菌数量变化趋势也是中间高两头低,而弧菌的数量变化则是无规律的波动;水源地AL3的可培养异养细菌数量变化大体呈下降趋势,弧菌数量放养初期最高.3）高位虾池的可培养异养细菌和弧菌数量虽多于水源地,但差异不显著,同一个位点,细菌和弧菌数量呈现同步增多或减少.虾场水体的可培养异养细菌和弧菌数量与多数环境因子之间相关性不显著,这与虾池养殖生态系统受人为因素影响较大有关,同时反映了高位虾池生态系统受多种因子作用的复杂性.     

大珠母贝组织蛋白酶D的cDNA克隆、序列特征分析及应激表达研究

通过同源克隆方法和cDNA末端快速扩增技术(RACE),获得了大珠母贝(Pinctada maxima)组织蛋白酶D基因(命名为pmCTSD)的cDNA全长序列1 742 bp,其中5'非翻译区(UTR)38 bp,3'UTR 534 bp,开放阅读框1 170 bp,编码390个氨基酸,分子量约为41.9 ku,等电...     

Characterization of two bacteriophages for specific treatment of biofilm formed by a Vibrio sp. isolated from an abalone farm

Vibrio harveyi and related vibrios are potential pathogens causing luminous vibriosis in marine aquaculture systems. In this study, two lytic phages P4A and P4F isolated using Vibrio strains B4A and B4F as indicator bacteria, respectively, were isolated from seawater of an abalone farm. Vibrio strain B4F belongs to the Harveyi clade of the genus Vibrio and was found to cause mortality of abalones in laboratory microcosms. Both phages were able to lyse Vibrio strain B4F. Electron microscopy revealed that phage P4A had an icosahedral head while P4F possessed an elongated hexagonal head. Both phages belong to the family Siphoviridae with long non‐contractile tails. Restriction endonuclease analysis indicated that both phages were double‐stranded DNA viruses and the genome sizes of P4A and P4F were estimated to be about 49 and 44 kb respectively. One‐step growth curves revealed that these two phages exhibited distinct latent periods, exponential periods and burst sizes by infecting the same Vibrio strain B4F. Both phages were able to significantly reduce Vibrio population density in biofilm formed by Vibrio strain B4F on the surface of polyethylene film. It is suggested that these two phages may be promising candidates as biocontrol agents of infections caused by Vibrio strains belonging to the Haveyi clade in marine aquaculture systems.     

Effect of Vibrio Bacterial Product CIBASTIM Administration on Productivity in Commercial Tiger Shrimp Penaeus monodon Culture Ponds in Gujarat

A study was conducted to evaluate the effect of an immunostimulant product prepared from vibrio bacterial components on production of Penaeus monodon culture in five commercial shrimp farmer’s ponds in the Navsari district of Gujarat. The product was administered at the concentration of 2 × 10⁸ cfu/kg pelleted feed as top dressing using a commercial binder for two consecutive days in a week. In general, we observed improvement of up to 33.33% in average body weight, up to 44.83% in survival rate, up to 11.11% in FCR, and up to 50.00% in production per ha over untreated control ponds on the same farm. Administration of this vibrio bacterial product could improve shrimp production in Gujarat.     

花鲈皮肤溃疡病病原菌的分离与鉴定

为了确定海水池塘养殖花鲈发生皮肤溃疡病的病原菌及其防治方法。利用莆田市人工饲养的花鲈中挑取具有皮肤溃疡病明显症状的幼鱼,从体表病灶和体内组织中分离出病原菌,通过人工感染试验、细菌形态观察及常规生理生化指标测定对其进行了分类学鉴定,同时进行药敏试验。结果表明：引起该养殖场皮肤溃疡病的病原菌为哈维氏弧菌,该菌对复方新诺明、阿莫西林、哌拉西林、替卡西林、头孢拉定、头孢噻肟、头孢吡肟、头孢他啶、头孢呋辛钠、美罗培南、头孢西丁、卡那霉素、奈替米星、阿米卡星等14种药物敏感,对米诺环素、链霉素等抗生素表现出耐药。在实际生产上采用饲料中添加复方新诺明和维生素C的方法来治疗花鲈皮肤溃疡病,连续用药10天后,取得了较好的疗效。     

Induction of apoptosis in sea bream fibroblasts by Vibrio harveyi haemolysin and evidence for an anti‐apoptotic role of heat shock protein 70

In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2–8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH‐exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH‐mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post‐heat shock recovery, and the protective effect of heat shock‐induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti‐apoptotic factor.     

刺参苗期附着基更换频率对刺参生长及其养殖系统菌群结构的影响

为了探究刺参(Apoasichopus japonicus)保苗阶段(7–9月)最佳的附着基更换频率(changing frequency,CF),本实验在夏季保苗期设置5个附着基更换频率组,即CF10、CF20、CF_(30)、CF40和CF_(50)。采用实验生态学的方法,并结合传统细菌培养法和16S r DNA细菌鉴定技术对上述不同实验组进行检测。结果表明:CF20组刺参整池增重和个体增重幅度最大,CF_(30)次之,CF_(50)组由于死亡率高,整池重量为负增长。CF20组的特定生长率和存活率分别为(5.986±0.135)%/d和(95.231±0.265)/%,且显著高于其他各组(P0.05),CF_(30)次之,而CF_(50)组的特定生长率和存活率最低,且显著低于其他各组(P0.05)。养殖用水中4NH+-N、2NO--N和COD随着附着基更换频率的降低而升高,并在第50天时分别达到0.53 mg/L、0.28 mg/L、0.18 mg/L。各实验组水体中异养细菌和弧菌数量随附着基更换频率变化不明显,而附着基上的异养细菌和弧菌数量随附着基更换频率的降低而升高,CF_(50)组异养细菌总数在第50天时达到1.38×105 cfu/cm2,弧菌数量达到1.5×104 cfu/cm2,皆明显高于其他各组。附着基上优势菌为溶藻弧菌(Vibrio algindyticus)、需钠弧菌(V.natriegens)、马胃葡萄球菌(Staphylococcus equorum)、枯草芽孢杆菌(Bacillus subtilis)、苏云金芽孢杆菌(B.thuringiensis)和副溶血弧菌(V.parahaemolyticus)。其中,溶藻弧菌为刺参条件致病菌,且一直存在于养殖系统中并逐步占据绝对优势。这与CF_(50)组在实验进行到43 d时开始出现化皮,50 d时开始出现死亡现象有一定的关系。同时,附着基长时间未更换,会滋生大量玻璃海鞘、日本毛壶、内刺盘管虫等敌害生物,争夺栖息空间和食物,导致刺参苗种生长减慢。综上,由实验结果显示,在7–9月高温季节每20 d更换一次附着基最佳。考虑到生产成本,附着基更换频率一般为20~30 d为宜。本研究结果为刺参苗种培育工艺的优化及刺参健康养殖提供了理论依据和参考。     

半滑舌鳎CD40基因克隆及其免疫功能分析

本研究通过RACE技术在半滑舌鳎(Cynoglossus semilaevis)中克隆得到1个CD40同源基因。该基因c DNA全长为2098 bp,开放阅读框为1011 bp,可编码由336个氨基酸组成的蛋白质。推导得到的CD40氨基酸序列包含1个信号肽、1个跨膜结构区、2个N-糖基化位点以及含4个富含半胱氨酸的保守结构区。不同物种氨基酸序列同源分析结果显示,该基因与哺乳类、两栖类以及硬骨鱼类相似性为28%~47%,其中与硬骨鱼类相似性较高。系统进化分析结果显示,半滑舌鳎CD40与其他硬骨鱼类CD40聚类为一支,哺乳类与两栖类CD40聚类为另一支。荧光定量PCR结果显示,CD40在健康半滑舌鳎不同组织中均有表达,其中在鳃、肝和脾等组织中表达量较高,在肠、肌肉和皮肤中表达量较低,在脑和肾中微量表达。经哈维氏弧菌(Vibrio harveyi)感染刺激后,与PBS处理的对照组相比,该基因m RNA在半滑舌鳎3个主要免疫组织(肝、脾和肾)中表达量均呈显著性升高,在肝中12 h到达峰值,在脾和肾中6 h达到峰值。上述结果表明CD40基因参与了半滑舌鳎抵抗哈维氏弧菌的免疫应答反应。     

哈维氏弧菌和白斑综合症病毒对墨吉明对虾仔虾的致病性

研究了不同浓度哈维氏弧菌与白斑综合症病毒单独、合并浸泡感染,以及不同温度和盐度条件下单独、合并浸泡感染对墨吉明对虾仔虾死亡情况和WSSV携带量的影响。结果表明:高浓度哈维氏弧菌浸泡感染仔虾,48 h死亡率可达100%,高浓度WSSV浸泡感染仔虾,96 h死亡率可达97%;盐度突变可以降低弧菌与病毒浸泡感染对仔虾的致病性;高温条件下(32℃)弧菌对仔虾的致病性较强,低温条件下(26℃)病毒对仔虾的致病性较强;盐度26、水温26℃条件下,合并感染组死亡率较WSSV单独感染组低;哈维氏弧菌感染可以抑制WSSV在对虾体内的复制。