创伤弧菌物种特异性检测靶标基因的发掘及评价

实验根据GenBank公布的细菌全基因组数据,采用比较基因组学分析创伤弧菌与其它细菌基因组间的差异,筛选出34个潜在的创伤弧菌物种特异性检测靶标基因,其中VV1_2692、VV2_0075和VV2_0939这3个基因已有功能注释.常规PCR扩增结果显示这3个基因均具有良好的特异性和灵敏度.进而以这3个基因和传统靶基因vvhA作为检测靶标,采用常规PCR技术检测来自杭州市农贸市场的137份海产品中创伤弧菌,发现VV2_0075、VV2_0939和vvhA基因的检测结果与传统生化鉴定方法一致,表明新发掘的VV2_0075和VV2_0939基因在对海产品中创伤弧菌检测时具有很好的稳定性和很强的抗干扰能力.在137份海产品中创伤弧菌的检出率为28.5％,其中牡蛎的检出率最高(68％),表明杭州市农贸市场售卖的海产品,尤其是牡蛎中的创伤弧菌污染现状严重,应加强对杭州市海产品中创伤弧菌污染状况的监测.     

杂色鲍同种移植炎症因子1的克隆及其在应激下的表达

同种移植炎症因子AIF-1 (allograft inflammatory factor 1,AIF-1)是一种由干扰素γ诱导的含有EF-hand结构域的钙离子结合蛋白,其功能主要是参与移植排斥、免疫炎症反应、自身炎性和非炎性的损伤等.首次克隆了杂色鲍AIF-1基因cDNA全序列,命名为HdAIF-1,其全长为942 bp,开放阅读框为456 bp,编码151个氨基酸.实时荧光定量PCR结果表明:HdAIF-1在杂色鲍各组织中均有表达,其中在血淋巴和鳃中表达量最高.高温应激下,HdAIF-1在鳃组织中各时相表达均显著上调,并在温度升至31℃时达到最高.而血淋巴和肝胰腺中HdAIF-1在高温应激前4个时相表达无显著差异,到了96 h均显著上调.缺氧应激下,HdAIF-1在血淋巴中表达变化没有显著差异,而鳃中24 h显著下调,192 h显著上调.副溶血弧菌感染实验表明HdAIF-1基因在感染后3、24和48 h均检测到HdAIF-1的表达量显著上调.高温和缺氧应激以及弧菌感染均显示HdAIF-1基因表达量发生显著变化,说明HdAIF-1可能作为免疫因子在杂色鲍应激等状况下发挥重要作用.     

免疫多糖对日本蟳抗溶藻弧菌能力的影响

通过对日本蟳(Charybdis japonica)分别注射生理盐水(对照组)、溶藻弧菌(攻毒组)、免疫多糖(免疫组)、免疫多糖+溶藻弧菌(免疫攻毒组)后,再测定4组肝胰腺及肌肉中溶菌酶(LZM)和超氧化物岐化酶(SOD)的活性变化,探讨免疫多糖对日本蟳抗溶藻弧菌能力的影响。结果表明:日本蟳肝胰腺中LZM和SOD的酶活均高于肌肉中的酶活,且免疫多糖对肝胰腺的免疫增强效应显著高于肌肉;免疫组肝胰腺和肌肉中LZM酶活,在注射后24h时显著升高至峰值(P<0.05),分别为(11.2±2.78)U/mg和(3.75±1.05)U/mg,肝胰腺和肌肉中的SOD酶活也在24h达到最高值,分别是对照组的2.2倍和1.4倍;免疫攻毒组在72h时,肝胰腺和肌肉中的LZM酶活分别高出攻毒组62.8%和75.2%,两组织中SOD酶活同样显著高于攻毒组(P<0.05),是攻毒组的3.5倍和4.2倍。由此可见,免疫多糖能提高日本蟳抗溶藻弧菌能力,且在72h达到最佳免疫保护效果。     

不同C:N条件下一株溶藻弧菌对不同种类氮源的吸收特性

在不同C:N(5、10、15、20)条件下研究了一株溶藻弧菌(Vibrio alginolyticus)Z5对单一氮源(有机氮源、NH₄-N、NO₂-N)及混合氮源(有机氮源与NH₄-N、有机氮源与NO₂-N、NH₄-N与NO₂-N)的吸收利用特性及蛋白酶、谷氨酰胺合成酶(GS)和亚硝酸还原酶(NiR)等3种氮代谢相关酶的活力响应。研究结果表明, 溶藻弧菌Z5对有机氮与无机氮的吸收均受到环境中碳含量的影响, 在C:N=20时, 对NH₄-N和NO₂-N的吸收率分别是C:N=5时吸收率的3.39倍和2.25倍。在C:N=15和C:N=20的水平下, 溶藻弧菌Z5对NH₄-N和NO₂-N的吸收均没有显著性差异(P>0.05)。溶藻弧菌(Z5)吸收利用NH₄-N和NO₂-N的途径分别为NH₄-N→细菌菌体蛋白和NO₂-N→NH₄-N→细菌菌体蛋白。在高C:N条件下, 溶藻弧菌Z5对无机氮源(NH₄-N和NO₂-N)的吸收增强, 细菌菌体蛋白中来自NH₄-N和NO₂-N的量分别由C:N=5时的25.0%和19.4%上升到C:N=20时的41.3%和43.0%。GS酶活力受NH₄-N调控明显, 相比之下, 蛋白酶和NiR酶受氮源种类调控不明显。

     

Identification of Vibrio parahaemolyticus in Seafood by Multiplex PCR

ABSTRACT

Vibrio parahaemolyticus is a human pathogen frequently found in seafood. Once the seafood is contaminated by V. parahaemolyticus, it can become a vehicle for foodborne illness. The conventional culture methods for detection of V. parahaemolyticus are time-consuming and cannot differentiate pathogenic strains from nonpathogenic ones. In this study, a multiplex polymerase chain reaction (PCR) technique was investigated for detecting tdh, chiA, and toxR of V. parahaemolyticus. The sensitivity of the multiplex PCR was determined by testing 28 strains of V. parahaemolyticus, 15 non-V. parahaemolyticus strains, and fresh seafood spiked with cells of V. parahaemolyticus. All the strains were analyzed for production of thermostable direct hemolysin (TDH) and chitinase. This study showed that both the chiA and toxR are excellent markers for detecting V. parahaemolyticus strains, and a multiplex PCR targeting chiA and tdh genes can be applied to simultaneously detect environmental and pathogenic V. parahaemolyticus.     

Occurrence and Control of Vibrio vulnificus in Shellfish

Abstract

Vibrio vulnificus is a ubiquitous marine bacterium frequently isolated from shellfish and associated with severe and often fatal disease in humans. Various control measures have been suggested in an effort to reduce the disease risk associated with V. vulnificus and aquatic food products. This paper reviews the current literature to describe the physiological and morphological characteristics of V. vulnifi-cus, the epidemiological aspects of the diseases caused by this organism, and the scientific and practical considerations associated with various strategies aimed at controlling V. vulnificus contamination in shellfish.     

Damage to the Membrane Permeability and Cell Death of Vibrio parahaemolyticus Caused by Phlorotannins with Low Molecular Weight from Sargassum thunbergii

ABSTRACT

To understand the mode of action of low molecular weight phlorotannins (LMPs) from Sargassum thunbergii against Vibrio parahaemolyticus, effects of LMPs on the growth curve and the content changes of some substances in culture media were investigated, and those on morphological and ultramicrostructure were also observed by scanning electron microscope and transmission electron microscopy. The results showed that LMPs inhibited thalli growth at logarithmic phase and damaged the cell membrane and cell wall of tested bacteria which led to the leakage of cytoplasm and the deconstruction of membrane permeability. The present study indicates that LMPs as natural products in seaweeds deserve further investigation for food safety control and aquacultural drugs.     

方斑东风螺“急性死亡症”的病原病理研究

对海南发生的方斑东风螺（Babylonia areolata）“急性死亡症”进行了病原分离纯化、人工感染试验,以及病原16SrDNA分析和生理生化鉴定。结果显示病原菌为哈维弧菌（Vibrioharveyi）;药敏试验结果显示,该病原菌对常见的抗菌药物如恩诺沙星、复合磺胺等敏感,而对青霉素表现为耐药;组织病理结果显示鳃叶、腹足、肝胰腺、消化道、肾等器官的细胞出现变性、坏死、血细胞浸润等现象。     

舟山海域海泥细菌的分离及抗水产病原弧菌的筛选

采集中国东海舟山海域海底不同层次9个海泥样品,通过稀释培养分离纯化得到海洋细菌78株。采用滤纸片琼脂扩散法测定上述分离所得海洋细菌对鳗弧菌、溶藻弧菌、哈维氏弧菌、创伤弧菌、费氏弧菌等5种水产病害常见病原弧菌的抑制作用,获得具有抑弧菌作用的海洋细菌17株,占21.8%。其中6株菌株对费氏弧菌有明显的抑菌作用,占7.7%。7株菌株对哈维氏弧菌的抑菌作用较显著,占9.0%。2株菌株对创伤弧菌有明显的抑菌作用,占2.6%。2株菌株对溶藻弧菌和鳗弧菌显示一定的抑制作用,各占1.3%。研究结果表明：通过稀释培养法能分离到一定数量和种类的海洋细菌;来自海底泥的海洋细菌中存在较多的具有水产病原弧菌拮抗活性的菌株。     

罗非鱼创伤弧菌的分离鉴定和药敏试验

2010年春季广东、海南的养殖罗非鱼苗种出现较大范围的死亡现象,在广东珠海某罗非鱼养殖场的发病罗非鱼体上分离到一株病原菌ZH1。人工感染试验显示该分离菌株具有较强毒力,该病原菌经ATB 32E细菌鉴定系统鉴定和16S rRNA基因序列分析,确定为创伤弧菌(Vibrio vulnificus)。药敏试验结果显示,该菌株对诺氟沙星、头孢克洛、氧氟沙星和壮观霉素等19种试验药物敏感。本研究病原的鉴定与药物敏感性试验结果为罗非鱼病害的有效防控提供参考。