南方九孔鲍鲍苗掉板病原菌的药敏测定*

为提高鲍鱼培苗的成活率,对分离自广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)的、经回归感染试验证明为致病菌的菌株进行了鉴定和药物敏感性测定。API鉴定表明,这些致病菌株由Vibrio alginolyticus,Vibrio cholerae,Vibrio parahaem olyticus等组成,其中弧菌17株,约占总分离菌株的50%,而溶藻弧菌则为弧菌的优势菌株,有11株,约占弧菌总数的70%。药敏结果显示,绝大多数菌株对链霉素、红霉素和庆大霉素敏感;相反,四环素和新生霉素则对它们没有作用或     

南方九孔鲍培苗过程中潜在致病菌胞外产物的分析

从广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)分离筛选到105株菌,并对之进行了致病毒力因子(胞外酶及溶血作用)的分析,同时应用PCR对溶血毒素的归属进行了初步的探讨,采用API对菌株进行了种类鉴定。结果表明,105株菌中,仅35株菌具有较强的分泌胞外蛋白酶、明胶酶和脂肪酶的能力,尤其以菌株1、2、3、5、9以及16相对强大。在此35株菌中,85.6%的菌株(3035株)表现出溶血现象,但仅16株菌的TlhPCR呈阳性。API鉴定表明,35株菌中弧菌约占50%,溶藻弧菌又占弧菌的70%。研究结果揭示,和其它菌株相比,分离自变白鲍苗的6株溶藻弧菌(菌株1、2、3、5,13和16)和2株副溶血弧菌(菌株9和21)具有较强的分泌胞外酶和或溶血的能力,意味着极有必要对其作进一步的研究,以观测它们对鲍苗的影响。     

半滑舌鳎病原菌轮虫弧菌(Vibrio rotiferianus)的分离与鉴定

从患有严重皮肤溃疡病的半滑舌鳎(Cynoglossus semilaevisGünther)病灶处分离出4株优势菌,经人工感染证实其中一株菌株BV1为养殖半滑舌鳎皮肤溃疡病的病原菌。通过Biolog系统、细菌常规形态特征和生理生化反应指标测定以及16S rRNA和gyrB序列分析对菌株BV1进行了综合鉴定,结果表明该致病菌为轮虫弧菌(Vibrio rotiferianus),其半致死量LD50为6.7×103CFU/mL。进一步的药敏试验表明其对链霉素、四环素等敏感,而对其他用于试验的抗生素敏感度低或具有一定的抗性。     

引起文蛤暴发性死亡病原菌的分离和鉴定

本文从江苏吕泗文蛤养殖区发病文蛤和养殖水体中分离到5株优势菌(病蛤中分离到3株优势菌, 养殖水体中分离到2株优势菌), 人工感染实验结果显示, 菌株WG1702能引起供试文蛤发病, 死亡率为100%, 且发病症状与原发病症状相同, 提示该菌是引起文蛤大面积死亡的主要致病菌, 对其形态、生理生化特征、分类地位及致病性进行了初步研究, 菌株WG1702为革兰氏阴性菌, 直杆状, 生理生化指标为：赖氨酸脱羧酶、鸟氨酸脱羧酶、硝酸盐还原、甘露糖、氧化酶、甲基红阳性, 精氨酸脱羧酶、精氨酸双水解酶、水杨素、V.P阴性。为确定该致病菌的分类学地位, 进行了16S rRNA分子鉴定, PCR扩增获得了大小约1.5 kb的16S rRNA部分基因片段, 对其序列进行了测定, 并进行同源性分析和系统进化树构建。结果表明, 该菌株与哈氏弧菌(DQ146937)同源性最高, 为97.4%, 结合形态、生理生化鉴定结果, 最后鉴定菌株WG1702为哈氏弧菌(Vibrio.harveyi)。     

一株分离自美国龙虾的麦氏弧菌的鉴定及其低温状态下细胞脂肪酸的变化

[目的]对从美国龙虾(Homarus americanus)中分离的菌株F5-1进行种属鉴定并分析其低温状态下脂肪酸组成变化.[方法]通过VITEK 2 Compact全自动微生物鉴定仪分析菌株的生理生化特征和进行药敏试验;16S rRNA基因序列同源性分析确定该菌株的系统发育学地位;通过气相色谱和质谱联用法(GC-MS)分析菌株的全细胞脂肪酸.[结果]菌株F5-1为革兰氏阴性菌,对弧菌抑制剂O/129敏感,对青霉素有耐药性;生理生化特征与麦氏弧菌(Vibrio metschnikovii)的相似性为96％,16S rRNA序列与麦氏弧菌(GenBank No.HQ658055)的相似性为99％;菌株的主要脂肪酸组成为C12∶0、C14∶0、C16∶0和C16∶1(n-7),不饱和脂肪酸(棕榈油酸)相对含量达34％,低温培养状态下,不饱和脂肪酸(棕榈油酸)相对含量增加至40％.[结论]将美国龙虾中分离的菌株F5-1鉴定为麦氏弧菌,该菌对多种药物敏感,菌细胞脂肪酸组成与来源于俄罗斯海参威某饮用水水库中分离的麦氏弧菌有较大差异.     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

2013‒2015年杭州地区急性腹泻患者来源 副溶血弧菌毒力基因及耐药性调查

摘要：目的 对2013?2015年杭州地区腹泻患者副溶血弧菌（Vibrio parahaemolyticus）毒力基因的携带和耐药进行调查研究。方法 可疑菌株用全自动细菌鉴定仪（VITEK2 Compact,生物梅里埃,法国）进行鉴定,用PCR方法检测其8种毒力基因,用纸片扩散法检测13种抗生素的敏感性。结果 120株副溶血弧菌中,tdh+或trh+的致病菌株占95.0%（114株）,tdh-且trh-的非致病株占5.0%（6株）。所有120株副溶血弧菌均含有T3SS1基因,T3SS2α基因主要存在于tdh+/trh-的菌株中,T3SS2β存在于trh+的菌株中,2株tdh-的菌株首次检出T3SS2α基因。本研究中大流行菌株占64.2%（77株）,非大流行菌株占35.8%（43株）。多达65.8%的副溶血弧菌对氨苄西林耐药,而90.0%以上的副溶血弧菌对其他抗生素包括喹诺酮类、第三代头孢菌素类、氨基糖苷类、四环素类和磺胺类抗菌药物较敏感。结论 杭州地区腹泻来源的副溶血弧菌多为大流行菌株,对常见抗菌药物敏感性高,但耐药率有所上升,要加强监测并引起临床重视。     

九孔鲍鲍苗脱板过程中细菌胞外产物的研究

自2002年后半年开始,汕尾出现大规模九孔鲍鲍苗脱板问题。为了解养殖环境中的细菌(主要是弧菌)对鲍苗的影响,于汕尾一养殖场跟踪观察多次鲍鱼育苗过程,取鲍苗正处于脱板过程中的水样和采苗板样用TCBS平板分离出25株菌,测定其分泌酪蛋白酶、胰蛋白酶和明胶酶3种胞外酶的能力,并进行其溶血现象分析。结果表明,产酶菌株达到17株,其中10株菌兼具分泌溶血素的能力。对这10株菌进行了种类鉴定,表明它们是副溶血弧菌(4株)、溶藻弧菌(2株)、腐败希瓦菌(3株)和杀鲑气单胞菌(1株)。     

268例化脓性中耳炎分泌物细菌培养及对三种滴耳液的筛选

对２６８例（２８２耳）急性化脓性中耳炎分泌物进行需氧菌培养鉴定。分离出致病菌２３８株,阳性率８４．３％。其中革兰氏阳性菌１４８株,占６２．２％,以金黄色葡萄球菌为主,革兰氏阴性菌９０株,占３２．８％,以假单胞菌属为主。单一菌生长２１２例,复合菌生长２６例。２３８株细菌共分离出１９个种类细菌。我们对其中１２０例分离菌株进行体外药敏测定。对氧氟沙星敏感者Ⅲ株（占９２．９％）,对环丙沙星敏感者１０７株（     

一株牙鲆皮肤溃烂症病原菌的鉴定

从山东荣成养鱼场发病牙鲆分离到一株病原菌M3,革兰氏阴性,杆状,能运动,菌落半透明,用BIOLOG细菌鉴定系统不能鉴定。通过16S rDNA序列分析和同源性检索发现M3菌株与弧菌属的同源性较高,为94%～98%。系统发育学分析表明菌株M3与鳗弧菌关系最近,相似性为996%,其生化性状也和鳗弧菌的特征相似,故可把M3定为鳗弧菌(Vibrio anguillarum)。     

九孔鲍养殖水体及其肠道不同菌群抗药性研究*

为更好地防治鲍鱼病害的发生和流行,对分离自广东汕尾健生鲍鱼养殖场九孔鲍养殖环境及其肠道中不同细菌菌群的耐药性进行了研究。结果表明,四环素、青霉素G、卡那霉素、丁胺卡那霉素和新生霉素均对绝大多数异养菌和弧菌菌株都不敏感或无作用;相反,氟哌酸、红霉素、氯霉素以及环丙沙星等则均对它们比较敏感;复方新诺明、链霉素和多粘霉素B对弧菌菌株均有作用,而且多粘霉素B也对水体中的异养菌群相当有效。     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

黄鳍鲷弧菌病病原特性及其全菌苗的研究*

自湛江市发病养殖黄鳍鲷分离一株致病菌。对该菌的基本特征、药物筛选、灭活全菌苗等进行了研究。结果表明该病原菌为溶藻弧菌,且对四环素、土霉素、氯霉素、氟哌酸、氧氟沙星等药物敏感,pH6．0比pH8．4时灭活效果好,加入福尔马林可以提高灭活效果;全菌苗能够提高鱼体的免疫能力,减少攻毒时的死亡率。     

Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica)

Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica , were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents. Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin. Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%). While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63·4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11. Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4·3×10⁻³ transconjugants per donor cell.     

Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis)

Twelve Vibrio vulnificus biotype 1 and 11 Vibrio alginolyticus isolated from mussels in Italy were analysed by antimicrobial resistance, plasmid profiles, random amplification of polymorphic DNA (RAPD), and single enzyme amplified fragment length polymorphism (sAFLP). Plasmid DNA was detected in three V. vulnificus and four V. alginolyticus cultures. All isolates were resistant to at least two antimicrobial agents: all isolates were resistant to ampicillin, carbenicillin and streptomycin, except one V. alginolyticus which was sensitive to carbenicillin and two V. alginolyticus which were sensitive to streptomycin. No association was detected between the presence of plasmid DNA and antimicrobial resistance. Seven of the twelve V. vulnificus and two of the eleven V. alginolyticus cultures were susceptible to the 10 microg of the vibriostatic compound O/129; all cultures were susceptible to the 150 microg of O/129. Both RAPD and sAFLP was found to be reproducible. Ten sAFLP and seven RAPD profiles were detected amongst the 12 V. vulnificus cultures: three cultures were identified as indistinguishable by both methods. RAPD and sAFLP analysis of V. alginolyticus generated nine and seven profiles respectively, and these two methods were independent. These results demonstrate extreme variability of V. vulnificus and V. alginolyticus isolated from mussels, and both RAPD and sAFLP provided information on intraspecific differences which will be useful for molecular epidemiological or ecological studies. A combination of methods gave optimal discrimination, although a single method could provide sufficient information to characterise V. vulnificus isolates.     

The implication of ambient temperature with the outbreak of vibriosis in cultured small abalone Haliotis diversicolor supertexta Lischke

(1) Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta Lischke with vibriosis occurred in May and September of 1998 in Kao-Hsiung, Taiwan. (2) The effect of different temperature treatments on the susceptibility of small abalone to vibrio and its extracellular products (ECP) was investigated. (3) Two bacterial strains, Vibrio alginolyticus H11 and V. parahaemolyticus B4 originally isolated from the haemolymph of the moribund small abalone at 28 and 32°C, respectively, were used in this study for susceptibility tests. (4) The results reveal that at higher temperatures, the small abalone were more susceptible to vibrio and ECP challenge indicating that the outbreak of vibriosis is associated with warm water conditions.     

Vibrio alginolyticus associated with white spot disease of Penaeus monodon

In February 2000, white spot disease outbreaks occurred among cultured Penaeus monodon in extensive shrimp farms on the southwest coast of India. Bacteria were isolated from infected shrimp that showed reddish body coloration and white spots in the cuticle. The isolates were screened on thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. The primary isolate (QS7) was characterized as V. alginolyticus based on morphological, biochemical and physiological characteristics. Antibiotic sensitivity tests of QS7 indicated that the isolate was highly sensitive to chloramphenicol, ciprofloxacin, nalidixic acid and streptomycin. Pathogenicity tests confirmed that the isolate was virulent for P. monodon. Based on the lethal dose (LD50) value (5 x 10(6) cfu per shrimp), it was inferred that shrimp weakened by white spot syndrome virus would succumb to secondary infection by QS7.     

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.