红鳍东方皮肤溃烂病病原菌的分离与鉴定

从体表溃烂的养殖红鳍东方鲀(Fugu obscurus)病灶处分离到1株优势生长菌,编号为H-06091.分别通过创伤浸泡和注射方式感染健康红鳍东方鲀,发现这两种途径均可引发皮肤溃烂,两种途径的感染率均为100%,2×108 cell/mL.菌浓度注射组死亡率为100%,4×107 cell/mL菌浓度注射组死亡率为50%,创伤浸泡感染未见死亡.药敏实验表明,复方新诺明、呋喃妥因、链霉素、环丙沙星、利福平、红霉素、氟哌酸、氟嗪酸、庆大霉素、阿米卡星、四环素、青霉素G等抗菌素对H-06091有较强的抑制作用.按照<常见细菌系统鉴定手册>进行菌种鉴定并测定其16S rRNA基因序列,结果表明,该菌呈革兰氏染色阴性,氧化酶阳性,接触酶阳性,V-P试验阴性,硝酸盐还原反应阳性等特征,符合哈氏弧菌(Vibrio harveyi)特征,其16s rRNA基因序列与GenBank中哈氏弧菌同源性为99%,因此将H-06091鉴定为哈氏弧菌.     

不同温度下哈维氏弧菌和白斑综合症病毒对凡纳滨对虾的致病性

在不同温度[(19±1)、(25±1)、(31±1)℃]条件下,研究了不同浓度哈维氏弧菌Vibrio harveyi和白斑综合症病毒(WSSV)对凡纳滨对虾Litopenaeus vannamei(体长8.12 cm±0.83 cm)的致病性。结果表明:温度为(19±1)℃时,整个试验过程中对虾死亡率均低于8.9%,各组病毒携带量低于3.9×104copy/g;温度为(25±1)℃时,病毒单独感染组对虾的死亡率和病毒携带量(最大值71.1%,1.9×105copy/g)和高浓度继发感染组的死亡率和病毒携带量(最大值77.8%,3.2×106copy/g)从试验中期开始明显高于其他组,死亡加快,病毒携带量迅速增加,单独感染细菌组死亡率随感染浓度的升高而升高(最大值为21.1%);温度为(31±1)℃时,病毒单独感染组的死亡率和病毒携带量(最大值47.8%,2.2×104copy/g)从试验中期开始明显低于继发感染组(最大值80.0%,6.1×106copy/g),单独感染细菌组的累积死亡率随细菌浓度的升高而升高(最大值24.4%)。研究表明,温度对WSSV和哈维氏弧菌的致病力影响显著,继发感染和细菌单独感染随温度的升高致病力升高,高温和低温对WSSV的致病力有抑制作用。     

Pathogenicity of halophilic Vibrio harveyi in giant freshwater prawns (Macrobrachium rosenbergii De Man)

In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non‐lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post‐larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the first‐injected infected prawns were re‐isolated and re‐injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non‐lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post‐injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic γ‐hemolytic VL19 and non‐lysogenic β‐hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and non‐lysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.     

虾源哈维氏弧菌的致病性与生物学特性比较分析

哈维氏弧菌(Vibrio harveyi)是导致养殖对虾暴发弧菌病的重要病原之一。从我国南方养殖凡纳滨对虾(Penaeus vannamei)分离、鉴定了10株哈维氏弧菌(Vh00947、Vh00949、Vh11011、Vh11014、Vh21217、Vh21218、Vh21220、Vh21229、Vh21231和Vh31487),分别肌注感染健康凡纳滨对虾后发现,菌株Vh21229致病性很弱,Vh00949其次,其它菌株毒力较强;对8种哈维氏弧菌常见毒力基因(Vh1、Vh2、Vh3、Vh4、tox S、hcp、zot和pap6)的检测显示,南方虾源哈维氏弧菌无论强弱毒株都很少携带zot、pap6、toxs和4种Vh基因(≤10%),表明这10个菌株的致病性与这7种常见毒力因子的相关度很低;hcp基因在所有菌株中均有检出,其中强毒株中检出率较高(50%),在较弱毒株(Vh00949)中也存在,表明hcp基因与这些菌株的致病性密切相关,但不是决定性因子。因此,这10株南方虾源哈维氏弧菌菌株的致病性差异应由这8种常见毒力因子以外的未知或未检测到的因子所决定。生化特征分析显示,所有菌株中只有弱毒株Vh21229不能利用D-甘露糖、蔗糖、D-海藻糖,而且只有其赖氨酸脱羧酶反应至阳性,而其它菌株均为阴性,推测导致哈维氏弧菌菌株生化特性改变的某种因子可能对菌株的致病性也产生了影响。药敏实验表明,10株哈维氏弧菌均对环丙沙星、氯霉素、恩诺沙星、美罗培南、头孢曲松、多西环素、头孢吡肟、诺氟沙星敏感,而对氨苄西林耐受性强,表明在虾类养殖过程中应当严格规范和控制抗菌药物尤其是青霉素类药物的使用。     

二种佐剂对哈氏弧菌OMPC-溶藻弧菌LPS偶联疫苗的增效作用

制备哈氏弧菌SpGY020601外膜蛋白复合物(OMPC)和溶藻弧菌EpGS021001脂多糖(LPS)偶联疫苗,设置偶联疫苗组、偶联疫苗 ISA763佐剂组、偶联疫苗 β-葡聚糖佐剂(DEAE)组以及生理盐水对照组,并以相同程序免疫卵型鲳鲹。溶菌酶活性检测、微量凝集反应试验结果显示:各疫苗组血清中的溶菌酶含量无显著差异(P>0.05),但均远高于生理盐水对照组(P<0.05);ISA763佐剂组抗哈氏弧菌SpGY020601抗体滴度较其他组出现的早,抗溶藻弧菌EpGS021001抗体滴度较其他组持续时间长;β-葡聚糖佐剂组次之,其次是无佐剂组,对照组始终未检测到抗体。各组分为腹腔注射攻毒与浸泡攻毒,对EpGS021001的攻击,ISA763佐剂组相对保护率分别为90%和70%,高于β-葡聚糖佐剂组的85%和55%,以及无佐剂组的80%和55%;对SpGY020601的攻击,ISA763佐剂组相对保护率分别为95%和80%,高于或相当于β-葡聚糖佐剂组的95%和75%,以及无佐剂组的90%和75%。     

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。     

3种抗生素对3种常见鱼类病原菌的抑菌活性研究

为研究盐酸恩诺沙星、乳酸恩诺沙星和乳酸诺氟沙星对嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌(Vibrio anguillarum)和哈维氏弧菌(Vibrioharveyi)的体外抑菌活性,本实验通过测定抑菌圈直径、最小抑菌浓度(MIC)、最小杀菌浓度(MBC)和杀菌曲线,研究3种常用喹诺酮类抗生素药物对3种水产病原菌的体外药效学特性.结果表明:乳酸诺氟沙星对嗜水气单胞菌和哈维氏弧菌抑菌效果最好,MIC分别为0.244μg/mL和1.954μg/mL:盐酸恩诺沙星对鳗弧菌抑菌效果最好,MIC为0.122μg/mL;8 MIC的乳酸诺氟沙星药液在12 h内完全杀灭嗜水气单胞菌,在24 h内也能全部杀灭哈维氏弧菌,8 MIC的盐酸恩诺沙星药液在24 h内全部杀灭鳗弧菌.本研究为3种抗生素在水产养殖中的应用提供实验依据.     

Immune responses of barramundi, Lates calcarifer (Bloch), after administration of an experimental Vibrio harveyi bacterin by intraperitoneal injection, anal intubation and immersion

Barramundi, Lates calcarifer (Bloch), were immunized with an experimental Vibrio harveyi bacterin via intraperitoneal injection, immersion and anal intubation. Both specific and non-specific immune parameters were measured to compare responses to bacterin after delivery by various methods. Elevated antibody activities in sera were found in all treatment groups with barramundi injected intraperitoneally displaying significantly higher antibody activity than the other groups. In addition, there was evidence of memory induction with a heightened antibody response in the intraperitoneally injected group only. Bacteriostatic assays indicated activity against V. harveyi in the sera of all bacterin-treated groups; again this activity was significantly higher in the intraperitoneally injected groups. There was no enhancement noted in head kidney macrophage phagocytic activity or in serum lysozyme levels.