生物净水栅对凡纳滨对虾肠道菌群组成的影响

利用Illumina Miseq高通量测序技术和生物信息学分析手段,采集传统养殖池塘和添加生物净水栅养殖池塘凡纳滨对虾肠道样品,比较分析两个池塘凡纳滨对虾肠道菌群组成及其多样性。试验结果表明,对照组和试验组凡纳滨对虾肠道测得的可操作分类单元分别为239和296。对照组和试验组对虾肠道样品菌群的物种丰度和优势菌种存在一定差异,试验组和对照组样品细菌Shannon指数分别为2.04、1.80,前者细菌Shannon指数高于后者13.33%(P0.05)。对照组和试验组对虾肠道样品所鉴定出的细菌门类组成基本相似,组成比例存在一定差异,前三大优势细菌门类均为变形菌门、拟杆菌门和软壁菌门,分别占试验组细菌总数和对照组细菌总数的97.56%和98.54%。在优势菌群方面,试验组对虾肠道的优势群为弧菌属、希瓦氏菌属、Cloacibacterium、黄质菌属、红杆菌属,分别占细菌总数的70.83%、18.29%、1.97%、1.38%和1.06%,对照组对虾肠道的优势群为弧菌属和希瓦氏菌属,分别占细菌总数的58.51%和37.67%。本研究分析了两种养殖池塘下对虾肠道菌群结构,试验组对虾菌群丰富度高于对照组,其多样性也高于对照组,这有助于生物净水栅更好地应用于池塘养殖。     

人工培育对虾苗种体内可培养细菌数量及组成分析

采用常规细菌分离、培养与纯化,结合细菌16S rDNA序列分析等方法,调查分析了山东、天津、浙江部分苗种场凡纳滨对虾(Litopenaeus vannamei)和中国明对虾(Fenneorpenaeus chinensis)苗种体内的可培养细菌总数以及优势菌的种类和数量,并用PCR的方法检测对虾苗种携带病毒情况.结果显示,凡纳滨对虾和中国明对虾苗种体内的可培养细菌总数均在105-107 CFU/g之间,分离出的细菌分属于弧菌属(Vibrio)、发光杆菌属(Photobacterium)、芽孢杆菌属(Bacillus)、盐单胞菌属(Halomonas)等8个属,其中,弧菌属在对虾苗种体内占绝对优势,达40.0％-90.23％.部分凡纳滨对虾和中国明对虾苗种样品检测为白斑综合征病毒(White spot syndrome virus,VSSV)和传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)阳性,WSSV检出率为50.0％,IHHNV检出率为37.5％,同时携带两种病毒的检出率为11.11％.只携带WSSV病毒以及同时携带两种病毒的对虾苗种体内的总菌数量在1.23×107-4.14×107 CFU/g之间,显著高于其他批次的样品(P＜0.05),且弧菌数量在107 CFU/g左右,显著高于其他批次的样品(P＜0.05).只携带IHHNV以及不携带病毒的对虾苗种体内的弧菌数量为104-106 CFU/g.研究表明,弧菌属在凡纳滨对虾和中国明对虾苗种体内普遍存在且为优势菌属,携带WSSV可能会引起对虾苗种体内的弧菌数量增长.     

高通量测序法分析两株益生菌对凡纳滨对虾肠道菌群结构的影响

为分析荚膜红细菌和蜡样芽孢杆菌2株益生菌对凡纳滨对虾肠道菌群结构的影响,本实验对凡纳滨对虾进行为期30 d的养殖饲喂实验,饲喂后期利用高通量测序凡纳滨对虾肠道微生物的16S rDNA V4区,来分析不同益生菌饲喂后凡纳滨对虾肠道菌群的结构特征,并结合对虾体质量增加率和攻毒后累计死亡率的宏观指标来进行分析。结果显示：①空白组（CK）、荚膜红细菌组（Rc）和蜡样芽孢杆菌组（Bc）样品OTU范围为374~506,其中CK组对虾肠道菌群OTU数量最低,饲喂益生菌后的2组对虾肠道菌群中OTU数量相对较高;②在门分类水平上,3组的变形菌门数均为最多,CK组主要为变形菌门和少量拟杆菌门,Bc组主要有变形菌门、拟杆菌门、无壁细菌门、厚壁细菌门和假单胞菌,Rc组主要是变形菌门、拟杆菌门、厚壁菌门、酸杆菌门、放线细菌、梭杆菌门和蓝细菌;③稀释曲线和Shannon指数结果可见,CK组样品物种丰度和复杂度最低,且Bc组样品丰度和复杂度相对较高;④PCA分析发现,Rc组和CK组样品微生物组成较为接近,结合宏观对虾体质量增加率和攻毒后累计死亡率的结果分析,可见相较于荚膜红细菌,蜡样芽孢杆菌对凡纳滨对虾肠道微生物菌群影响更显著,且益生效果更佳。研究表明,饲喂益生菌可以扩增对虾肠道微生物菌群丰度,并能抑制弧菌属等有害菌群的生长,提高对虾体质量增加率并降低死亡率,从而达到益生效果,其中以饲喂蜡样芽孢杆菌菌株效果更佳。     

虾池有机污染物降解细菌的筛选

在实验室条件下对612株海洋细菌和10株海水驯化淡水菌进行筛选,检测其快速降解水体中富营养有机物的能力。利用对虾饵料培养基、BOD仪、胞外酶检测等方法进行筛选,最后筛选到10株对富营养有机物具有 较高降解性能的细菌。所有的细菌均能产生明胶酶和脂酶（Tween-80),其中9株细菌能产生淀粉酶,8株产生卵磷脂酶,2株产生酪蛋白酶,1株产生褐藻胶酶。通过测量BOD来衡量10株细菌利用对虾饵料的效果,2天时能消化46.6%-59.5%的对虾饵料,5天时能消化50.8%-70.2%的对虾饵料。用常规生理生化方法将细菌鉴定到属,其中3株为弧菌属细菌（Vibrio spp.),3株为假单胞菌属细菌（Pseudomonas spp.),2株为发光杆菌属细菌（Photobacterium spp.),1株为气单胞菌属细菌（Aeromonas spp.),1株为枯草杆菌（Bacillus subtilis)。     

养殖花鲈（Lateolabrax maculatus）肠道菌群的多样性分析

为探究养殖花鲈(Lateolabrax maculatus)肠道菌群的结构特征,采用Illumina Hiseq2500高通量测序技术,对福建宁德三都澳网箱养殖花鲈的肠道内容物及肠壁菌群16S r DNA V3-V4区进行测序。肠道内容物和肠壁样品分别获得456和293个OTUs(operational taxonomic units),分属22个门120个属。花鲈肠道菌群以厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和梭杆菌门(Fusobacteria)细菌为主要优势菌群,分别占其肠道菌群数量的47.2%、25.8%、14.4%和10.3%。在属分类水平上,发光杆菌属(Photobacterium)、梭菌属(Clostridium)及鲸杆菌属(Cetobacterium)等属细菌是主要类别。对养殖花鲈的肠道内容物与肠壁菌群进行比较分析显示,肠壁菌群多样性略高于肠道内容物。肠道内容物中,发光杆菌属、梭菌属及鲸杆菌属等是主要菌属,其中鲸杆菌属和梭菌属分别占肠道内容物菌群总菌数的17.8%和12.5%,为主要标志性差异种类;在肠壁样品的菌群中,乳球菌属(Lactococcus)、真杆菌属(Eubacterium)、Lachnoclostridium、弓形菌属(Arcobacter)和Flavonifractor为丰度较高的菌属,其中格氏乳球菌(Lactococcus garvieae)为主要标志性物种,占肠壁菌群总数的5.6%。在花鲈肠壁上定殖着格氏乳球菌(L. garvieae)和美人鱼发光杆菌(Photobacterium damselae subsp. damselae)等条件致病菌,因此,在养殖中需要注重饵料及环境的管理,避免致病菌大量增殖引起暴发性鱼病。     

红鳍东方鲀稚鱼肠道可培养细菌的多样性

使用2216E平板涂布法从肠道样品分离可培养细菌,通过16S r DNA测序鉴定细菌。所有分离的细菌分为变形菌门(包含γ-变形细菌纲和α-变形细菌纲)和厚壁菌门。其中以γ-变形细菌纲为优势(83.3%)。在属的水平,从肠道中共分离出弧菌属、希瓦氏菌属、假交替单胞菌属、亚硫酸杆菌属、芽孢杆菌属、Aliivibrio、发光杆菌属、科尔韦尔氏菌属8个属,其中弧菌属、希瓦氏菌属和假交替单胞菌属的种类占总数的70%。首次发现亚硫酸杆菌属和科尔韦尔氏菌属作为红鳍东方鲀稚鱼肠道菌群的一部分。     

健康和患病卵形鲳鲹肠道菌群结构的差异

为研究病害发生后卵形鲳鲹肠道菌群结构的差异及其与养殖环境的关系,分析不同样品中的菌群组成和细菌多样性。实验采用Illumina HiSeq高通量测序技术和生物信息学分析手段,分别构建了健康鱼水样、患病鱼水样、健康鱼肠道、患病鱼肠道和饵料颗粒5个样品中菌群的16S rRNA基因测序文库。研究表明,与健康鱼肠道细菌组成相比,患病鱼肠道中螺旋体门相对丰度显著升高,厚壁菌门和拟杆菌门相对丰度显著降低;患病后肠道中的细菌种类仅为健康鱼肠道细菌种类总数的54.94%;健康卵形鲳鲹肠道菌群中有73.46%的可操作分类单元(OTUs)与水体样品中OTUs一致,有70.58%与饲料样品一致,而患病后分别只有17.98%和38.95%的OTUs与水体和饲料样品一致。健康卵形鲳鲹肠道中黑海弧菌的相对丰度为17.19%,患病后该菌的相对丰度大幅提高,达到78.90%;健康鱼肠道中鳆发光杆菌占比54.53%,患病鱼肠道中不含鳆发光杆菌。健康和患病鱼肠道菌群种类组成类似,但存在一定差异,病害的发生会导致肠道细菌多样性显著降低。健康鱼肠道菌群与养殖环境和颗粒饲料中菌群组成关系密切,而患病后鱼肠道细菌多样性受环境中菌群的影响较小。黑海弧菌在患病鱼肠道中的大量增殖可能是引起其患病的重要原因。     

牙鲆(Paralichthys olivaceus)仔稚幼鱼肠道菌群结构比较分析

采用MiSeq 16S rRNA高通量测序技术和生物信息学分析方法,构建了牙鲆(Paralichthys olivaceus)工厂化人工育苗模式下仔稚幼鱼阶段6个不同发育时期18个样品的16SrRNA基因测序文库,共获得7462个OTU (Operational Taxonomic Unit),分类为42个菌门972个菌属.对肠道菌群的形成过程及结构多样性变化分析显示,牙鲆初孵仔鱼的菌群组成多样性丰富,体内的优势菌为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在9日龄和21日龄摄食轮虫(Rotifer)和卤虫(Artemia sp.)幼体样品中,肠道的优势菌群结构较单一,变形菌门成为此时期肠道的优势菌群;45日龄摄食配合饲料后,肠道中变形菌门的相对丰度显著降低,厚壁菌门和拟杆菌门的相对丰度明显增大,成为肠道菌群的优势菌群.在属水平的菌群结构中发现,牙鲆仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化,在9日龄和21日龄时期肠道中弧菌属(Vibrio)相对丰度最高,到45日龄后相对丰度锐减到最低水平;拟杆菌属(Bacteroides)和普氏菌属(Prevotella)在80日龄后达到较高水平,成为肠道优势菌属;厚壁菌门的8个菌属在80-115日龄时期均发展成为优势菌属,定植于牙鲆的肠道.本研究揭示了工厂化人工育苗模式下牙鲆仔稚幼鱼肠道菌群结构及演替规律.     

不同健康程度和抗生素氟苯尼考干预下斑石鲷肠道菌群的结构差异

为了研究不同健康程度和抗生素氟苯尼考干预下斑石鲷肠道菌群结构的差异及其与养殖环境中菌群结构的相关性,采用Illumina Hi Seq PE250高通量测序的方法对健康、亚健康、典型黑身病和口服氟苯尼考条件下的斑石鲷肠道、养殖水体和颗粒饵料中的细菌多样性及群落结构进行了分析比较。结果显示,养殖水体中细菌多样性高于肠道和颗粒饵料。不同健康程度及氟苯尼考干预下斑石鲷肠道中细菌均以变形菌门、厚壁菌门和软壁菌门为主,且对应的操作分类单元(OTU)占样品全部OTU的比例均达到85%以上。黑身病的发生可影响斑石鲷肠道中丰度最高的前20种优势细菌种类的排名次序,其中变形菌门中的弧菌属的相对丰度显著增加,且随着弧菌属丰度的增加,斑石鲷的黑身病症状也逐渐加重。饵料中添加氟苯尼考投喂斑石鲷能使患病鱼肠道弧菌属的丰度从60.33%下降到1.29%,较大程度改变了肠道的菌群结构,并证实氟苯尼考有效防治黑身病。其次,养殖水体和颗粒饵料对斑石鲷肠道菌群也有一定影响,且养殖水体的影响高于颗粒饵料。本研究首次报道了斑石鲷肠道菌群结构,其研究结果为今后斑石鲷的健康养殖、疾病防控及其微生态学研究提供了参考依据和技术支撑。     

鲵鱼消化道细菌群落结构及多样性初步研究

采用常规分离纯化与构建16S rDNA克隆文库相结合的方法对（鲵）鱼（Miichthys miiuy）消化道细菌多样性进行了初步研究.结果表明,（鲵）鱼消化道各部位可培养细菌的菌落数量从大到小依次为前肠＞幽门胃＞中肠＞后肠＞前胃＞后胃＞口咽腔,其中肠道部位菌落数量最多.分离细菌菌株主要为杆状细菌和革兰氏阴性菌,对其中的50株典型菌株进行分子鉴定,发现它们主要属于变形菌门的γ-变形菌纲（占52.7％）和β-变形菌纲（占36.8％）,以及厚壁菌门的芽孢杆菌纲（占10.5％）;不可培养细菌16S rDNA文库克隆子主要属于γ-变形菌纲、α-变形菌纲和脱铁杆菌纲,部分序列与已报道的物种相似性较低,说明消化道中可能存在新的有待开发的肠道菌株.     

Effect of Chitosan on Intestinal Bacteria of Allogynogenetic Crucian Carp,Carassius auratus gibelio,as Depicted by polymerase chain reaction‐denaturing Gradient Gel Electrophoresis

A 16S rDNA‐based polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) method was applied to detect intestinal bacterial communities of juvenile allogynogenetic crucian carp, Carassius auratus gibelio, fed with chitosan‐containing diet. This is the first time to use the molecular method to analyze the bacterial communities in the allogynogenetic crucian carp intestine. The DGGE profile with universal bacterial primers revealed simple communities in all treatment groups. Sequencing and phylogenetic analysis of excised DGGE bands showed that the dominant bacteria belonged to class γ‐Proteobacteria and Fusobacteria. The relative abundance and diversity of detected bacteria suggested that 0.5 and 0.75% of chitosan in diet were optimum for juvenile allogynogenetic crucian carp. As in these concentrations, some detected pathogen bacteria either disappeared or decreased. However, the DGGE profile with Aeromonas‐specific primers showed a similar composition among all treatment groups, which suggested that Aeromonas was one of relative stable bacteria components in the intestine of juvenile allogynogenetic crucian carp.     

Intestinal microbiota of mangrove red snapper (Lutjanus argentimaculatus Forsskål, 1775) reared in sea cages

Using polymerase chain reaction amplification of 16S rDNA coupled to denaturing gradient gel electrophoresis (DGGE) and sequencing of isolated amplicons, we investigated the microbiota of the intestinal digesta and mucosal surface in mangrove red snapper cultured in a cage aquaculture area in Daya Bay. A total of 14 sequences were characterized by phylogenetic analysis. Among the bacterial species determinated from sequences, the γ‐Proteobacteria group (64.25%, nine species) dominated absolutely in fish intestines. Others belonged to Spirochaetes (14.3%, two species), Cyanobacteria (14.3%, two species) and Firmicutes (7.15%, one species). However, the bacteria were identified as uncultured accounting for 28.6% (four species). The apparent bacterial richness (calculated as the numbers of DGGE bands) was significantly higher in digesta than that in mucosal tissue samples (P<0.05). There existed five dominant individual populations including one unknown species of Firmicutes, Arthrospira sp., Vibrio metschnikovii, Vibrio harveyi and Vibrio sp. in intestinal digesta, and in contrast, only three dominant individual populations including Vibrio natriegens, V. harveyi and Vibrio sp. in intestinal mucosal surface. The results indicated that the microbiota in intestinal digesta was significantly different from that in mucosal surface.     

养殖刀鲚与生长环境菌群PCR-DGGE指纹图谱及多样性分析

为了分析养殖刀鲚体内与生长环境菌群结构,利用PCR-DGGE技术,对养殖刀鲚鳃、胃、肠壁及肠内容物和养殖水体菌群结构进行了初步分析。PCR-DGGE指纹图谱分离显示,42条清晰条带,其中养殖水体(27)、鳃(9)、胃(13)、肠道壁(19)、肠道内容物(18)的香农指数分别为3.037、1.883、2.193、2.825、2.683;养殖水体与刀鲚鳃、胃、肠道壁及肠道内容物分别具有6、9、11、8共有带。UPGMA聚类分析显示,样品3个重复相似度都在95%以上,差异不明显;不同样品之间,养殖刀鲚鳃和胃聚为一支,具有较高的相似度(76%),同时与养殖水体相似度达29%;养殖刀鲚肠道壁和肠道内容物聚为一支,相似度为38%。回收测定所有显示条带,主要包含变形菌、放线菌、拟杆菌、柔膜菌、蓝藻细菌、厚壁菌、梭杆菌及少量未定义菌种。研究表明,PCR-DGGE技术能区分养殖刀鲚主要部位及水体微生物的结构差异和多样性,澄清养殖刀鲚及生长水体微生物区系,可为定植益生菌的开发提供参考。     

长毛对虾海水养殖环境以及虾肠道微生物群落结构研究

为了研究长毛对虾养殖环境以及对虾肠道微生物种群结构的特征,实验分别采集养殖区进水口水体、养殖池底泥、养殖池水体以及长毛对虾肠道样品,采用构建16S rRNA基因克隆文库的方法对不同样品间的微生物群落组成进行了研究。结果表明,4组样品中共获得621条序列,操作分类单元(OTU)总数达212个,表明养殖环境微生物群落结构具有高度的多样性。从遗传进化树分析发现,进水口水体中细菌优势种群为蓝细菌(53.97%)、α-变形杆菌(13.76%)和γ-变形杆菌(10.58%);养殖池水体细菌优势种群为蓝细菌(33.55%)、γ-变形杆菌(14.84%)、厚壁菌(14.19%)、拟杆菌(12.26%)和α-变形杆菌(9.68%);养殖池底泥细菌优势种群大部分属于厚壁细菌(79.12%);对虾肠道细菌优势种群为厚壁细菌(75.79%)、梭杆菌(13.68%)和γ-变形杆菌(10.53%)。在目分类水平上,养殖池底泥、养殖池水体和对虾肠道中芽孢杆菌占有较高的比例,分别占克隆数的69.78%、13.55%和72.63%;进水口水体和养殖池水体中红细菌的比例较高,分别占克隆数的10.05%和9.68%。本研究分析了养殖环境以及对虾肠道微生物的群落结构,揭示微生物从水源到对虾肠道内的演替规律。总体上,本养殖系统微生物群落结构良好,但在养殖池水体和对虾肠道中也检测到黄杆菌类群和少量的弧菌。本研究有助于了解养殖环境对于对虾肠道微生物组成的影响,并为长毛对虾病害的预防提供参考。     

Molecular analysis of the intestinal bacterial flora in cage‐cultured adult small abalone,Haliotis diversicolor

To characterize the composition of the intestinal bacterial flora in cage‐cultured adult small abalone, Haliotis diversicolor, we used both culture‐dependent and culture‐independent approaches. In addition, in order to investigate the presence of probable pathogens, the intestinal bacterial community structures of healthy and diseased adult abalones were compared using the denaturing gradient gel electrophoresis (DGGE) fingerprinting technique. Bacteria isolated from the healthy abalone intestine belonged to the genera Vibrio, Flammeovirga, Shewanella, Persicobacter and Paraferrimonas. From the 16S rRNA gene clone library analysis, the intestinal bacterial community comprised two groups: the Proteobacteria and Tenericutes. Betaproteobacteria (18.48%), Deltaproteobacteria (30.43%) and Mollicutes (35.87%) were the most dominant components in the clones and the Mollicutes were mainly Mycoplasma‐affiliated clones. The DGGE band pattern demonstrated that more bands appeared in the healthy abalones (H group) than in the diseased abalones (D group). Although few potential pathogens were detected from the D group, a clustering dendrogram indicated that individuals of the H and D groups (except for D6) formed two separate clusters. An unidentified Deltaproteobacteria species and Mycoplasma sp. were the dominant components in the intestine of both the H and the D group.     

Seasonal dynamics in a coastal Vibrio community examinedby a rapid clustering method based on 16S rDNA

ABSTRACT:   A rapid clustering scheme for Vibrio specieswas developed based on the analysis of 16S rDNA, which compriseda group-specific polymerase chain reaction (PCR) and restriction fragmentlength polymorphisms (RFLP) of the PCR-amplified 16S rDNA. The group-specificPCR, using a specific primer Vib1F, clustered the Vibrio speciesinto two large groups: Vib1F⁺ group and Vib1F^– group.The PCR-RFLP, using Sca I and Bln I, further dividedthese groups into smaller groups. Finally, 46 Vibrio speciesand eight related species could be clustered into 14 groups usingthis rapid clustering method. Seasonal dynamics of the Vibrio communityin Yoshimi Bay, Hibiki-nada Sea, Japan, were examined based on therapid clustering method. In both seawater and sediments, the groupcomprised Vibrio parahaemolyticus , Vibrio alginolyticus , Vibriocampbellii , Vibrio carchariae , Vibrio harveyi and Vibrionatriegens and was predominant when the seawater temperaturewas above 20°C, whereas the group of Vibrio splendidus biotypeI and Vibrio lentus was abundant when the temperature wasaround or below 20°C.     

Assessment of the bacterial community diversity associated with the queen conch Strombus gigas (Linnaeus, 1758) from the Caribbean coast of Colombia using denaturing gradient gel electrophoresis and culturing

The biotic diversity of Strombus gigas has not been thoroughly studied despite the status of the queen conch as an important ecological resource. The bacteria associated with the conch influence their host in several ways, including through the metabolism of nutrients, protection against invasive bacteria and the regulation of the physical conditions. In this study, conventional microbiological methods and molecular tools such as denaturing gradient gel electrophoresis (DGGE) were used to assess the composition of the bacterial communities associated with the queen conch (S. gigas) in wild and captive habitats in Colombia. A genetic analysis of the bacterial communities revealed a high level of diversity based on the large number of bands detected using DGGE. In addition, differences in bacterial community structure were found between the conchs in captivity and the wild populations. The dominant phylogenetic affiliations of the bacteria, as determined using 16S rRNA gene sequencing, were grouped into four classes, namely, Betaproteobacteria (16%), Gammaproteobacteria (70%), Firmicutes (7%) and Actinobacteria (2%). These groups are related to host defence processes and the decomposition of organic matter. The 16S rDNA sequence analysis of the cultured bacteria and the resulting DGGE profiles are useful tools for characterizing the diversity of the bacteria associated with the analyzed conchs.     

Characterization of Vibrio spp. Associated with Diseased Shrimp from Culture Ponds of Andhra Pradesh (India)

Abstract.— Surveys undertaken on diseases caused by Vibrio spp. in Penaeus monodon from culture ponds of coastal Andhra Pradesh recorded the occurrence of five types of diseases: tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD). Among these, LSS, WGD, and red disease caused mass mortalities in shrimp culture ponds. Six species of Vibrio—V. harveyi , V. parahaemolyticus, V. alginolyticus, V. anguillarum , V. vulnificus , and V. splendidus —are associated with the diseased shrimp. The number of Vibrio spp. associated with each disease ranged from two to five. Additionally, shrimp with red disease had concurrent infections with white spot syndrome virus. Vibrio harveyi in the case of LSS and WGD, V. parahaemolyticus for red disease, and V. alginolyticus for shell disease are the major etiologcal agents. Differences occur in the degree of virulence of different species of Vibrio and also different isolates of the same species. Vibrio harveyi isolated from LSS shrimp is the most virulent. In general, all the Vibrio isolates from LSS shrimp tend to be more virulent as compared to their counterparts from other diseased shrimp. It is apparent that the degree of virulence of various Vibrio isolates depends on its source and the pond environmental conditions. Most of the Vibrio isolates showed susceptibility to oxytetracycline, norfloxacin, and ciprofloxacin. The luminous V. harveyi exhibited resistance to many antibiotics and susceptibility to only three drugs. Considering the emergence of antimicrobial resistant strains of Vibrio , the need for using probiotics in place of antibiotics for disease control is stressed.     

Comparison of the Intestinal Bacterial Flora in Healthy and Intestinal‐diseased Seahorses Hippocampus trimaculatus,Hippocampus erectus,and Hippocampus spinosissimus

This investigation examined the intestinal microbial flora among healthy and intestinal‐diseased seahorses Hippocampus trimaculatus, Hippocampus erectus, and Hippocampus spinosissimus by utilizing polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) and densitometric analysis. Results demonstrated that 16 disparate DGGE bands belong to six major bacterial groups, which were Vibrionace, Enterobacteriacea, Rhodobacteraceae, Sphingomonadaceae, Flavobacteriaceae, and Alcaligenaceae. It was found that Vibrionaceae was the dominant population among the healthy and intestinal‐diseased seahorses. Vibrio ponticus strain XJ3 and Vibrio neptunius strain WT82, especially V. ponticus strain XJ3 of high abundance, were identified for the first time in seahorses and concluded to be intestinal disease pathogens because of their co‐existence in three intestinal‐diseased seahorse species and other reported fish or oyster. In comparison, uncultured bacterium clone Alcaligenaceae, Vibrio sp., uncultured bacterium clone Rhodobacteraceae, Serratia nematodiphila strain, and Serratia marcescens strain comprised the basic intestinal bacterial flora of all healthy seahorses. This study is the first to report the presence of S. nematodiphila in seahorses.     

凡纳滨对虾虾苗细菌性玻化症(BVS)的病原、病理分析

为进一步查明对虾"玻璃苗"的主要致病原,实验通过对河北省沧州市凡纳滨对虾苗种玻化症进行了流行病学调查及病原、病理分析。结果发现,患病虾苗表现为活力降低、厌食直至空肠、空胃,虾体消瘦、暗浊;肝胰腺组织坏死性萎缩、轮廓模糊、颜色变浅呈淡黄色,甚至肝胰腺区由正常的饱满褐色组织变为无组织结构的"玻璃化"状态。组织病理观察结果显示,患病对虾肝小管上皮细胞坏死、脱落,肝小管中充斥大量的碎片组织,并逐步褐化、黑化,甚至肝小管组织大面积坏死,留有连片玻璃样均质化区域;肠道内充斥大量的组织碎片,绒毛膜脱落消失。超微组织病理观察发现,患病对虾肝小管上皮细胞的细胞膜消融,细胞器解体,细胞核固化;其后细胞解体、脱落,甚至肝小管组织结构解体消融;肝胰腺、肠道、胃黏膜周围发现大量细菌,优势菌株为杆状菌且呈弧形,未发现病毒粒子的存在。从患病虾苗分离出2株优势菌(Lv-A和Lv-B),经人工浸染实验发现,Lv-A和Lv-B可致凡纳滨对虾PL7苗种出现与自然患病相同的玻璃化症状,其半致死浓度分别为1.62×10~3和5.38×10~3 CFU/mL,致病力强。根据16S rDNA和gyrB序列分析结果发现,Lv-A和Lv-B与溶藻弧菌、新喀里多尼亚弧菌和副溶血弧菌均有较高相似性。初步将该病命名为虾苗细菌性玻化症(shrimp postlarva bacterial vitrified syndrome,BVS)。药敏实验结果显示,Lv-A和Lv-B均对米诺环素、多西环素、萘啶酸等敏感,而对新霉素、吡哌酸、利福平等耐药。本研究为BVS的有效防控、保障对虾行业健康发展提供理论基础和技术支撑。