两种弧菌对拟穴青蟹溞状幼体致病性研究

于5L试验容器内,以副溶血弧菌和溶藻弧菌通过菌浴和口服法感染拟穴青蟹溞状幼体,比较2种弧菌对拟穴青蟹溞状幼体感染的致病力。海水水温25～26℃,盐度30.1,紫外线消毒。试验结果表明,副溶血弧菌菌浴感染拟穴青蟹幼体,24h对Z1至Z5幼体半致死密度分别为4.03×105、7.67×105、8.56×105、1.98×106、8.99×106cfu/mL;溶藻弧菌菌浴拟穴青蟹幼体,24h对Z1至Z5幼体半致死密度为5.76×106、7.64×106、7.92×106、1.02×107、6.02×107 cfu/mL;口服感染试验的幼体死亡率随投喂次数和时间增加而提高。在5×106cfu/mL密度下2种弧菌的混合感染组(密度比1∶1)24h幼体死亡率低于对照副溶血弧菌感染组,高于对照溶藻弧菌组。     

江苏南部河蟹幼苗培育系统中致病性弧菌的分离诊断

从急性死亡的河蟹大眼幼体体内分离得到3q8T－2、3q7T－4等菌株，经细菌学鉴定为副溶血性弧菌，回归试验表明这几种弧菌均能感染健康动物致死亡。通过药敢试验，发现试转药1号、悉复欢等对其有较强抑制作用。     

对太平洋牡蛎弧菌病的初步研究

1991年6月大连金州海珍品增殖站育苗室,在太平洋牡蛎人工育苗期间发生了严重的流行病,幼体发育到D形幼虫阶段大量下沉,连续几批幼体出现了类似现象。显微镜观察,D形幼虫面盘肿胀,严重者面盘脱落,取发病幼体经表面并研碎后,接种在TCBS弧菌生长培养基上,经25℃、15小时恒温培养后,分离培养出3种弧菌。我们分别对3种弧菌的群体形态特征、个体形态特征做了观察,井用3种弧菌分别感染正常的太平洋牡蛎D形幼体均获得成功,同时使用十几种抗菌药物分别对3种弧菌做了药物敏感性试验。结果表明,抗菌药物土霉素、吡哌酸、氯霉素、红霉素对3种弧菌均有明显的抑制作用。     

蜡样芽孢杆菌对凡纳滨对虾幼体变态的影响

研究了1株蜡样芽孢杆菌zou8对数期细胞对凡纳滨对虾无节幼体Ⅲ期到溞状幼体Ⅱ期幼体变态的影响;并通过zou8与弧菌的平板拮抗、共培养试验探讨其促进幼体变态的机制是否与拮抗病原弧菌有关。结果表明:高数量zou8细胞对凡纳滨对虾无节幼体Ⅲ期到溞状幼体Ⅱ期幼体的变态具有极显著(P<0.01)促进作用,尤其是多次加菌;zou8促进幼体变态作用的发挥并非通过产生拮抗物质直接抑制病原弧菌,而是通过其他可能机制。     

导致对虾幼体大量死亡病原及其传播途径的初步研究

从发病育苗单位采集亲虾、卵子、各期幼体及水样进行分离培养,取可疑致病菌进行浸浴感染试验,对确定的致病菌进行生化鉴定。结果表明导致对虾幼体大量死亡的病原菌为一种荧光弧菌,其感染流行是由亲虾垂直传播造成的。     

蚤状幼体副溶血性弧菌病病原体的鉴定及防治

从患病河蟹蚤状幼体体内分离出的病原菌为副溶血性弧菌；人工回感染实验表明副溶血性弧菌是导致河蟹蚤状幼体发病的病原；其对氧哌嗪青霉素、呋哺西林高度敏感。副溶血性弧菌可以通过食物和水体感染河蟹蚤状幼体。加强消毒措施有利于对该病的预防。     

弧菌对斑节对虾虾苗致病性的研究

本文用6种弧菌对斑节对虾虾苗进行致病性试验。发现6种弧菌对各阶段幼和仔虾都有很强的致病作用。其中,对蚤状幼体致病性最强,其次是无节幼体、糠虾和仔虾。     

多种药物控制对虾育苗中的弧菌病

在对虾人工育苗期间常常出现对虾幼体因感染弧菌病而大量死亡的现象。人们为了防治该病通常投一些土霉素等药物。笔者于1990年对虾人工育苗期间在辽宁省瓦房店横山乡育苗现场通过分离幼体培育池水中主要优势弧菌种类,并针对分离出的优势弧菌做药物敏感性试验。得出以下结果。     

仿刺参幼体烂胃病及其致病原鉴定

山东、辽宁两省在仿刺参（Apostichopus japonicus）苗种培育期间的5-7月，耳状幼体易发生一种以胃壁增厚、萎缩、溃烂为主要特征的“烂胃病”。从患烂胃病的耳状幼体中分离到1株细菌LW-1，人工回接感染实验证实其具有较强的致病性，并与自然发病症状相同。通过API半自动化鉴定和常规的生理生化试验的结果表明，LW-1具有弧菌属的特征，其表型特征与灿烂弧菌（Vibrio splendidus）相似。对该菌株16S rRNA基因序列分析和构建系统发生树。结果显示，菌株LW-1与灿烂弧菌的亲缘关系最近，相似率达到99．8％。菌株LW-1可鉴定为灿烂弧菌，并视为耳状幼体烂胃病的致病原之一。通过对多起仿刺参苗期烂胃病的调查研究表明，烂胃病病原也具有多样性和复杂性，并与投喂老化腐败的单胞藻饵料有关。     

运载亲虾水体弧菌的分离与相关试验

作者由运载亲虾水体分离出弧菌119株,发现9个种,并用所得菌株做了弧菌对虾卵孵化影响及虾苗的致病性试验。结果表明:弧菌严重地影响草虾卵的孵化,对虾苗各阶段幼体都表现出很强的致病性,尤其是对蚤状幼体的致病性最强。     

厦门对虾养殖水域异养细菌和弧菌的研究

本文对厦门三个主要对虾养殖区水体中异养细菌和弧菌的数量进行了研究。结果表明，在对虾养殖水环境中，异养细菌和弧菌的数量与水温、盐度、氨氮、化学耗氧量的水体营养指数密切相关，弧菌可作为高温季节有机污染的一个指标，弧菌的数量与对虾对病有一定的关系。     

杂色鲍养殖环境中致病性弧菌分布调查

从深圳不同的杂色鲍人工养殖场采集并分类环境生物样品。应用TCBS平板分离并作生理生化初步鉴定弧菌,平板涂布法统计水样和各类生物每mL(或每g)所含弧菌总数,并应用基于16S~23SrDNA间区序列设计的4种水产病原弧菌特异性引物进行PCR,定性检测各类环境样品所携带致病性弧菌的分布状况。结果显示,弧菌广泛存在于杂色鲍养殖环境中,且杂色鲍养殖池池水中的弧菌密度大于进水口海水。在养殖环境生物中,不同环境生物中每克生物所携带的弧菌数差别很大,其中盘管虫、海蟑螂、等足类所携带的弧菌数最多,而海鞘携带的弧菌数最少。在常见的4种致病性弧菌的检测结果上,创伤弧菌和溶藻弧菌阳性率均为3.4%。通过研究弧菌在杂色鲍养殖环境中弧菌的分布特征,为杂色鲍养殖中流行性弧菌病的预防提供科学依据。     

养参池底泥弧菌数量变动及微生态制剂对其影响

2010年和2011年连续对放养相同密度刺参池塘底泥中弧菌总数的变化进行了监测,探讨微生态制剂对刺参养殖池塘底泥弧菌数量的影响.结果显示,新池塘弧菌总数在同一时期显著低于老池塘,投施微生态制剂试验组池塘底泥中弧菌总数显著低于对照组.活菌浓度为1.25×106 cfu/L时,微生态制剂效应周期为12d.微生态制剂具有很强的抑制弧菌的作用.     

大黄鱼感染致病弧菌的检测及其病害的预测预警

为预防与控制大黄鱼溃疡病的发生,本研究利用特异多重PCR技术,对象山县养殖大黄鱼的3种致病弧菌感染情况进行了流行病学调查。每月在各定点网箱组随机采集大黄鱼肝、肾、脾、肌肉等组织进行感染情况的检测。结果显示,3种致病弧菌对大黄鱼的感染率存在一定的差异性。溶藻弧菌、哈维弧菌对大黄鱼的感染率整体高于副溶血弧菌;从感染组织分析,与肝脏、肾脏相比3种致病弧菌更易感染肌肉、脾脏;3种致病弧菌对不同鱼龄的大黄鱼感染率各不相同。研究还表明,3种致病弧菌在各个采样时间点均有感染,其中7—9月为弧菌感染高峰期,而台风后的感染率较台风前都有一定提高。另外,根据大黄鱼溃疡病发生与3种致病弧菌感染率关系的研究可知,在大黄鱼溃疡病暴发前几天,弧菌的感染率都会显示出较高数值。因此,可以通过3种致病弧菌对大黄鱼感染的分子流行病学调查与分析来预测预警病害的发生和流行。     

锯缘青蟹苗池微生态环境影响因素的初步探讨

通过对锯缘青蟹苗池微生态环境和水质环境的全过程检测,探讨了总菌数、弧菌数及弧菌数/总菌数的消长规律,并对苗种培育阶段涉及的各种外源因素及控制苗池微生态环境的相关手段的有效性作了初步的探讨。结果表明:苗池水体中总菌数在Z1~Z2少量添加水不换水阶段,呈相对稳定状态,进入Z3后总菌数逐渐增加,最高达3.5×106cfu/m l;弧菌数在Z1培育阶段出现2×102cfu/m l激增至5×104cfu/m l的峰值,第二峰值出现在Z5阶段,达6×105cfu/m l;弧菌数/总菌数比值在育苗起始时仅为0.4%,之后迅速升至30%~40%,在Z5阶段,由于弧菌数量急剧上升,达到50%的高比值;NH4-N与COD检测值在育苗过程中变动较大,两者与弧菌数/总菌数比值呈正相关关系。现行育苗方式中对苗池微生态环境产生影响的主要因素是苗种培育密度,海水、饵料的处理方式和抗生素;净化功能菌和蛭弧菌应用于育苗水体具有抑制弧菌繁殖的效果。     

Comparative activity and stability under salinity conditions of different antimicrobial peptides isolated from aquatic animals

This study reports the in vitro activity of six antimicrobial peptides (AMPs) produced by aquatic animals (most marine invertebrates): tachyplesin (Tach), magainin (Mag), clavanin (Clav), penaeidin (Pen), mytilin (Myt) and antilipopolysaccharide factor (ALF) against marine vibrios, filamentous fungi and yeast. Their stability under salinity conditions and seawater was also examined. The results showed that Mag, Myt and especially Tach and ALF (minimum inhibitory concentration<1.5 μM) had a potent activity against all tested vibrio species, whereas Clav and Pen were ineffective (up to 50 μM). With respect to the antifungal activity, each AMP had a different potency according to the fungal species. In general terms, Tach was the most potent peptide, followed by Mag. Interestingly, Tach, Myt and ALF had a significant effect on the filamentous fungus Fusarium solani that could be pathogenic to marine organisms. All AMPs had a tendency to decrease or lose their activity at high salinity (>225 mM NaCl). Tach and Myt were the most stable peptides, maintaining significant activity under seawater salinity (450 mM). Curiously, all peptides lost their effect under seawater conditions. The results suggest that Tach, ALF and Myt are the most promising candidates for potential therapeutic use in farmed-marine species, because all have a significant and broad antimicrobial activity maintained at high salinity.     

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.     

Mortality event involving larvae of the carpet shell clam Ruditapes decussatus in a hatchery: isolation of the pathogen Vibrio tubiashii subsp. europaeus

Diseases caused by bacteria belonging to the genus Vibrio are a common, as yet unresolved, cause of mortality in shellfish hatcheries. In this study, we report the results of routine microbiological monitoring of larval cultures of the carpet shell clam Ruditapes decussatus in a hatchery in Galicia (NW Spain). Previous episodes of mortality with signs similar to those of vibriosis affecting other species in the installation indicated the possibility of bacterial infection and led to division of the culture at the early D‐veliger larval stage. One batch was cultured under routine conditions, and the other was experimentally treated with antibiotic (chloramphenicol). Differences in larval survival were assessed, and culturable bacterial population in clams and sea water was evaluated, with particular attention given to vibrios. Severe mortalities were recorded from the first stages of culture onwards. The pathogen Vibrio tubiashii subsp. europaeus was detected in both batches, mainly associated with larvae. Moreover, initial detection of the pathogen in the eggs suggested the vertical transmission from broodstock as a possible source. Experimental use of antibiotic reduced the presence and diversity of vibrios in sea water, but proved inefficient in controlling vibrios associated with larvae from early stages and it did not stop mortalities.     

Glycosphingolipid receptors for pathogenic vibrios in intestines of mariculture fish

Vibrios are highly motile, facultatively anaerobic bacteria that are ubiquitous in aquatic environments and part of the normal intestinal microflora of healthy fish, but some species can cause vibriosis. The adherence of vibrios to host fish intestines is a significant event not only for their survival and growth, but also in terms of pathogenicity. However, the molecular mechanism underlying the adhesion of vibrios to the intestinal tract of fish is not fully understood. We report here the identification of intestinal glycosphingolipid (GSL) receptors to which pathogenic vibrios attach in typical mariculture fish. Thin-layer chromatography overlay assays using five species of ³⁵S-labeled vibrios and intestinal glycosphingolipids of seven species of mariculture fish revealed that all of the fish tested possessed GM3 (NeuAcα2-3Galβ1-4Glcβ1-1′Cer) and/or GM4 (NeuAcα2-3Galβ1-1′Cer) as major acidic intestinal GSLs and that all of the vibrios tested specifically adhered to GM3 and/or GM4. Our results demonstrate that these GSLs were able to function as a receptor for the various vibrios tested. Analysis of the relationship between sugar structure and receptor activity for vibrios revealed that ‘NeuAcα2-3Galβ1-’ is required at the non-reducing end of glycosphingolipids for the bacteria to attach.     

Effects of Bdellovibrio and Like Organisms on Survival and Growth Performance of Juvenile Turbot,Scophthalmus maximus

In this study, the impact of Bdellovibrio and like organisms (BALOs) on juvenile turbot rearing was investigated in a static water rearing system with weekly partial change of water. A 60‐d‐long rearing experiment showed that, compared with control, survival rate, percent weight gain, percent length gain, and average daily growth were 92 ± 2.8%, 387.1 ± 4.6%, 78.6 ± 1.5%, and 1.87 ± 0.79 g/d, respectively, significantly higher than those in the control, viz., 81 ± 3.2%, 248.2 ± 5.3%, 56.7 ± 2.1%, and 1.16 ± 0.68 g/d, respectively. Water quality results demonstrated that no significant difference (P > 0.05) was observed between test and control with the addition of BDW03. Compared with the control, total culturable bacteria and total vibrio numbers in rearing water and in intestines were significantly (P < 0.05) reduced by 0.8–1.4 log CFU/mL/ log CFU/g, with the addition of BDW03. Furthermore, denaturing gradient gel electrophoresis profiles in rearing waters collected at a 1.5‐d interval over a week revealed that BDW03 simplified bacterial community structures with time, and some vibrios disappeared with the addition of BDW03. To the best of our knowledge, this is the first report that BALOs can promote growth and survival of juvenile turbots and be beneficial to coldwater fish aquaculture at the production level.